Antigen-binding proteins targeting alpha-toxin of Staphylococcus aureus and their 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 targeting the alpha-toxin of Staphylococcus aureus. [Background technology]

[0002] Staphylococcus aureus belongs to the genus Staphylococcus and is an important Gram-positive pathogen that can cause 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., tracheotomy, intravenous catheter insertion, etc.). According to data from the China Drug Resistance Surveillance Network (http: / / www.carss.cn / ) in 2019, the clinical detection rate of Staphylococcus aureus ranked 4th among all pathogens, and G + It ranks first in the detection rate of pathogenic bacteria. With the continuous development and mass application of antibacterial drugs, Staphylococcus aureus is constantly mutating, and the phenomenon of drug resistance is becoming increasingly serious.

[0003] Alpha toxin is the most important exotoxin released by Staphylococcus aureus, of which alpha hemolysis (Hla) plays the most important role in the pathogenesis. Alpha toxin is a secreted toxin protein encoded by the HLA gene of Staphylococcus aureus, is expressed in almost all strains, and is the most important virulence factor affecting the pathogenicity of Staphylococcus aureus. Alpha toxin binds to cholesterol and sphingomyelin in the host cell membrane to form a heptamer, which then folds to form a beta barrel transmembrane structure with a diameter of about 1.5 nm, rapidly lysing the host's red blood cells and other tissue cells. At the same time, alpha toxin can destroy white blood cells in the infected tissue and prevent the host from eliminating the infected Staphylococcus aureus. With the action of the host's immune system and antibiotics, Staphylococcus aureus at the site of infection releases a large amount of alpha toxin, which, when they enter the blood, activate the host's immune system and release excessive inflammatory factors, resulting in sepsis. Summary of the Invention [Problem to be solved by the invention]

[0004] Currently, the increasing resistance of S. aureus to antibiotics has complicated the problem of S. aureus infections, and in the clinical treatment of S. aureus infections, small molecule anti-infective drugs cannot effectively treat toxin-induced body damage and sepsis, so there is an urgent need to develop effective alternative methods for diagnosing and treating S. aureus infections. [Means for solving the problem]

[0005] Summary of the Invention The present application provides isolated antigen-binding proteins targeted to S. aureus alpha-toxin, and having one or more properties selected from: 1) the ability to specifically bind to S. aureus alpha-toxin; 2) the ability to neutralize the biological activity of S. aureus alpha-toxin; 3) the ability to have a pharmacodynamic effect on diseases or symptoms caused by S. aureus infection; and 4) a biological function superior to currently disclosed S. aureus antibody molecules. The present application further provides nucleic acid molecules encoding the isolated antigen-binding proteins, expression vectors, host cells, immunoconjugates comprising the antigen-binding proteins, pharmaceutical compositions, methods of preparing the isolated antigen-binding proteins, and applications of the isolated antigen-binding proteins described in the present application.

[0006] Meanwhile, the present invention provides an isolated antigen binding protein, comprising an HCDR3, wherein the 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 an HCDR2, wherein the HCDR2 comprises the amino acid sequence depicted 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 an HCDR1, wherein the HCDR1 comprises the amino acid sequence depicted 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 the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:53 or SEQ ID NO:28, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:52 or SEQ ID NO:27, and the HCDR3 comprises the amino acid sequence set forth 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 said HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:16 and SEQ ID NO:28, said HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:15 and SEQ ID NO:27, and said HCDR3 comprises the amino acid sequence set forth 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 an 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 set forth in SEQ ID NO: 16, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, and the HCDR3 comprises the amino acid sequence set forth 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, wherein the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, 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 set forth 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.

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

[0017] In some embodiments, the H-FR2 comprises an amino acid sequence set forth 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.

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

[0019] In some embodiments, the H-FR3 comprises an amino acid sequence set forth 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 an H-FR4, wherein the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and wherein the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:59.

[0021] In some embodiments, the H-FR4 comprises an amino acid sequence set forth 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, wherein said H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:54, said H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:55 or SEQ ID NO:56, said H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:57 or SEQ ID NO:58, and said H-FR4 comprises the amino acid sequence set forth 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 said H-FR1 comprises the amino acid sequence set forth 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, said H-FR2 comprises the amino acid sequence set forth 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, said H-FR3 comprises the amino acid sequence set forth 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 said H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NO:7, SEQ ID NO: 11, comprising the amino acid sequence shown in any one of 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) said H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 4, said H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 5, said H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 6, and said H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 7; 2) said H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, said H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 9, said H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 10, and said H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 11; 3) said H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, said H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 18, said H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 19, and said H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 20; 4) said H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, said H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, said H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 22, and said H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 23; 5) said H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, said H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, said H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 31, and said H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 32; and 6) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 33, 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: 35, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 36.

[0025] In some embodiments, the isolated antigen binding protein comprises a VH, wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:60 or SEQ ID NO:61.

[0026] In some embodiments, the VH comprises an amino acid sequence set forth 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.

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

[0028] In some embodiments, the VHH comprises an amino acid sequence as set forth 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.

[0029] In some embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region.

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

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

[0032] In some embodiments, the isolated antigen binding protein comprises an amino acid sequence set forth 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.

[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 a Fab, a Fab', an Fv fragment, a Bs-Fv, F(ab')2, F(ab)2, a scFv, a di-scFv, and / or a dAb.

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

[0036] In some embodiments, the isolated antigen binding protein is capable of specifically binding to S. aureus alpha-toxin.

[0037] In some embodiments, the isolated antigen binding protein is capable of neutralizing the biological activity of S. aureus alpha-toxin.

[0038] In some embodiments, the isolated antigen binding protein is capable of preventing and / or treating a disease and / or condition and its complications.

[0039] In some embodiments, the disease and / or condition and its complications are caused or mediated by S. aureus alpha-toxin.

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

[0041] However, the present application further provides a polypeptide molecule comprising said isolated antigen binding protein.

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

[0043] In some embodiments, the polypeptide molecule comprises the amino acid sequence set forth in any one of SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67.

[0044] However, the present application further provides an immunoconjugate comprising said isolated antigen-binding protein.

[0045] Meanwhile, the present application further provides a nucleic acid molecule encoding said isolated antigen binding protein or said polypeptide molecule.

[0046] On the other hand, the present application further provides a vector comprising said nucleic acid molecule. Meanwhile, the present application further provides a cell comprising said nucleic acid molecule or said vector.

[0047] Meanwhile, the present application further provides a pharmaceutical composition comprising said isolated antigen-binding protein, said polypeptide molecule, said immunoconjugate, said nucleic acid molecule, said vector and / or said cell, and optionally a pharma- ceutically acceptable carrier.

[0048] Meanwhile, the present application further provides a method of preparing said isolated antigen binding protein, comprising culturing said cell under conditions to express said antigen binding protein.

[0049] Meanwhile, the present application further provides the use of said isolated antigen-binding protein, said polypeptide molecule, said immunoconjugate, said nucleic acid molecule, said vector, said cell and / or said pharmaceutical composition in the manufacture of a medicament for the prevention and / or treatment of a disease and / or condition and complications thereof.

