Staphylococcus aureus alpha-toxin specific antibodies and uses thereof

Abstract

The present disclosure relates to an antibody or antigen-binding fragment that specifically binds to Staphylococcus aureus alpha-toxin. The present disclosure also relates to a pharmaceutical composition, a use of the antibody or antigen-binding fragment for the manufacture of a medicament for treating and / or preventing a disease and / or a disorder caused by Staphylococcus aureus infection in a subject in need thereof, a kit for detecting Staphylococcus aureus alpha-toxin in a sample, a method for detecting Staphylococcus aureus alpha-toxin in a sample, and a kit for detecting Staphylococcus aureus alpha-toxin in a sample.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to Staphylococcus aureus alpha-toxin specific antibodies or antigen-binding fragments and uses thereof. BACKGROUND

[0002] Staphylococcus aureus is an opportunistic pathogen that is often carried asymptomatically in humans. Pathogenic strains often promote infection by producing potent protein toxins and other virulence factors that evade the human immune system. S. aureus can cause a range of diseases, from mild skin infections to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia, and sepsis. It remains one of the five most common causes of nosocomial infections and is often the cause of post-surgical wound infections.

[0003] The emergence of antibiotic-resistant S. aureus (MRSA) is a worldwide clinical medical problem. Current concepts of the virulence mechanisms of MRSA include a series of prominent cell surface and secreted virulence factors. Cell surface virulence factors include microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), iron-regulated proteins, intercellular polysaccharide adhesins, and capsular polysaccharides. The secreted virulence factors are usually produced in the late exponential and stationary phases, including exoenzymes, exotoxins alpha, beta, gamma, and delta toxins, Panton-Valentine leukocidin (PVL), superantigens, toxic shock syndrome toxin-1 (TSST-1), and exfoliative toxins A and B.

[0004] Alpha-toxin (AT) is a cytolysic pore-forming toxin that is conserved among S. aureus clinical isolates and has been shown to play a role in pneumonia, skin necrosis, endocarditis, and sepsis. AT is a soluble monomeric protein that can assemble into a ring-like structure on the surface of eukaryotic cells. The assembled toxin can insert into the cell membrane, forming a pore that disrupts the integrity of the cell membrane, leading to cell damage and death. For decades, toxins have been targeted for immunoprophylaxis and have been successful as vaccines or as part of passive immunotherapy for bacterial diseases such as diphtheria, tetanus, and botulism. Unlike active immunization, which sometimes requires repeated boosts and a long time to generate the maximum immune response, passive immunization provides immediate treatment for unvaccinated patients to help reduce the severity of acute S. aureus disease.

[0005] Accordingly, there is a need to develop a new method for treating or preventing S. aureus infection. SUMMARY

[0006] The present disclosure provides an antibody or antigen-binding fragment that specifically binds to an S. aureus alpha-toxin epitope or fragment thereof. The antibody according to the present disclosure is capable of neutralizing S. aureus alpha-toxin, and thus can be used for treating and / or preventing a disease and / or disorder caused by S. aureus infection. The antibody of the present disclosure can also be used for detecting S. aureus alpha-toxin.

[0007] The present disclosure provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment and a pharmaceutically acceptable carrier or excipient.

[0008] The present disclosure provides a method for treating and / or preventing a disease and / or disorder caused by S. aureus infection in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment.

[0009] The present disclosure provides a method for detecting S. aureus alpha-toxin in a sample, comprising contacting the sample with the above-mentioned antibody or antigen-binding fragment.

[0010] The present disclosure provides a kit for detecting S. aureus alpha-toxin in a sample, comprising the above-mentioned antibody or antigen-binding fragment.

[0011] The present disclosure will be described in detail in the following paragraphs, and other features, objects, and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description and the claims. BRIEF DESCRIPTION OF DRAWINGS

[0012] Brief description of the drawings

[0013] Figure 1 Figure 6: Anti-alpha-toxin monoclonal antibodies increase A549 cell viability in the presence of alpha-toxin. Purified antibodies at concentrations of 4 and 40 μg / ml were added to the cytolytic assay in the presence of 8 μg / ml alpha-toxin to obtain an alpha-toxin to antibody ratio of 1 :0.1 (4 μg / ml) or 1 :1 (40 μg / ml). Cell viability was measured by MTT colorimetric assay. The asterisk indicates that the antibody has the ability to prevent alpha-toxin induced cytolysis.

[0014] Figure 2 Figure 8: Mouse 25A1 monoclonal antibody binds to alpha-toxin. Captured 25A1 antigen binding profile on a Biacore T100 active flow cell. The line represents the binding response of 25A1 to alpha-toxin over a concentration range of 1.56 to 50 nM.

[0015] Figure 3 (A) Amino acid sequences of various anti-alpha-toxin VH and VL domains. Human germline IGHV1-2 and IGVK3-11 sequences were used for grafting. Differences in amino acid sequences between mouse antibody and human germline are underlined; CDR residues are marked with an outer box; back mutations are marked with bold italic. Figure 3 (B) SPR sensorgram of antibody binding kinetics to recombinant alpha-toxin. Binding kinetics were determined using BIAcore T200 single-cycle kinetics method. Figure 3 (C) Amino acid sequence of 25A1-B5B6AQT. Amino acid sequence of signal peptide is underlined and bold; variable region is marked with gray.