[0050] In some embodiments, the disease and / or condition and its complications are caused or mediated by S. aureus alpha-toxin.

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

[0052] Alternatively, the antigen-binding proteins, the polypeptide molecules, the immunoconjugates, the nucleic acid molecules, the vectors, the cells and / or the pharmaceutical compositions described in the present application may be used alone or in combination with other molecules.

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

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

[0055] Meanwhile, the present application further provides the use of said isolated antigen-binding protein, said polypeptide molecule, said immune complex, said nucleic acid molecule, said vector, said cell and / or said pharmaceutical composition in the manufacture of a reagent kit for detecting the presence and / or content of Staphylococcus aureus alpha-toxin in a sample.

[0056] Those skilled in the art can readily recognize other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As will be understood by those skilled in the art, the contents of this application may enable those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the drawings and description of the present application are merely illustrative and not restrictive.

[0057] BRIEF DESCRIPTION OF THE DRAWINGS Specific features of the invention related to this application are set forth in the appended claims. The features and advantages of the invention related to this application can be better understood with reference to the exemplary embodiments described in detail below and the accompanying drawings, the brief description of which is as follows: [Brief description of the drawings]

[0058] [Figure 1] FIG. 1 shows the detection of the binding ability of the antigen-binding proteins described in this application to Staphylococcus aureus alpha-toxin protein by ELISA assay. [Diagram 2] FIG. 2 shows the neutralizing effect of antigen-binding proteins described in this application on the hemolytic activity of recombinant S. aureus alpha-toxin. [Diagram 3] FIG. 3 shows the therapeutic effect of the antigen binding protein described in this application on S. aureus alpha-toxin induced sepsis in mice. [Figure 4] FIG. 4 shows the therapeutic effect of antigen binding proteins described in this application against S. aureus alpha-toxin induced bacteremia in mice. [Diagram 5] FIG. 5 shows the detection of the binding ability of the antigen-binding proteins described in this application to Staphylococcus aureus alpha-toxin protein by ELISA assay. [Figure 6] FIG. 6 shows the neutralizing effect of antigen-binding proteins described in this application on the hemolytic activity of recombinant S. aureus alpha-toxin. [Figure 7] FIG. 7 shows the therapeutic effect of the antigen binding proteins described in this application on S. aureus alpha-toxin induced sepsis in mice. [Figure 8] FIG. 8 shows the therapeutic effect of antigen binding proteins described in this application against S. aureus alpha-toxin induced bacteremia in mice. [Figure 9] FIG. 9 shows the neutralizing effect of the tetravalent antigen binding proteins described in this application on the hemolytic activity of S. aureus alpha-toxin. [Figure 10] FIG. 10 shows the neutralizing effect of the tetravalent antigen binding proteins described in this application on the hemolytic activity of S. aureus alpha-toxin. [Figure 11] FIG. 11 shows the neutralizing effect of the tetravalent antigen binding proteins described in this application on the hemolytic activity of S. aureus alpha-toxin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present application will be described in detail with reference to specific examples, so that those skilled in the art can easily understand the advantages and effects of the present invention based on the contents disclosed in the present specification.

[0060] Term definition In this application, the term "Staphylococcus aureus" can be used interchangeably with "Staphylococcus aureus" and "S. aureus", which generally belong to Gram-positive bacteria. In this application, the term covers all types and forms of Staphylococcus aureus and its variants, homologues, and functionally active fragments.

[0061] In this application, the term "S. aureus alpha-toxin", also referred to as "alpha-toxin", "alpha-hemolysin", "hemolysin A", "Hla", or "hemolysin A toxin", generally refers to the pore-forming protein hemolysin produced by S. aureus. In this application, the S. aureus alpha-toxin may include wild-type S. aureus alpha-toxin and its mutants, derivatives, homologs, recombinant proteins, monomers, oligomers, and other forms. The oligomeric forms may include forms other than the monomeric form of alpha-toxin, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, and other forms of alpha-toxin, or multimeric forms such as the heptameric prepore form.

[0062] In this application, the term "isolated" generally means obtained in a natural state by artificial means. When a particular "isolated" substance or component occurs in nature, the natural environment in which it exists may have been altered, or the substance may have been isolated from the natural environment, or both. For example, a non-isolated polynucleotide or polypeptide may naturally occur in an animal organism, and the same polynucleotide or polypeptide of high purity isolated in this naturally occurring state is called isolated. The term "isolated" does not exclude the presence of artificial or synthetic mixtures, or other impurities that do not affect the activity of the substance.

[0063] In this application, the term "isolated antigen-binding protein" refers to a protein having antigen-binding ability, generally free from its naturally occurring state. The "isolated antigen-binding protein" may include an antigen-binding portion and, optionally, a framework or framework portion that allows the antigen-binding portion to adopt a conformation that promotes binding to an antigen. The antigen-binding protein may include, for example, a protein framework region (FR) derived from an antibody, or an alternative protein framework region or an artificial framework region with grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to, framework regions derived from antibodies in which mutations have been introduced to stabilize the three-dimensional structure of the antigen-binding protein, and completely synthetic framework regions, including, for example, biocompatible polymers. See, e.g., Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Illustrative 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 fragments thereof.

[0064] In this application, the term "CDR", also referred to as "complementarity determining region", generally refers to a region in the variable domain of an antibody, which is highly variable in sequence 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, a naturally occurring camelid antibody consisting of only heavy chains may function normally and be stable 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, such as CCG, Kabat, AbM, Chothia, IMGT, and the combined consideration of Kabat / Chothia. These coding systems are well known in the art, see in particular http: / / www.bioinf.org.uk / abs / index.html#kabatnum. For example, the numbering of the amino acid sequence of the antigen-binding protein can be determined according to 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 CDRs of the antigen-binding protein can be determined by the Kabat numbering system (see, e.g., 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 the more highly conserved parts of antibody variable domains, known as framework regions. Typically, naturally occurring heavy and light chain variable domains each contain four FR regions, four in VH (H-FR1, H-FR2, H-FR3 and H-FR4) and four in VL (L-FR1, L-FR2, L-FR3 and L-FR4).

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

[0067] In this application, the term "variable" generally refers to the fact that certain segments of the variable domains may differ substantially in sequence between antibodies. The variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains. 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 portions of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions, which usually adopt a β-pleated sheet structure, forming a cyclic link and sometimes forming part of the β-pleated sheet structure. The CDRs in each chain are held in close proximity by the FR regions and, together with the CDRs in the other chain, facilitate the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

[0068] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, and encompasses 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, polyspecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. For purposes of the present invention, unless modified by the term "intact", such as "intact antibody", the term "antibody" also includes antibody fragments, such as, for example, Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function (e.g., specifically binds to Staphylococcus aureus alpha-toxin). Typically, such fragments include an antigen-binding domain. The basic four-chain 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 heterotetramer units and another polypeptide called J chain and contain 10 antigen-binding sites, while IgA antibodies contain 2-5 basic 4-chain units that can combine with J chains and polymerize to form multivalent aggregates. In the case of IgG, the 4-chain unit is usually about 150,000 daltons. Each L chain is linked to a H chain by a covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the isotype of the H chain. Each H and L chain also has regularly spaced interchain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for the α and γ chains, respectively, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains. The pairing of VH and VL forms an 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. L chains from any vertebrate species can be classified into one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domain. Immunoglobulins can be assigned to different classes or isotypes depending on the amino acid sequence of the heavy chain (CH) constant domain. There are currently five types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains called α, δ, ε, γ and μ, respectively.