[0016] Figure 4 Figure 5: 25A1, 25A1-B2B4AQT and 25A1-B5B6AQT inhibit recombinant alpha-toxin or native alpha-toxin induced lysis of rabbit red blood cells. Antibody serial dilutions (100, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 mg / mL) were incubated with recombinant alpha-toxin (400 ng / mL) or crude bacterial supernatant (1:8 to 1:16 dilutions) with rabbit red blood cells. Hemolysis was measured by the amount of hemoglobin released in the supernatant. Hemolysis inhibition was calculated as ((OD450 of 2% Triton X-100 - OD450 of test antibody) / OD450 of 2% Triton X-100) x 100%.

[0017] Figure 5 Figure 6: Kaplan-Meir survival curves of mice infected with S. aureus BAA-1717 given SYN100. CD-1 mice were intravenously injected with USA300 MRSA, BAA-1717 at 8 x 10 7 CFU / mouse. 25A1 was intraperitoneally injected at 50, 25, 10 and 5 mg / kg twenty-four (24) hours prior to infection. Control antibody was intraperitoneally injected at 25 and 10 mg / kg twenty-four (24) hours prior to infection. Animal mortality was monitored for 10 days. A 50% or greater (50%) survival of animals compared to the vehicle control group indicates significant anti-infective activity.

[0018] Figure 6 Figure 7: Kaplan-Meir survival curves of mice infected with S. aureus ATCC29213 given SYN100. CD-1 mice were intraperitoneally injected with ATCC29213 at 2.0 x 10 7 CFU / mouse. Three groups of mice were intraperitoneally injected (IP) with SYN100 at 100, 50 and 10 mg / kg twenty-four (24) hours prior to infection. Survival of infected animals was monitored for 4 days.

[0019] Figure 7 Kaplan-Meir survival curves of mice infected with S. aureus BAA-1556, NRS261, and SF8300 given SYN100. SYN100 prophylactic treatment increased survival in a mouse pneumonia model. C57BL / 6J mice were intranasally inoculated with 1.62 x 10 7 BAA-1556 at 3.3 x 10 7 NRS261 at 2.82 x 10 7 SF8300 at 2.82 x 10 C57BL / 6J mice were intranasally inoculated with 1.62 x 10

[0020] Figure 8 Kaplan-Meir survival curves of mice infected with S. aureus NRS261 given SYN100 and / or vancomycin. C57BL / 6J mice were intranasally inoculated with 5.0 x 10 7 CFU / mouse. Four groups of mice were given 10 mg / kg SYN100 intraperitoneally (IP) twenty-four (24) hours prior to infection. Vancomycin was administered at 30, 15, and 7.5 mg / kg at two hours post-infection. Three groups of mice received both vancomycin and SYN100. Survival of infected animals was monitored for 5 days.

[0021] Figure 9 Kaplan-Meir survival curves of rabbits infected with S. aureus ST20120426 given SYN100. New Zealand rabbits were intranasally inoculated with 3.2-5.2 x 10 7 CFU / rabbit. SYN100 was injected intravenously at 125, 100, 75, 50, and 25 mg / kg twenty-four (24) hours prior to infection. Survival of infected animals was monitored for 7 days.

[0022] Kaplan-Meir survival curves of rabbits infected with S. aureus ST20120426 given SYN100 and / or linezolid. New Zealand rabbits were intranasally inoculated with 2.9-4.1 x 10 7CFU / rabbit were intranasally inoculated with ST20120426. SYN100 was injected intravenously at 30 mg / kg twenty-four (24) hours prior to infection. Dosing was at 50 mg / kg / 8h at 4 hours post infection. Survival of infected animals was monitored for 48 hours. Figure 10(B) Lung inflammation was assessed in all treatment groups with macroscopic scoring, with higher scores indicating more severe damage from bacterial infection. Open circles represent animals that died prior to 30 hours post infection. Filled circles represent animals that were alive at 30 hours post infection. Figure 10(C) Lung weight (LW) to body weight (BW) ratio. Figure 10(D) Bacterial counts in lung tissue. p value < 0.0083 indicates significance. DETAILED DESCRIPTION

[0023] The disclosure provides an antibody or antigen-binding fragment that specifically binds to a Staphylococcus aureus alpha-toxin epitope or fragment thereof.

[0024] In the following description, numerous specific details are set forth to provide a thorough understanding of the claimed subject matter. However, it will be apparent that the claimed subject matter can be practiced without the specific details. In other instances, well-known methods have not been described in detail in order to avoid obscuring the claimed subject matter.

[0025] Unless otherwise indicated, "a" or "an" means "one or more"

[0026] As used herein, the term "epitope" refers to a site on an antigen to which an antibody binds.

[0027] The term "antibody", as used herein, refers to any antigen-binding molecule or molecular complex comprising at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., alpha-toxin). The term "antibody" includes immunoglobulin molecules that are composed of four polypeptide chains, two heavy (H) and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region. The heavy chain constant region includes three domains, C H1 , C H2 , and C H3 . Each light chain includes a light chain variable region (abbreviated herein as LCVR or V L ) and a light chain constant region. The light chain constant region includes one domain (C L1 ). The V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each V H and V LComposed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the disclosure, the FRs of an anti-alpha-toxin antibody (or antigen binding portion thereof) can be identical to the human germline sequence, or can be naturally or artificially modified. Amino acid consensus sequences can be defined based on side-by-side analysis of two or more CDRs.

[0028] The term "monoclonal antibody" as used herein does not refer to an antibody produced by a hybridoma. A monoclonal antibody is one which is produced by any existing or yet to be developed means from a single clone, including any eukaryotic, prokaryotic, or phage clone.

[0029] The term "chimeric" antibody, as used herein, refers to an antibody having variable sequences from a non-human immunoglobulin and constant regions of a human immunoglobulin, typically selected from a human immunoglobulin template.