[0069] In the present application, the term "antigen-binding fragment" generally refers to one or more fragments capable of specifically binding to an antigen (e.g., S. aureus alpha-toxin). In the present application, said 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 an antigen-binding fragment of an antibody. As described above, intact antibodies can be digested with papain. After an antibody is digested with papain, two identical antigen-binding fragments are produced: a "Fab" fragment and a remaining "Fc" fragment (i.e., as above for the Fc region). The Fab fragment can consist of the complete light and heavy chain variable regions, as well as the first constant region (CH1) of the heavy chain (VH).

[0071] In this application, the term "Fab' fragment" generally 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 contain all of the light chain, all of the variable domain of the heavy chain, and the first and second constant domains of all or part of the heavy chain. For example, a Fab' fragment may contain some or all of amino acid residues 220-330 of the heavy chain.

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

[0073] In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which contains all or a portion of the heavy and light chain variable regions and lacks the heavy and light chain constant regions. The heavy and light chain variable regions include, for example, the CDRs. For example, the Fv fragment contains all or a portion of the amino-terminal variable regions of the heavy and light chains, about 110 amino acids.

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

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

[0076] In the present application, the term "VHH" generally relates to variable antigen-binding domains derived from heavy chain antibodies of the Camelidae family (camel, dromedary, llama, alpaca, etc.) (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and for review Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHHs are also referred to as Nanobodies (Nbs).

[0077] In this application, the term "monoclonal antibody" generally refers to a preparation of antibody molecules of single molecular composition. Generally, monoclonal antibodies are highly specific for a single antigenic site. And, unlike conventional polyclonal antibody preparations (which usually have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by the culture of a hybridoma, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies as used in this application can be produced in hybridoma cells or can be produced by recombinant DNA methods.

[0078] In this application, the term "chimeric antibody" generally refers to an antibody whose variable regions are derived from one species and whose constant regions are derived from another species. Usually, the variable regions are derived from an antibody of a laboratory animal such as a rodent (the "parent antibody"), and the constant regions are derived from a human antibody, thus reducing the likelihood that the resulting chimeric antibody will provoke an adverse immune response in a human individual compared to the parent (e.g., from an alpaca) antibody.

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

[0080] The term "fully humanized antibody" generally refers to an antibody that contains only human immunoglobulin sequences. If produced in a mouse, mouse cell, or a hybridoma derived from a mouse cell, a fully humanized antibody may contain mouse glycosylation. Similarly, a "mouse antibody" or a "rat antibody" refers to an antibody that contains only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans, in transgenic animals with human immunoglobulin germline sequences, by phage display or other molecular biology 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 using libraries, are well known in the art.

[0081] In this application, the terms "polypeptide molecule", "polypeptide" and "peptide" can be used interchangeably and generally refer to a polymer of amino acid residues. The term "fusion protein" generally refers to a polypeptide having at least two covalently linked portions, each of which can be a polypeptide with different properties. The properties can be biological properties, such as in vitro or in vivo activity. The properties can also be simple chemical or physical properties, such as binding to a target molecule, catalysis of a reaction, etc. The two portions can be directly linked by a single peptide bond or by a peptide linker.

[0082] In this application, the term "nucleic acid molecule" generally refers to nucleotides of any length, either deoxyribonucleotides or ribonucleotides, in an isolated form or analogues either isolated from their natural environment or artificially synthesized.

[0083] In this application, the term "vector" generally refers to a nucleic acid delivery tool into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed. A vector can transform, transduce, or transfect a host cell to express the genetic material element that it carries in the host cell. By way of example, 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 phage or M13 phage, and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (such as SV40). A vector may contain multiple elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. A vector may also contain an origin of replication. Vectors may further comprise components that aid in entry into cells, such as, but not limited to, a viral particle, a liposome, or a protein membrane.

[0084] In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that can be or has been a recipient of a subject's plasmid or vector, including a nucleic acid molecule described in the present invention or a vector described in the present invention. A cell may include the progeny of a single cell. Due to natural, accidental, or deliberate mutations, the progeny is not necessarily completely identical (either in the form of the total DNA complement or in the genome) to the original parent cell. A cell may include a cell that has been transfected in vitro with a vector of the present application. A cell may be a bacterial cell (e.g., E. coli), a yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NS0 cell, etc.

[0085] In this application, the term "immunoconjugate" generally refers to a complex formed by conjugating (e.g., covalently binding by a binding molecule) said other reagent (e.g., chemotherapeutic agents, radioactive elements, cytostatic agents and cytotoxic agents) to said antibody or antigen-binding fragment thereof, which is capable of delivering said other reagent to a target cell (e.g., a tumor cell) by specifically binding said antibody or antigen-binding fragment thereof 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 condition. The pharmaceutical composition may comprise an isolated antigen-binding protein described herein, a nucleic acid molecule described herein, a vector described herein, and / or a cell described herein, and an optional pharma- ceutically acceptable adjuvant. The pharmaceutical composition may further comprise an appropriate formulation of one or more (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 dosages and concentrations used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid compositions, frozen compositions, and lyophilized compositions.

[0087] In this application, the term "pharmaceutically acceptable carrier" generally includes pharma- ceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed at the dosages 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 non-ionic surfactants.

[0088] In this application, the term "specifically binds" or "specific" generally refers to a measurable and reproducible interaction, such as binding of an antibody with a target, that allows the presence of the target to be determined 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 be an antibody that binds to that target with higher affinity, avidity, more readily, and / or for a longer period of time than it binds to other targets. In some embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In some embodiments, specific binding may include exclusive binding, but this is not required.

[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, cow, sheep, rabbit, mouse, rat, or monkey.

[0090] In the present application, such proteins, polypeptides and / or amino acid sequences should be understood to include variants or homologues having at least the same or similar function as the protein or polypeptide in question.

[0091] In the present application, the variant may be, for example, a protein or polypeptide obtained by substitution, deletion, or addition of one or more amino acids in the amino acid sequence of the protein and / or polypeptide (e.g., an antibody or fragment thereof that specifically binds to S. aureus alpha-toxin). For example, the functional variant may include a protein or polypeptide having an amino acid modification by substitution, deletion, and / or insertion of at least 1, for example 1-30, 1-20, or 1-10, even for example 1, 2, 3, 4, or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or polypeptide before modification (e.g., substitution, deletion, or addition). For example, the functional variant may 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 the present 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 S. aureus alpha-toxin).