[0030] "Humanized" forms of non-human antibodies are chimeric immunoglobulins which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.

[0031] The term "complementarity determining region" (CDR), as used herein, refers to the non-contiguous antigen combining sites within the variable region of both heavy and light chain polypeptides. CDRs are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared to each other.

[0032] The terms "antigen binding portion" of an antibody, "antigen binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, artificially synthesized, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.

[0033] As used herein, the term "treatment" covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in an individual which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0034] In the present context, the terms "individual", "subject", "host" and "patient" refer to a mammal, including, but not limited to, a mouse (rat, mouse), a non-human primate, a human, a canine, a feline, an ungulate (e.g., a horse, a cow, a sheep, a pig, a goat), and the like.

[0035] As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of an antibody that, when administered to a mammal or other subject for treatment of a disease, is sufficient to effect such treatment for that disease.

[0036] As used herein, the term "sample" includes various sample types obtained from an individual, subject, or patient, which can be used for diagnostic or monitoring assays. This definition includes blood and other biological source fluid samples, solid tissue samples, such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny.

[0037] The present disclosure develops a monoclonal antibody that specifically neutralizes alpha-toxin, thereby providing passive immunotherapy for Staphylococcus aureus infection. Passive immunization will provide immediate treatment for unvaccinated patients to help reduce the severity of acute Staphylococcus aureus disease. Functionally, the antibody of the present disclosure exhibits significant inhibitory activity against AT-induced cytotoxicity and strong in vivo efficacy in preventing, prophylactically treating, and / or treating infection of Staphylococcus aureus and / or pneumonia.

[0038] Preferably, the antibody or antigen-binding fragment comprises: a complementarity determining region of a heavy chain variable region and a complementarity determining region of a light chain variable region, wherein the complementarity determining region of the heavy chain variable region comprises a CDRH1, a CDRH2, and a CDRH3 region, and the complementarity determining region of the light chain variable region comprises a CDRL1, a CDRL2, and a CDRL3 region, wherein:

[0039] the CDRH1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2 or a substantially similar sequence thereof; the CDRH2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6 and 31 or a substantially similar sequence thereof; the CDRH3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9 or a substantially similar sequence thereof; and

[0040] The CDRL1 region comprises an amino acid sequence selected from the group of SEQ ID NOs: 10 to 13, or a substantially similar sequence thereof; the CDRL2 region comprises an amino acid sequence selected from the group of SEQ ID NOs: 14 to 15, or a substantially similar sequence thereof; and the CDRL3 region comprises an amino acid sequence selected from the group of SEQ ID NOs: 16 to 18, or a substantially similar sequence thereof.

[0041] The sequence listing is shown in Table 1.

[0042] Table 1:

[0043]

[0044]

[0045] Antibodies according to the present disclosure can be full-length (e.g., IgGl or IgG4 antibodies), or can comprise only an antigen-binding portion (e.g., a Fab, F(ab')2, or scFv fragment), and can be modified as desired to affect functionality.

[0046] Antibodies or antigen-binding fragments according to the present disclosure specifically bind to alpha-toxin of S. aureus. Alpha-toxin is a cytolysic pore-forming toxin that is conserved among clinical isolates of S. aureus. Alpha-toxin is a 33 kDa soluble monomeric protein that is able to assemble into a ring-like structure on the surface of eukaryotic cells, and then the assembled toxin is inserted into the cell membrane to form a pore, leading to cell damage and death by disrupting the integrity of the cell membrane.

[0047] The present disclosure includes an anti-alpha-toxin antibody and antigen-binding fragments thereof that bind to the monomer or ring-like structure of the alpha-toxin molecule with high affinity.

[0048] Various techniques known to those of ordinary skill in the art can be used to determine whether an antibody "specifically binds to one or more amino acids in a polypeptide or protein." Exemplary techniques include, for example, routine cross-blocking experiments, as described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.), alanine scanning mutational analysis, peptide blotting analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can also be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids in a polypeptide to which an antibody specifically binds is hydrogen / deuterium exchange detected by mass spectrometry. Generally, the hydrogen / deuterium exchange method involves labeling a protein of interest with deuterium, and then binding the antibody to the deuterium-labeled protein. Next, the protein / antibody complex is transferred to water, allowing hydrogen-deuterium exchange to occur on all residues except those protected by the antibody (retaining deuterium labeling). After the antibody is isolated, protease cleavage and mass spectrometry analysis of the target protein reveals deuterium-labeled residues corresponding to specific amino acids that interact with the antibody. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0049] The disclosure also includes anti-alpha-toxin antibodies that specifically bind to the same epitope.

[0050] Whether an antibody specifically binds to the same epitope as a reference anti-α-toxin antibody, or competes for binding with a reference anti-α-toxin antibody, can be readily determined by using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference anti-α-toxin antibody of the present disclosure, the reference antibody is allowed to bind to an α-toxin protein (e.g., a monomer or a ring structure of α-toxin). Next, the ability of the test antibody to bind to the α-toxin molecule is assessed. If the test antibody is able to bind to the α-toxin after the α-toxin has been saturated with the reference anti-α-toxin antibody, then it can be concluded that the test antibody binds to a different epitope than the reference anti-α-toxin antibody. On the other hand, if the test antibody is not able to bind to the α-toxin molecule after the α-toxin has been saturated with the reference anti-α-toxin antibody, then the binding epitope of the test antibody can be the same as the binding epitope of the reference anti-α-toxin antibody of the present disclosure. Additional routine assays (e.g., peptide mutagenesis and binding assays) can then be performed to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody, or whether steric blocking (or other phenomena) is responsible for the observed lack of binding. Such assays can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present disclosure, two antibodies bind to the same (or overlapping) epitope if a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 50%, but most preferably by 75%, 90%, or even 99%. Alternatively, two antibodies are considered to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. If only a subset of amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody, then the two antibodies are considered to have an "overlapping epitope."