[0093] In this application, the homology generally refers to the closeness, similarity, or relatedness between two or more sequences. The method of calculating "percent sequence homology" is to compare two sequences aligned within a comparison window, determine the number of positions 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 in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions within the comparison window (i.e., the window size), and multiply the result by 100 to calculate the percent sequence homology. Alignment for determining percent sequence homology can be achieved in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared or within a region of the target sequence. The homology may be measured by FASTA and BLAST. For a description of the FASTA algorithm, see W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Comparison," Proc. Natl. Acad. Sci., 85:2444-2448, 1988; and D.J. Lipman and W.R. Pearson, "Fast and Sensitive Protein Similarity Searching," Science, 227:1435-1441, 1989. For a description of the BLAST algorithm, see S. Altschul, W. Gish, W. Miller, E.W. Myers and D. Lipman, "Basic Local Alignment Search Tools," Journal of Molecular Biology, 215:403-410, 1990.

[0094] In this application, the term "comprise" generally means "comprise", "contain", or "include". In some cases, it also means "is" or "consists of".

[0095] In this application, the term "about" generally means a variation of 0.5%-10% above and below the specified value, for example, a variation 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 and below the specified value.

[0096] Detailed Description of the Invention Isolated antigen-binding proteins The CDR of an antibody is also called the complementarity determining region, and is a part of the variable region. The amino acid residues of the region may contact the antigen or the epitope of the antigen. The antibody CDR can be determined by various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and the 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 example. Those skilled in the art can determine the CDR region using different coding systems according to the sequence and structure of the antibody. The CDR region may differ depending on the coding system. In the present application, CDR encompasses the CDR sequence divided according to any CDR division method; it also encompasses variants of the amino acid sequence of the CDR in which one or more amino acids are substituted, deleted, and / or added. For example, 1-30, 1-20, or 1-10, further for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids are substituted, deleted, and / or inserted; further including homologs thereof, which can 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 higher) sequence identity to the amino acid sequence of the CDR. In some embodiments, the isolated antigen binding proteins described herein are defined by the Chothia coding system.

[0097] Alternatively, the present application provides an isolated antigen binding protein comprising an HCDR3. In some embodiments, the HCDR3 may comprise the amino acid sequence depicted in SEQ ID NO: 1. In some embodiments, the HCDR3 may comprise the amino acid sequence depicted in SEQ ID NO: 14. In some embodiments, the HCDR3 may comprise the amino acid sequence depicted in SEQ ID NO: 26.

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

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

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

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

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

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

[0104] For example, 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.

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

[0106] For example, 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.

[0107] In the present application, the isolated antigen-binding protein may comprise 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, and the CDR may include CDRs obtained by splitting in any manner, including but not limited to the CDRs obtained by the splitting methods shown in the table below. CDRs split in any manner are all within the scope of protection of the claims of the present application as long as the sequence is identical to 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 dividing the amino acid sequence set forth in SEQ ID NO:12 or SEQ ID NO:13 in the manner set forth in Table 1, an isolated antigen binding protein according to the present application may comprise the CDRs set forth in the table below.

[0109] [Table 1]

[0110] For example, by dividing the amino acid sequence set forth in SEQ ID NO:24 or SEQ ID NO:25 in the manner set forth in Table 2, an isolated antigen binding protein according to the present application may comprise the CDRs set forth in the table below.

[0111] [Table 2]

[0112] For example, by dividing the amino acid sequence set forth in SEQ ID NO:37 or SEQ ID NO:38 in the manner set forth in Table 3, an isolated antigen binding protein according to the present application may comprise the CDRs set forth in the table below.

[0113] [Table 3]

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

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

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

[0117] In the present application, the isolated antigen binding protein can comprise an H-FR2, and the H-FR2 can be located between the HCDR1 and the HCDR2. In some embodiments, the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 56.

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

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

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

[0121] In the present application, the isolated antigen binding protein can comprise H-FR4, and the N-terminus of the H-FR4 can be linked to the C-terminus of the HCDR3. In some embodiments, the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO:59.

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

[0123] In the present application, the isolated antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4. For example, the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO:54, the H-FR2 may comprise the amino acid sequence set forth in SEQ ID NO:55 or SEQ ID NO:56, the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO:57 or SEQ ID NO:58, and the H-FR4 may comprise the amino acid sequence set forth 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, wherein the H-FR1 may comprise the amino acid sequence set forth 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 may comprise the amino acid sequence set forth 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 may comprise the amino acid sequence set forth 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 may comprise the amino acid sequence set forth in any one of SEQ ID NO:7 ... It may comprise the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:32 and SEQ ID NO:36.

[0125] In the present application, the isolated antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4. For example, the H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen binding protein may comprise the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively. For example, the H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen binding protein may comprise the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively. For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen binding protein may comprise the amino acid sequences set forth in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, respectively. For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen binding protein may comprise the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, respectively. For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen binding protein may comprise the amino acid sequences set forth in SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, respectively. For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen binding protein can comprise the amino acid sequences set forth in SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, respectively.

[0126] In the present application, the isolated antigen binding protein may comprise a VH. In some embodiments, the VH may comprise the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the VH may comprise the amino acid sequence set forth in SEQ ID NO: 61.

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

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

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

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

[0131] In the present 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 comprise a Fab, a Fab', an Fv fragment, a F(ab')2, a F(ab)2, a scFv, a bs-Fv, a di-scFv, and / or a dAb.

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

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

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

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

[0137] In the present application, the isolated antigen-binding protein may comprise a tetravalent antigen-binding protein and / or a tetravalent antigen-binding protein Fc fusion protein. For example, two sequences encoding anti-alpha toxin antigen-binding protein variable regions (e.g., two huA3 molecules or two huG1 molecules, or one huA3 molecule and one huG1 molecule, as described in the present application; e.g., two A3 molecules or two G1 molecules, or one A3 molecule and one G1 molecule, as described in the present application) can be tandemly connected and fused to a human IgG1 Fc fragment. For example, the C-terminus of the first VHH can be connected to the N-terminus of the second VHH, and the C-terminus of the second VHH can be connected to the N-terminus of the Fc fragment to obtain a bivalent Fc fusion protein, which can be subcloned into a plasmid and transfected into a cell for recombinant expression to obtain a tetravalent antigen-binding protein as described in the present application. For example, the isolated antigen binding protein may comprise a huA3-huG1-Fc antigen binding protein. For example, the isolated antigen binding protein may comprise a huA3-huA3-Fc antigen binding protein. For example, the isolated antigen binding protein may comprise a huG1-huG1-Fc antigen binding protein. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:65. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:66. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:67.