[0051] The term "antibody", as used herein, also includes antigen-binding fragments of full antibody molecules. Antigen-binding fragments of an antibody can be derived from full antibody molecules using, for example, any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and, where needed, constant domains. Such DNA is known and / or is readily available from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA sequencing and manipulation can employ chemical methods or molecular biological techniques, e.g., to arrange one or more variable and / or constant domains into an appropriate structure, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0052] Non-limiting examples of antigen binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR), such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, Nanobodies® (e.g., monovalent Nanobodies, bivalent Nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen binding fragment" as used herein.

[0053] Antigen binding fragments of antibodies generally include at least one variable domain. The variable domain can be of any size or amino acid composition and is typically comprised of at least one CDR, which is adjacent to or in frame with one or more framework sequences. In certain embodiments, a V L Antigen binding fragments of antibodies that have V H domains associated with them can be of any suitable arrangement. For instance, the variable region can be dimeric and contain V H -V L or V H -V H dimer. Alternatively, an antigen binding fragment of an antibody can include a monomeric V H or V L domain. L -V L or V H domain. L

[0054] In certain embodiments, an antigen binding fragment of an antibody can include at least one variable domain covalently linked to at least one constant domain. Variable and constant domains which can be found within the antigen binding fragments of the antibodies of the present disclosure include: (i) V H -C H1 ; (ii) V H -C H2 ; (iii) V H -C H3 ; (iv) V H -C H1 -C H2 ; (v) V H -C H1 -C H2 -C H3 ​; (vi) V H -C H2 -C H3 ; (vii) V H -C l ; (viii) V L -C H1 ; (ix) V L - C H2 ; (x) V L -C H3 ; (xi) V L -C H1 -C H2 ; (xii) V L -C H1 -C H2 -C H3 ; (xiii) V L -C H2 - C H3 ; and (xiv) V L -C L In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be directly connected to one another or connected via a full or partial hinge or linker region. Hinge regions can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that form a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, an antibody or antigen-binding fragment of the present disclosure can comprise a homo- or hetero-dimer (or other multimer) of one or more of any of the above variable and constant domain configurations, and are associated with one another and / or with one or more monomeric V H or V L domains via non-covalent binding (e.g., via disulfide bonds).

[0055] As with full antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically include at least two different variable domains, where each variable domain is capable of specifically binding to a separate antigen or to a different epitide on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats of the present disclosure, can be adapted for antigen-binding fragments of the antibodies of the present disclosure using routine techniques available in the art.

[0056] Preferably, the antibody or antigen-binding fragment is a mammalian antibody.

[0057] The term "mammalian antibody", as used herein, is intended to include antibodies having variable and constant regions derived from mammalian germline immunoglobulin sequences. The mammalian antibodies of the disclosure can include amino acid residues which are not encoded by mammalian germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs, and especially in the CDR3.

[0058] The term "recombinant mammalian antibody", as used herein, is intended to include all mammalian antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transformed or transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial mammalian antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for mammalian immunoglobulin genes or antibodies prepared, expressed, created or isolated by any other means that involves splicing of mammalian immunoglobulin gene sequences to other DNA sequences. Such recombinant mammalian antibodies have variable and constant regions derived from and related to mammalian germline immunoglobulin sequences. In certain embodiments, however, such recombinant mammalian antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis), and thus the amino acid sequences of the V H and V L sequences derived from and related to human germline V

[0059] Mammalian antibodies, such as human antibodies, can exist in two forms that are related to the heterogeneity of the hinge. In one form, the immunoglobulin molecule comprises a stable, approximately 150-160 kDa, four-chain structure in which dimers are held together by interchain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, and a covalently coupled light and heavy chain (half-antibody) forms a molecule of approximately 75-80 kDa. These forms are difficult to separate even after affinity purification.

[0060] The anti-a-toxin antibodies of the present disclosure can include one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily determined by comparing the amino acid sequences of the present disclosure to germline sequences available from, for example, public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof that are derived from any of the amino acid sequences of the present disclosure, wherein one or more amino acid mutations within one or more framework and / or CDR regions are to the corresponding residue of the germline sequence from which the antibody was derived, or to the corresponding residue of another mammalian germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (collectively, these sequence changes are referred to herein as "germline mutations"). From the heavy and light chain variable region sequences of the present disclosure, one of skill in the art can readily produce a number of antibodies and antigen-binding fragments that comprise one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V L all of the framework and / or CDR residues within the domain are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., the mutated residues are found only in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or the mutated residues are found only in CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present disclosure can comprise any combination of two or more germline mutations within the framework and / or CDR regions, e.g., where certain individual residues are mutated to the corresponding residue of a particular germline sequence, while certain other residues that differ from the original germline sequence are retained or mutated to the corresponding residue of a different germline sequence. Once prepared, the antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, e.g., improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties as the case can be, reduced immunogenicity, in this general manner, the antibodies and antigen-binding fragments obtained in this manner are also encompassed by the present disclosure.

[0061] The present disclosure also includes an anti-a-toxin antibody comprising a variant of a V H , V L and / or CDR amino acid sequence of the present disclosure having one or more conservative substitutions. For example, the present disclosure includes an anti-a-toxin antibody having a V H , V L and / or CDR amino acid sequence, the V H , V Land / or CDR amino acid sequences have, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions.

[0062] The term "substantial identity" or "substantially identical" when applied to a nucleic acid or fragment thereof indicates that, when aligned for maximum sequence comparison purposes with another nucleic acid (or its complementary strand), at least about 95 percent of the nucleotide bases are identical with the other nucleic acid, and more preferably at least about 96, 97, 98, or 99 percent of the nucleotide bases are identical over a specified region as determined by any of the known sequence comparison algorithms (e.g., FASTA, BLAST or Gap) as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can in some instances encode a polypeptide having the same or substantially similar amino acid sequence to the polypeptide encoded by the reference nucleic acid molecule.

[0063] The term "substantial similarity" or "substantially similar" when applied to a polypeptide means that two peptide sequences, when optimally aligned, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, non-identical residue positions are varied by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) of similar chemical properties (e.g., charge or hydrophobicity). In general, conservative substitutions will not dramatically change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the variation. Means for making this adjustment are well known to those of ordinary skill in the art. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic- aspartic acid, and asparagine-glutamine. Alternatively, conservative substitutions are those changes that result in a PAM250 log-likelihood matrix, disclosed in Gonnet et al. (1992) Science 256:1443-1445, incorporated herein by reference, which has positive values. "Moderately conservative" substitutions are those changes that result in a PAM250 log-likelihood matrix which has non-negative values.

[0064] Sequence similarity of polypeptides, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit that can be used with the default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species or between a wild-type protein and a mutein thereof. Polypeptide sequences also can be compared using FASTA, a program in GCG version 6.1, using default parameters or recommended parameters. FASTA (for example, FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a published sequence to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, incorporated herein by reference.

[0065] In a preferred embodiment of the disclosure, the antibody or antigen-binding fragment comprises: a complementarity determining region of a heavy chain variable region and a complementarity determining region of a light chain variable region, wherein the complementarity determining region of the heavy chain variable region comprises a CDRH1, a CDRH2, and a CDRH3 region, and the complementarity determining region of the light chain variable region comprises a CDRL1, a CDRL2, and a CDRL3 region, wherein:

[0066] the CDRH1 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;

[0067] the CDRH2 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 6 and 31, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;

[0068] the CDRH3 region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 9, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity;

[0069] The CDRL1 region comprises an amino acid sequence selected from the group of SEQ ID NOs: 10 to 13, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity;

[0070] The CDRL2 region comprises an amino acid sequence selected from the group of SEQ ID NOs: 14 to 15, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity;

[0071] The CDRL3 region comprises an amino acid sequence selected from the group of SEQ ID NOs: 16 to 18, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0072] In one preferred embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the complementarity determining regions of a heavy chain variable region and the complementarity determining regions of a light chain variable region, as shown in Table 2.

[0073] Table 2:

[0074] Clone CDRH1 (SEQ ID NO. CDRH2 (SEQ ID NO. CDRH3 (SEQ ID NO. 25A1 GYSFTDYNMN (1) SINPYYGITSYNQTFKG (3) IYYGDSLGLDY (7) 25A10 GYSFTDYNMN (1) SINPYYGITSYNQTFKG (3) IYYGDSLGLDY (7) 25E4 GYSFTDYNMN (1) SINPYYGITSYNQTFRG (31) IYYGDSLGLDY (7) 25E12 GYSFTDYNMN (1) SINPHYGITSYNQTFKG (4) IYYGDSLGLDY (7) 25H3 GYSFTDYNMN (1) SINPYYGITTYNQTFKG (5) IYYGDSLGLDY (7) 25B7 GYSFTDYNMN (1) SINPYYGITSYNQTFKG (3) IYYGDSLGLDY (7) 25G1 GYSFTGYFMN (2) RINPYNGDTLYKQNFKD (6) DGDGYYYAMDY (8) 25G4 GYSFTDYNMN (1) SINPYYGITSYNQTFKG (3) IYYGDSLGLDY (7) 5H9 GYSFTDYNMN (1) SINPYYGITSYNQTFKG (3) VYYGDSLGLDY (9) N2F6 GYSFTDYNMN (1) SINPYYGITSYNQTFKG (3) IYYGDSLGLDY (7)

[0075]

[0076]

[0077] In one preferred embodiment of the disclosure, the 25A1 antibody or antigen-binding fragment thereof comprises a CDRH1 region comprising the amino acid sequence of SEQ ID NO: 1 or a substantially similar sequence thereof; a CDRH2 region comprising the amino acid sequence of SEQ ID NO: 3 or a substantially similar sequence thereof; a CDRH3 region comprising the amino acid sequence of SEQ ID NO: 7 or a substantially similar sequence thereof; a CDRL1 region comprising the amino acid sequence of SEQ ID NO: 10 or a substantially similar sequence thereof; a CDRL2 region comprising the amino acid sequence of SEQ ID NO: 14 or a substantially similar sequence thereof; and a CDRL3 region comprising the amino acid sequence of SEQ ID NO: 16 or a substantially similar sequence thereof. Preferably, the 25A1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. Preferably, the 25A1 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0078] In another aspect, the antibody according to the present disclosure is preferably a humanized antibody. A "humanized antibody" is a recombinant protein in which the CDRs from an antibody of one species, such as a rodent antibody, are transferred from the heavy and light chain variable regions of the rodent antibody to human heavy and light chain variable domains, including human framework region (FR) sequences. The constant domains of the antibody molecule are derived from a human antibody.

[0079] According to the present disclosure, in order to improve the binding affinity of the humanized antibody, some amino acid residues in the human framework region are replaced by the corresponding amino acid residues of the CDRs species, such as rodents.