[0138] It should be noted that the isolated antigen binding proteins described in the present application may comprise 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 change the binding characteristics of the antibody. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications may be introduced into the antibodies of the present invention by standard techniques known in the art, such as by site-directed mutagenesis or PCR-mediated directed mutagenesis. Conservative amino acid substitutions are those that replace an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are already known in the art. These families include amino acids containing alkaline side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDR regions of the isolated antigen binding proteins described in the present application can be replaced with other amino acid residues from the same side chain family. As will be known to those of skill 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 S. aureus alpha-toxin antigen binding proteins described herein can be identified, screened, or characterized by a variety of detection methods known in the art.

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

[0141] In the present application, the isolated antigen binding protein can specifically bind to S. aureus alpha-toxin. In some embodiments, the binding of the isolated antigen binding protein to S. aureus alpha-toxin can be detected by ELISA. For example, the antigen binding proteins described in the present application have an EC of about 0.010 μg / mL or less, about 0.009 μg / mL or less, about 0.008 μg / mL or less, about 0.007 μg / mL or less, about 0.006 μg / mL or less, about 0.005 μg / mL or less, about 0.004 μg / mL or less, about 0.003 μg / mL or less, about 0.002 μg / mL or less, and about 0.001 μg / mL or less. 50 It can bind to Staphylococcus aureus alpha-toxin at this value.

[0142] In the present application, the isolated antigen-binding protein can neutralize the biological activity of S. aureus alpha-toxin. For example, rabbit red blood cells can be added to S. aureus alpha-toxin and the antigen-binding protein, and the anti-hemolytic activity can be measured using a microplate reader. For example, the antigen-binding protein described in the present application can completely neutralize 18.75 ng of recombinant S. aureus alpha-toxin at an amount of about 200 ng or less, about 100 ng or less, about 75 ng or less, about 60 ng or less, about 50 ng or less, about 45 ng or less, about 40 ng or less, or about 35 ng or less.

[0143] In the present application, the isolated antigen binding protein can prevent and / or treat a disease and / or condition and its complications. In some embodiments, the disease and / or condition and its complications can be caused or mediated by S. aureus alpha-toxin. In some embodiments, the disease and / or condition and its complications can include sepsis and / or bacteremia.

[0144] Polypeptide molecules, nucleic acid molecules, vectors, cells, immunoconjugates and pharmaceutical compositions On the other hand, the present application provides polypeptide molecules that can comprise the isolated antigen binding proteins described in the present application.

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

[0146] In some embodiments, the polypeptide molecule comprises two or more of the isolated antigen binding proteins described in any one of the present applications, and a fusion protein consisting of two or more of the isolated antigen binding proteins described in any one of the present applications 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 which the first antigen-binding domain may comprise an isolated antigen-binding protein as described in any one of the claims herein and the second antigen-binding domain may comprise an isolated antigen-binding protein as described in any one of the claims herein.

[0148] In some embodiments, the polypeptide molecule may comprise the amino acid sequence set forth in any one of SEQ ID NO:65, SEQ ID NO:66, and SEQ ID NO:67.

[0149] In contrast, the present application provides an isolated nucleic acid molecule capable of encoding an isolated antigen binding protein described herein, e.g., that has been (i) amplified in vitro, e.g., by polymerase chain reaction (PCR), (ii) recombinantly produced, e.g., by cloning, (iii) purified, e.g., by enzymatic cleavage and gel electrophoretic fractionation, or (iv) synthetically produced or synthesized, e.g., chemically synthesized.

[0150] On the other hand, the present application provides a vector that may contain the nucleic acid molecule described in the present application. The vector may also contain other genes, such as, for example, marker genes that allow the selection of the vector under suitable conditions in a suitable host cell. In addition, the vector may also contain expression control elements that allow the appropriate expression of the coding region in a suitable host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, etc. The vector may transform, transduce, or transfect a host cell to express the genetic material elements that it carries in the host cell. The vector may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used, for example, in genetic engineering. For example, the vector is an expression vector. The vector may also include, but is not limited to, components that aid in cell entry, such as, for example, viral particles, liposomes, or protein membranes.

[0151] Alternatively, the present application provides cells that may include the nucleic acid molecules described herein or the vectors described herein. In some embodiments, each or each host cell may include one or more of the nucleic acid molecules or vectors described herein. In some embodiments, each or each host cell may include a plurality (e.g., two or more) or a plurality (e.g., two or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein may be introduced into the host cell, e.g., a eukaryotic cell, e.g., a cell derived from a plant, a fungus or a yeast cell. In some embodiments, the cell may be a bacterial cell (e.g., E. coli), a yeast cell, or other eukaryotic cell, e.g., a COS cell, a Chinese hamster ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a 293T cell, a COS-1 cell, a SP2 / 0 cell, a NS0 cell, or a myeloma cell. The vectors described herein may be introduced into the host cell by methods known in the art, e.g., electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0152] On the other hand, the present application provides immunoconjugates that can include the isolated antigen-binding proteins described in the present application.

[0153] In some embodiments, the isolated antigen binding protein or fragment thereof described herein can be conjugated to another agent, such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, a spectroscopic probe, etc. The conjugation can be via one or more covalent or non-covalent interactions and can include chelation. Various linkers known in the art can be used to form the immunoconjugate. Furthermore, the immunoconjugate can be provided in the form of a fusion protein that can be expressed by a polynucleotide encoding the immunoconjugate. The immunoconjugate can further include, for example, an antibody-drug conjugate (ADC). Suitable drugs can include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA crosslinking agents, 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 an ADC, the antibody and the therapeutic agent can be crosslinked by a cleavable linker, such as a peptide linker, a disulfide linker, or a hydrazone linker.

[0154] Meanwhile, the present application further provides pharmaceutical compositions, which may comprise an isolated antigen binding protein described in the present application, a polypeptide molecule described in the present application, an immunoconjugate described in the present application, a nucleic acid molecule described in the present application, a vector described in the present application, and / or a cell described in the present application, and an optional pharma- ceutically acceptable carrier.

[0155] In some embodiments, the pharmaceutical composition may further comprise a suitable formulation of one or more (pharmaceutical effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. Preferably, the acceptable components of the composition are non-toxic to recipients at the dosages and concentrations used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid compositions, frozen compositions, and lyophilized compositions.

[0156] In some embodiments, the pharmaceutical composition may contain two or more active compounds, which are generally active compounds that have complementary activities 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, and on the clinical parameters of the subject.

[0157] In some embodiments, the pharma- ceutically acceptable carrier includes any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents compatible with drug administration, and are generally safe and non-toxic.

[0158] In some embodiments, the pharmaceutical composition may include parenteral, transdermal, intracavitary, intraarterial, intrathecal and / or intranasal administration, or direct injection into tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, administration of the pharmaceutical composition may be accomplished in a variety of ways, for example, intravenously, intraperitoneally, subcutaneously, intramuscularly, topically, or intradermally. In some embodiments, the pharmaceutical composition may be administered without interruption. Uninterrupted (or continuous) administration may be accomplished by a miniature pump system worn by the patient, which may meter the flow of therapeutic agent into the patient's body, for example as described in WO2015 / 036583.

[0159] Preparation method Meanwhile, the present application provides a method for preparing said isolated antigen-binding protein, said method comprising culturing a host cell described in the present application under conditions for expressing said antigen-binding protein. For example, methods such as a suitable medium, a suitable temperature and a suitable incubation time 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 the present application. For example, animals can be immunized with conjugated or naturally occurring S. aureus alpha-toxin or fragments thereof. Any suitable immunization method can be used, including adjuvants, immunostimulants, and repeated inoculations for immune enhancement, and one or more methods can be used. In some embodiments, immunized alpaca peripheral blood lymphocytes are extracted, and nucleic acid fragments of the cells are extracted and cloned into vectors, and the isolated antigen-binding proteins against S. aureus alpha-toxin are screened and enriched by a phage surface display system.

[0161] Any suitable form of S. aureus alpha-toxin can also be used as an immunogen (antigen) to generate non-human antibodies specific to S. aureus alpha-toxin and screen the biological activity of the antibodies. For example, the eliciting immunogen can be full-length S. aureus alpha-toxin, including the natural homodimer, or single / multiple epitope-containing peptides. The immunogen can be used alone or in combination with one or more immunogenicity enhancing agents known in the art.

[0162] Methods and Applications Meanwhile, the present application provides the use of said isolated antigen-binding protein, said polypeptide molecule, said nucleic acid molecule, said vector, said cell, said immunoconjugate and / or said pharmaceutical composition in the manufacture of a medicament for the prevention and / or treatment of a disease and / or condition and complications thereof.

[0163] Meanwhile, the present application further provides a method for preventing and / or treating a disease and / or condition and complications thereof comprising administering to a subject in need thereof an isolated antigen binding protein, said polypeptide molecule, said nucleic acid molecule, said vector, said cell, said immunoconjugate and / or said pharmaceutical composition described herein.

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

[0165] In the present application, said administration can be performed in various ways, for example, intravenously, intratumorally, intraperitoneally, subcutaneously, intramuscularly, topically or intradermally.

[0166] On the other hand, the isolated antigen-binding proteins, said polypeptide molecules, said nucleic acid molecules, said vectors, said cells, said immunoconjugates and / or said pharmaceutical compositions described in the present application can be used for the prevention and / or treatment of diseases and / or conditions.

[0167] In the present application, said diseases and / or conditions can be caused or mediated by S. aureus alpha-toxin.

[0168] In the present application, said disease and / or condition may be a complication of a disease and / or condition caused or mediated by Staphylococcus aureus.

[0169] In the present application, said diseases and / or conditions and complications thereof include bacterial infections.

[0170] In the present application, said disease and / or condition and complications thereof include bacteremia and / or sepsis.

[0171] In some embodiments, the isolated antigen binding proteins, the polypeptide molecules, the nucleic acid molecules, the vectors, the cells, the immunoconjugates and / or the pharmaceutical compositions described in the present 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 alpha-toxin in a sample, the detection method comprising administering the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell, the immune complex and / or the pharmaceutical composition.

[0173] In some embodiments, the method for detecting S. aureus alpha-toxin in a sample can be an in vitro method. For example, an isolated antigen binding protein described herein is contacted with an ex vivo sample to detect the presence and / or content of S. aureus alpha-toxin in the sample. In some embodiments, the method for detecting S. aureus alpha-toxin in a sample is not for therapeutic purposes. In some embodiments, the method for detecting S. aureus alpha-toxin in a 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 alpha-toxin in a sample, comprising said isolated antigen-binding protein, said polypeptide molecule, said nucleic acid molecule, said vector, said cell, said immune complex and / or said pharmaceutical composition.

[0175] Meanwhile, the present application further provides an application of said isolated antigen-binding protein, said polypeptide molecule, said nucleic acid molecule, said vector, said cell, said immune complex and / or said pharmaceutical composition in the manufacture of a reagent kit for detecting the presence and / or content of Staphylococcus aureus alpha-toxin in a sample.

[0176] The following examples are not limited by theory, but are merely intended to illustrate the antigen-binding proteins, preparation methods and applications of the present application, and are not intended to limit the scope of the present invention. EXAMPLES

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

[0178] [Table 4]

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

[0180] Example 3 Panning against Staphylococcus aureus alpha-toxin antigen-binding protein A 96-well plate was coated with α-toxin fusion protein and stored at 4°C overnight. The next day, it was blocked with 5% BSA and 100 μL of phage was added. It was allowed to react at room temperature for 1 h and then panned with PBST to remove unbound phages. Finally, the phages that specifically bound to α-toxin were dissociated with triethylamine groups and infected with logarithmic growth phase E. coli TG1 to generate and purify the phages for the next screening. The same screening process was repeated 3-4 times. This enriched the positive clones and achieved the goal of screening α-toxin-specific antibodies in the antibody library using phage display technology.

[0181] Example 4 Screening of specific single positive clones by phage enzyme-linked immunosorbent assay (ELISA) The obtained phages positive for S. aureus alpha-toxin binding were infected with E. coli and spread on a plate. Then, 210 single colonies were selected and cultured to produce and purify the phages. The alpha-toxin fusion protein was coated on a 96-well plate and stored at 4°C overnight, and the obtained sample phages (blank phages were used as a control) 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 color development solution was added and the absorbance at a wavelength of 405 nm was read. If the OD value of the sample well was more than 3 times that of the control well, it was marked as a positive clone well. The bacteria in the positive clone well were transferred to 100 μL of LB solution containing ampicillin and cultured for plasmid extraction and sequence measurement. Finally, the sequence of the antigen-binding protein was obtained.

[0182] Among them, the sequences of the antigen-binding proteins determined are as follows: A3 SEQ ID NO: 12 QVQLVESGGGLVQAGGSLTLSCVAS GFTFSRH 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 YRNFGNFCVMGRYEYDEWGQGTQVTVSS where the underlined portions indicate the CDR regions divided using the Chothia numbering scheme.

[0183] Example 5 Expression and purification of Fc fusion antigen binding proteins in Expi293 cells The variable region coding sequences of antigen-binding proteins A3, G1 and F6 obtained by sequence determination and analysis were fused with the Fc fragment of human immunoglobulin gamma 1 (IgG1) respectively and subcloned into expression vector PCDNA3.4. Expi293 cells were transfected with PEI for recombinant expression, and the cell supernatant was collected after 7 days and purified using Protein A magnetic beads. The antigen-binding proteins with a purity of more than 95% were collected, and A3-Fc antigen-binding protein, G1-Fc antigen-binding protein and F6-Fc antigen-binding protein were obtained. Among them, 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 binding between antigen-binding protein and Staphylococcus aureus alpha-toxin by ELISA Staphylococcus aureus alpha 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), incubated overnight at 4°C, and the next day the 96-well plate was removed and washed with PBST (containing 0.5% PBS), soaking for 1 min each time, and then 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, after which the plate was washed with PBST and the water in the wells was dried. 100 μL of the test sample was added to each 96-well plate and incubated overnight at 4°C. The 96-well plate was removed and 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 h. After washing five times with PBST, 100 μL of substrate solution (Invitrogen) was added to each well and incubated at 37°C for 10 min. The reaction was stopped by adding 50 μL of 1 mol sulfuric acid to each well, and the absorbance at a wavelength of 450 nm was detected using a microplate reader (Multiscin FC, Thermo). Human IgG was used as a negative control.