[0080] Preferably, the humanized antibody or antigen-binding fragment includes a heavy chain variable region including an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 23 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. The humanized antibody or antigen-binding fragment includes a light chain variable region including an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 to 26 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0081] In a preferred embodiment of the present disclosure, the humanized antibody 25A1-B2B4AQT or antigen-binding fragment includes a heavy chain variable region including the amino acid sequence of SEQ ID NO: 27 or a substantially similar sequence thereof; and a light chain variable region including the amino acid sequence of SEQ ID NO: 28 or a substantially similar sequence thereof.

[0082] In a preferred embodiment of the present disclosure, the humanized antibody 25A1-B5B6AQT or antigen-binding fragment includes a heavy chain variable region including the amino acid sequence of SEQ ID NO: 29 or a substantially similar sequence thereof; and a light chain variable region including the amino acid sequence of SEQ ID NO: 30 or a substantially similar sequence thereof.

[0083] Preferably, the antibody according to the present disclosure is a monoclonal antibody.

[0084] Antibodies of the present disclosure can be monospecific, bispecific, or multispecific. Multispecific antibodies can be specific for different epitopes of one target polypeptide, or can comprise antigen binding domains specific for more than one target polypeptide. Anti-a-toxin antibodies of the present disclosure can be linked to or co-expressed with another functional molecule (e.g., another peptide or protein). For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent binding or other means) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or a multispecific antibody with a second binding specificity. For example, the present disclosure includes bispecific antibodies in which one arm of the immunoglobulin is specific for a-toxin or a fragment thereof, and the other arm of the immunoglobulin is specific for a second therapeutic target or is conjugated to a therapeutic moiety.

[0085] In a preferred embodiment of the present disclosure, the antibody or antigen binding fragment thereof is conjugated to a therapeutic agent.

[0086] One example of a therapeutic agent is an antibiotic. Examples of antibiotics include, but are not limited to, daunorubicin, bleomycin, mitramycin, anthramycin, streptozotocin, gramicidin D, or a mitomycin.

[0087] In a preferred embodiment of the present disclosure, the antibody or antigen binding fragment thereof can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is a mammalian cell expression, such as a hybridoma or CHO cell expression system. Numerous such systems are widely available from commercial suppliers. In some embodiments, the expression system is a bacterial expression system, such as a bacterial cell expression system. In some embodiments, the expression system is a yeast expression system, such as a yeast cell expression system. In some embodiments, the expression system is a plant expression system, such as a plant cell expression system. In some embodiments, the expression system is an insect cell expression system, such as a baculovirus expression system. In some embodiments, the expression system is a viral expression system, such as a viral cell expression system. In some embodiments, the expression system is a mammalian cell expression system, such as a mammalian cell expression system. In some embodiments, the expression system is a hybridoma cell expression system, such as a hybridoma cell expression system. In some embodiments, the expression system is a CHO cell expression system, such as a CHO cell expression system. H and V L In some embodiments, the antibody comprises a V H and V L In some embodiments, the antibody comprises a V H and V L In some embodiments, the antibody comprises a V H or V L In some embodiments, the antibody comprises a V

[0088] Genes encoding the heavy and light chains of a desired antibody can be cloned from a cell, for example, genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce recombinant monoclonal antibodies. Gene pools encoding the heavy and light chains of a monoclonal antibody can also be obtained from a hybridoma or a plasma cell. Random combinations of heavy and light chains generate a large number of antibodies with different antigenic specificities (see Kuby, Immunology (3.sup.rd ed. 1997)).

[0089] Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Pat. No. 4,946,778; U.S. Pat. No. 4,816,567) can be used to generate the polypeptide antibodies of the present application. In addition, transgenic mice or other organisms (such as other mammals) can be used to express humanized or human antibodies (see U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0090] In one preferred embodiment of the disclosure, the antibody or antigen binding fragment is expressed on the surface of a cell. More preferably, the cell is a T cell.

[0091] The present disclosure provides pharmaceutical compositions comprising the antibodies or antigen-binding fragments of the present disclosure. The pharmaceutical compositions of the present disclosure are formulated with suitable diluents, carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like. The compositions can be formulated for specific uses, such as veterinary use or pharmaceutical use in humans. The form of the composition and the excipients, diluents, and / or carriers used will depend on the intended use of the antibody, and for therapeutic use, the mode of administration. Many suitable formulations are found in the standard references known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) such as liposomes (TM, Life Technologies, Carlsbad, California.), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, paraffin emulsions (polymers of various molecular weight polyethylene glycols), semi-solid gels, and semi-solid mixtures with carbon waxes. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0092] The dosage of the antibody administered to a patient can vary depending on the age and size of the patient, the target disease, condition, route of administration, and the like. The preferred dosage is usually calculated according to body weight or body surface area. Intravenous injection of the antibody of the present disclosure can be advantageous when the antibody is used to treat a condition or disease associated with S. aureus infection in adult patients. The frequency and duration of treatment can be adjusted according to the severity of the condition. The effective dosage and administration schedule of the antibody can be determined empirically; for example, the progress of the patient's condition can be monitored by periodic assessment, and the dosage adjusted accordingly. In addition, methods known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351) can be used.

[0093] Various delivery systems are known to be used for the administration of the pharmaceutical compositions of the disclosure, e.g., liposomes, microparticles, microcapsules, recombmant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., the linings of the mouth, rectum, and intestine, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0094] The pharmaceutical compositions of the disclosure can be administered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, pen delivery devices are readily adapted to deliver the pharmaceutical compositions of the disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically use a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device is prefilled with the pharmaceutical composition, which is stored in a reservoir within the device. Once the pharmaceutical composition in the container is depleted, the entire device is discarded.

[0095] In certain instances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment, a controlled release system can be placed in proximity to the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other systems are discussed in Langer, 1990, Science 249:1527-1533.

[0096] The injectable preparations can include dosage forms for intravenous injection, subcutaneous injection, intradermal injection, and intramuscular injection, drip infusion, etc. These injectable preparations can be prepared by the methods disclosed. For example, the injectable preparations can be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or salt thereof in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc. can be used in combination with an appropriate solubilizing agent (e.g., ethanol), a polyol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant, etc. [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are sesame oil, soybean oil, etc., which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[0097] It is advantageous to formulate the aforementioned pharmaceutical compositions in unit dosage form for oral or parenteral administration. Such unit dosage forms can be prepared by the methods disclosed. For example, tablets, pills, capsules, injections (ampoules), suppositories, etc. are included in the unit dosage forms.

[0098] The present disclosure provides a method of neutralizing S. aureus alpha-toxin, comprising administering to an individual an antibody or antigen-binding fragment thereof of the present disclosure, or a pharmaceutical composition of the present disclosure. In one embodiment, the antibody binds S. aureus alpha-toxin with a KD ranging from 1 x 10 -7 to 1 x 10 -10 M; more preferably, the KD ranges from 1 x 10 -8 to 1 x 10 -10 M; more preferably, the KD ranges from 1 x 10 -9 to 1 x 10 -10 M. In one embodiment, the method provides passive immunotherapy for S. aureus infection.

[0099] The present disclosure provides a method for treating and / or preventing a disease and / or disorder caused by S. aureus infection in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an antibody or antigen-binding fragment described above. In one embodiment, the S. aureus infection is pneumonia.

[0100] The term "treatment" as used herein refers to administering an agent or formulation to an individual who has clinical symptoms of an adverse condition, disorder, or disease, to reduce the severity and / or frequency of symptoms, to eliminate symptoms and / or their underlying cause, and / or to promote healing or remediation of damage. The term "prevention" refers to administering an agent or composition to an individual who is clinically asymptomatic but susceptible to a particular adverse condition, disorder, or disease, and thus involves preventing the occurrence of symptoms and / or their underlying cause. As understood by those skilled in the art, prevention or prophylaxis need not achieve an absolute (complete) blocking or avoidance of the condition. Rather, prevention can achieve a substantial (e.g., more than 50%) reduction or avoidance of the disease or condition to be prevented. Unless otherwise expressly specified or implied herein, if the term "treatment" (or "treating") is used without reference to possible prevention, prevention is also included.

[0101] The present disclosure provides a method of detecting S. aureus alpha-toxin in a sample, comprising contacting the sample with an antibody or antigen-binding fragment as described above.

[0102] The present disclosure provides a kit for detecting S. aureus alpha-toxin in a sample, comprising an antibody or antigen-binding fragment as described above.

[0103] Anti-alpha-toxin antibodies of the present disclosure can also be used to detect and / or measure alpha-toxin or alpha-toxin-expressing cells in a sample, e.g., for diagnostic purposes. For example, anti-alpha-toxin antibodies or fragments thereof can be used to diagnose a condition or disease characterized by abnormal expression (e.g., overexpression, underexpression, lack of expression, etc.) of alpha-toxin. Exemplary diagnostic assays for alpha-toxin can include, e.g., contacting a sample obtained from a patient with an anti-alpha-toxin antibody of the present disclosure, wherein the anti-alpha-toxin antibody is labeled with a detectable label or reporter molecule. Alternatively, unlabeled anti-alpha-toxin antibodies can be bound to a secondary antibody that is itself detectably labeled for diagnostic use. The detectable label or reporter molecule can be a radioisotope, such as 3 H、 14 C、 32 P、 35 S or 125 I; a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine; or an enzyme, such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Particular exemplary assays that can be used to detect or measure alpha-toxin in a sample include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence-activated cell sorting (FACS).

[0104] The following examples are provided to aid one skilled in the art in practicing the present disclosure.

[0105] Example

[0106] Materials and Methods

[0107] Antigen preparation

[0108] Non-toxic α-toxin mutant AT H35L The sequence was constructed into the pET27b vector (pET27b-TAC1pα-hemolysin-6His) with a C-terminal 6X-His tag. The α-toxin AT was expressed and purified from Escherichia coli strain BL21. H35L In short, at 37℃ and OD 600 750 mL of fresh culture was cultured at 0.67 °C. IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.25 mM, and protein expression was induced at 30 °C for 5 h. Cells were then harvested and resuspended in 100 mL of lysis buffer, followed by homogenization for 7 cycles using a French press. The cell lysate was clarified and then mixed with 2 mL of Ni-NTA. Recombinant His-tag α-toxin AT H35L After 2 hours of binding, the sample was eluted with 20–150 mM imidazole solution and dialyzed into a phosphate buffer solution.

[0109] Immunity and phage library generation

[0110] α-toxin AT H35L Eight to ten-week-old BABL / c mice were immunized with an incomplete Freud adjuvant every two weeks for 10 weeks. Additional stimulants were administered daily for three days prior to sacrifice, following the final two-week injection. An anti-AT-scFv (single-stranded variable region) phage library was prepared from mouse spleen cells. In short, using… Mouse spleens were homogenized and lysed using reagents to extract RNA, which was then synthesized using SuperScipt III™. The heavy chain variable region (VH) and light chain variable region (VL) of the DNA fragment were amplified using scFv primers. The VH region was then amplified by PCR. L V H A mixture of flexible connectors was used to prepare V L Connector V H V L -Connector-V H Fragments were digested with Sfi, and the excised fragments were ligated into phage vectors. The ligation mixture was electroporated into viable *E. coli* TG1 cells to generate a phage library. Diversity was estimated at 1.24 × 10⁻⁶. 9 A transformant.