[0185] The binding detection results of A3-Fc antigen-binding protein, G1-Fc antigen-binding protein, and F6-Fc antigen-binding protein with Staphylococcus aureus alpha-toxin are shown in Figure 1. The results show that the above three antigen-binding proteins have strong affinity for Staphylococcus aureus alpha-toxin, and the EC of A3-Fc antigen-binding protein is 50 The EC value was 0.009 μg / mL, which is the EC value for the G1-Fc antigen-binding protein. 50 The EC value was 0.007 μg / mL, which is the EC value for the F6-Fc antigen-binding protein. 50 The value is 0.003 μg / mL.

[0186] Example 7 Evaluation of neutralization of hemolytic activity of Staphylococcus aureus alpha-toxin by antigen-binding proteins AR-301 is a fully human antibody drug (Salvecin, tosatoxumab) developed by Aridis Pharmaceuticals that targets alpha-toxin. Using a rabbit erythrocyte alpha-toxin-induced hemolysis model, the neutralizing effect of AR301 against the hemolytic activity of S. aureus alpha-toxin was evaluated. 18.75ng of recombinant S. aureus alpha-toxin protein was mixed with antigen-binding protein or AR-301 at a certain ratio and added to a 96-well plate (Thermo) and incubated at 37°C for 10min, then 5% rabbit erythrocytes were added and incubated at 37°C for 1h. The samples were centrifuged at 3000rpm for 5min, and the anti-hemolytic activity was measured by detecting the absorbance at 405nm using a microplate reader (Multiscin FC, Thermo).

[0187] Using the rabbit erythrocyte hemolysis model by the above alpha toxin, the neutralizing effect of A3-Fc antigen-binding protein, G1-Fc antigen-binding protein and F6-Fc antigen-binding protein against S. aureus alpha toxin was detected, and the results are shown in Figure 2. 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) have a complete neutralizing effect against 18.75 ng of recombinant alpha toxin; 50 ng of the control antibody AR-301 cannot completely neutralize 18.75 ng of recombinant S. aureus alpha toxin (D), and the neutralizing effect of the A3-Fc antigen-binding protein, G1-Fc antigen-binding protein and F6-Fc antigen-binding protein disclosed by the present invention against S. aureus alpha toxin is significantly better than that of the control antibody AR-301.

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

[0189] Using a mouse sepsis model induced by Staphylococcus aureus alpha-toxin, the therapeutic effects of A3-Fc antigen-binding protein, G1-Fc antigen-binding protein and F6-Fc antigen-binding protein were tested, and the results are shown in Figure 3. 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 complete protective effects.

[0190] Example 9 Reproduction of a mouse bacteremia model caused by Staphylococcus aureus infection and evaluation of the therapeutic effect of antigen-binding proteins Staphylococcus aureus USA300 was activated from a -80°C refrigerator, activated for two generations in TSA medium, inoculated into 2 ml TSB, and cultured for 12 h. After washing three times with PBS, 6 × 10 7 Mice were infected with CFU via the tail vein, and 2 h after infection, the antigen-binding proteins were injected and the survival of the mice was monitored using human IgG as a control.

[0191] The anti-infective pharmacodynamic effects of A3-Fc antigen-binding protein and G1-Fc antigen-binding protein were evaluated using a mouse bacteremia model caused by Staphylococcus aureus, and the results are shown in Figure 4. The results showed that A3-Fc antigen-binding protein and G1-Fc antigen-binding protein had significant protective effects against a mouse bacteremia model caused by Staphylococcus aureus, and their pharmacodynamic effects were superior to those of the control antibody AR-301.

[0192] Example 10 Humanization of Staphylococcus aureus alpha-toxin antigen-binding protein The human germline sequence closest to the camel antibody was selected as the template based on the NCBI database alignment ( https: / / www.ncbi.nlm.nih.gov / igblast / ), and A3, G1 and F6 were humanized, and the post-translational modification sites of the antigen-binding protein were optimized.

[0193] Among them, the humanized sequence of the obtained antigen-binding protein is as follows: huA3 SEQ ID NO: 13 QVQLVESGGGLVQPGGSLRLSCAAS GFTFSRH 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 where the underlined portions indicate the CDR regions divided using the Chothia numbering scheme.

[0194] The variable region coding sequences of the above antigen-binding proteins huA3, huG1 and huF6 were fused with the Fc fragment of human immunoglobulin gamma 1 (IgG1) respectively and subcloned into the expression vector PCDNA3.4. Expi293 cells were transfected with PEI for recombinant expression, and the cell supernatant was collected after 7 days and purified using Protein A magnetic beads. Antigen-binding proteins with a purity of 95% or more were collected to obtain huA3-Fc antigen-binding protein, huG1-Fc antigen-binding protein and huF6-Fc antigen-binding protein. The humanized sequences of the above antigen-binding proteins are as follows: huA3-Fc SEQ ID NO: 68 QVQLVESGGGLVQPGGSLRLSCAAS GFTFSRH GMTWVRQAPGKGLEYVGEI NIPGDD VDYDISVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARAGENMRLVTRGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK huG1-Fc SEQ ID NO: 69 QVQLVESGGGLVQPGGSLRLSCAASGFTISSTGMTWVRQAPGKGLEWVGDINIPGTDTDYDISVKGRFTISKDHAKNTVYLQMNSLRAEDTAVYYCAKQGANLRQGVQGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK huF6-Fc SEQ ID NO: 70 EVQLVESGGGLVQPGGSLRLSCAASGSTLDSYTVAWVRQAPGKGLEWVSCASRSGASTNYANSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAAYRNFGNFCVMGRYEYDEWGQGTLVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK The binding activity of the above antigen-binding proteins with Staphylococcus aureus alpha-toxin was detected by the method of Example 6. The experimental results are shown in Figure 5. It was found that the above three antigen-binding proteins have strong affinity for Staphylococcus aureus alpha-toxin, except for the EC of huA3-Fc antigen-binding protein. 50 The EC value was 0.005970 μg / mL, which is the EC 50 The EC value was 0.006353 μg / mL, which is the EC 50 The value is 0.002721 μg / mL.

[0195] The neutralizing effect of the antigen-binding proteins against the hemolytic activity of S. aureus alpha-toxin was evaluated using the rabbit erythrocyte hemolysis model by alpha-toxin of Example 7. The experimental results are shown in Figure 6, and the neutralizing effect of huA3-Fc, huG1-Fc, and huF6-Fc against S. aureus alpha-toxin is 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 alpha-toxin. Different doses of antigen-binding proteins were injected into C57BL / 6 mice in advance, and 30 min later, 2.5 μg of S. aureus alpha-toxin recombinant protein was injected into the tail vein, and human IgG was used as a control. The survival time of the mice was observed. The therapeutic effects of huA3-Fc antigen-binding protein and huG1-Fc antigen-binding protein were tested using a mouse sepsis model induced by S. aureus alpha-toxin, and the results are shown in Figure 7. The results show that the protective effects of the three antigen-binding proteins described in this application, A3-Fc antigen-binding protein and G1-Fc antigen-binding protein, are all superior to AR301.

[0197] The therapeutic effect of antigen-binding proteins was evaluated using a mouse bacteremia model caused 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 TSB, and cultured for 12 h. After washing three times with PBS, 6 × 10 7 CFU mice were infected via the tail vein. 2 h after infection, the antigen-binding proteins were injected, and human IgG was used as a control to observe the survival of the mice. The pharmacodynamic effects of huA3-Fc and huG1-Fc antigen-binding proteins 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 and huG1-Fc antigen-binding proteins have significant protective effects 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 tetravalent Staphylococcus aureus alpha-toxin antigen binding protein Fc fusion protein Two anti-S. aureus alpha-toxin antigen-binding protein sequences (e.g., two huA3 molecules, two huG1 molecules, or one huA3 molecule and one huG1 molecule) and a nucleic acid fragment encoding human immunoglobulin G1 (IgG1) Fc were tandemly linked and subcloned into pcDNA3.4 plasmid, and expressed and purified as described in Example 5. The resulting recombinantly expressed molecule has four binding domains for S. aureus alpha-toxin, and is therefore called a tetravalent anti-S. aureus alpha-toxin Fc antigen-binding protein. 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 by S. aureus alpha-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 against S. aureus alpha-toxin were tested, and the results are shown in Figure 9. The results show that against 18.75 ng of recombinant S. aureus alpha-toxin, the neutralizing effect of the tetravalent huA3-huG1-Fc fusion protein at the same dose is superior to that of the bivalent huA3-Fc antigen-binding protein and huG1-Fc antigen-binding protein.

[0200] Using the rabbit erythrocyte hemolysis model induced by S. aureus alpha-toxin in Experimental Example 7, the neutralizing effects of tetravalent huA3-huA3-Fc antigen-binding protein and huA3-Fc antigen-binding protein against S. aureus alpha-toxin were tested, and the results are shown in Figure 10. The results show that against 18.75 ng of recombinant S. aureus alpha-toxin, the neutralizing effect of the tetravalent huA3-huA3-Fc fusion protein at the same dose is superior to that of the bivalent huA3-Fc antigen-binding protein.

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

Claims

1. An isolated antigen binding protein, comprising HCDR1, HCDR2 and HCDR3, wherein said HCDR1 comprises an amino acid sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:16 and SEQ ID NO:28, said HCDR2 comprises an amino acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:15 and SEQ ID NO:27, and said HCDR3 comprises an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:14 and SEQ ID NO:

26.

2. The isolated antigen binding protein of claim 1, comprising HCDR1, HCDR2, and HCDR3 selected from the group consisting of: 1) said HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, said HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and said HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1; 2) said HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16, said HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, and said HCDR3 comprises the amino acid sequence set forth 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.

3. The isolated antigen binding protein of claim 1, comprising H-FR1, H-FR2, H-FR3 and H-FR4, wherein said H-FR1 comprises an amino acid sequence set forth 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, said H-FR2 comprises an amino acid sequence set forth 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 said H-FR3 comprises an amino acid sequence set forth in any one of SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, and said 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.

4. The isolated antigen binding protein of claim 1, comprising H-FR1, H-FR2, H-FR3, and H-FR4 selected from the group consisting of: 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) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 17, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 18, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 19, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 20; 4) 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: 21, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 22, and the H-FR4 comprises 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) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 33, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 34, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 35, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

36.

5. The isolated antigen binding protein of claim 1, comprising a VH, said VH comprising an amino acid sequence set forth 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. The isolated antigen binding protein of claim 1, comprising a VHH, wherein the VHH comprises an amino acid sequence set forth 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.

7. The isolated antigen-binding protein of claim 1, comprising an antibody heavy chain constant region.

8. The isolated antigen binding protein of claim 1, comprising an amino acid sequence set forth 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.

9. The isolated antigen-binding protein of claim 1, comprising an antibody or an antigen-binding fragment thereof.

10. A polypeptide molecule comprising the isolated antigen-binding protein of claim 1.

11. The polypeptide molecule of claim 10, wherein the polypeptide molecule is a fusion protein comprising two or more of the isolated antigen-binding proteins and an antibody heavy chain constant region.

12. The polypeptide molecule of claim 10, comprising an amino acid sequence set forth in any one of SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:

67.

13. An immune complex comprising the isolated antigen-binding protein of claim 1.

14. A nucleic acid molecule encoding the isolated antigen-binding protein of claim 1 or a polypeptide molecule comprising said isolated antigen-binding protein.

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

16. A pharmaceutical composition comprising the isolated antigen-binding protein of claim 1, a polypeptide molecule comprising said isolated antigen-binding protein, an immune complex comprising said isolated antigen-binding protein, a nucleic acid molecule encoding said isolated antigen-binding protein, a vector comprising said nucleic acid molecule and / or a cell comprising said nucleic acid molecule or a vector comprising said nucleic acid molecule, and optionally a pharma- ceutically acceptable carrier.

17. Use of the isolated antigen-binding protein of claim 1, a polypeptide molecule comprising said isolated antigen-binding protein, an immune complex comprising said isolated antigen-binding protein, a nucleic acid molecule encoding said isolated antigen-binding protein, a vector comprising said nucleic acid molecule, a cell comprising said nucleic acid molecule or said vector and / or a pharmaceutical composition comprising said isolated antigen-binding protein, in the manufacture of a medicament for the prevention and / or treatment of a disease and / or condition and complications thereof in the human and / or animal.

18. In the application described in claim 17, the disease and / or symptoms and their complications are caused or mediated by Staphylococcus aureus.

19. A method for detecting Staphylococcus aureus alpha-toxin in a sample comprising using an isolated antigen-binding protein as described in claim 1, a polypeptide molecule comprising said isolated antigen-binding protein, an immune complex comprising said isolated antigen-binding protein, a nucleic acid molecule encoding said isolated antigen-binding protein, a vector comprising said nucleic acid molecule, a cell comprising said nucleic acid molecule or said vector and / or a pharmaceutical composition comprising said isolated antigen-binding protein.

20. A reagent or reagent kit for detecting Staphylococcus aureus alpha-toxin in a sample, comprising the isolated antigen-binding protein of claim 1, a polypeptide molecule comprising said isolated antigen-binding protein, an immune complex comprising said isolated antigen-binding protein, a nucleic acid molecule encoding said isolated antigen-binding protein, a vector comprising said nucleic acid molecule, a cell comprising said nucleic acid molecule or said vector and / or a pharmaceutical composition comprising said isolated antigen-binding protein.

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