[0111] Phage library affinity screening

[0112] Antibodies against AT were then selected from the phage library using both solution-based and plate-based approaches. For solution-based panning, biotinylated-α-toxin was incubated with the phage library and streptavidin magnetic beads. After washing, the bound phage antibodies were eluted. The eluted phage was amplified and used for the next round of panning. Three rounds of panning were performed. For plate panning, α-toxin was coated onto microplates and incubated with the phage library, and three rounds of plate panning were performed. The ability of the phage particles to bind α-toxin was analyzed by phage ELISA, and DNA sequencing was performed.

[0113] Phage ELISA

[0114] Ten microliters of individual phage were grown overnight in 150 μL of 2YT-A medium and incubated in a 96-well microplate for 2 hours at 37°C. The culture medium was then infected with 50 μL of helper phage (2 x 10 10 PFU / ml) and incubated with shaking at 37°C for 2 hours. Fifty microliters of 2YT-A medium supplemented with 125 μg / ml kanamycin was added to the culture plate and shaken overnight at 30°C. The culture supernatant containing the phage of interest was obtained by centrifugation at 3300 x g for 30 minutes and used for phage ELISA screening. One hundred μL of the phage supernatant was added to the ELISA plate coated with α-toxin. Positive binders were detected with mouse anti-M13-HRP and TMB substrate. The absorbance was measured at 450 nm by enzyme-linked immunosorbent assay.

[0115] Construction and expression of full-length antibodies

[0116] The light and heavy chains were treated with Dralll / BsiWI and Mlul / Nhel, respectively. The inserts were ligated into vectors containing the light and heavy chain constant regions and transfected into F293 cells for expression. The purified full-length antibodies were tested for neutralization of α-toxin-induced A549 cell lysis and ranked by IC 50 .

[0117] SPR with recombinant α-toxin

[0118] Surface plasmon resonance (SPR) was used to determine the binding kinetics of 25A1 to recombinant AT. Briefly, approximately 200 response units (RU) of 25A1 were immobilized on a CM5 chip using standard amine coupling procedures. Subsequently, 25A1 was serially diluted at concentrations of 1.5625, 3.125, 6.25, 12.5, 25, and 50 nM and injected at a rate of 30 μL / min for 180 seconds, followed by a 480-second dissociation period. Kinetic parameters (K on and K off) and affinity (K D ) and affinity (K

[0119] Humanization of parental mouse monoclonal antibody 25A1

[0120] Humanization was performed by complementarity determining region (CDR) grafting. Mouse 25A1 protein sequence was aligned with human germline sequences and human sequences with high homology were identified. Light chain family IGVK3-11 and heavy chain family IGHV1-2 were used as framework sequences. In addition to CDR grafting, further analysis of the sequence for potential free cysteines, deamination, lysine clipping and protease cleavage sites was performed. The fusion and humanized 25A1 antibodies were transiently expressed in F293 cells and purified.

[0121] Neutralization of alpha-toxin induced A549 cell lysis

[0122] A functional assay method for anti-alpha-toxin antibodies was established. A549 cells were seeded in microplates at 2 x 10 4 cells / well and incubated overnight at 37°C and 5% CO2. The next day, the culture medium was removed and the cells were washed with culture medium. Purified antibodies were added to the cells at concentrations of 0.4, 4 and 40 pg / mL and incubated with 8 pg / mL of AT. After incubation, cell viability was analyzed using the colorimetric MTT assay kit. The results were determined using the background absorbance at OD 690 nm and subtracted from the OD 570 nm measurements. Figure 1 ) and affinity (K

[0123] Rabbit red blood cell lysis assay

[0124] S. aureus crude supernatant was collected from 3 ml tryptic soy broth (TSB) overnight cultures by centrifugation at 6000 rpm for 10 minutes. The supernatant of various strains was then filter sterilized and stored at -80°C until further use.

[0125] Red blood cell hemolysis was assessed for the ability of antibodies to neutralize. Specifically, 25 μl of antibody at concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 were added to wells with 100 μl of 10% rabbit red blood cells, followed by the addition of 25 μl of 1:8-1:16 dilution of S. aureus culture supernatant from different strains. After incubation at 37°C for 45-60 minutes, the plates were centrifuged for 5 minutes, and 50 μl of supernatant was gently moved to a new microtiter plate, and the absorbance was read at 450 nm. Antibody titers were defined as the concentration of antibody at which 50% of the alpha-toxin-induced hemolysis was inhibited. 2% Triton X-100 was used as the 100% hemolysis control. Hemolysis inhibition was calculated as ((2% Triton X-100 OD450 - test antibody OD450) / 2% Triton X-100 OD450) x 100%.

[0126] Murine bacteremia model

[0127] Six female BALB / c mice were injected intraperitoneally with control antibody or 25A1 antibody, and 24 hours later were injected intravenously with a lethal dose of 90% S. aureus BAA-1717. Animals were observed for 10 days for mortality. Survival was recorded using GraphPad Prism and results were analyzed. Statistical significance was analyzed using Kaplan-Meir survival analysis and Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests.

[0128] Murine pneumonia model

[0129] Ten 7-9 week old female C57BL / 6J mice (Jackson Labs, Bar Harbor, MI) were passively immunized with SYN100 (25A1-B5B6AQT) by intraperitoneal injection, and then 24 hours later were injected intranasally (IN) with a lethal dose of S. aureus. Animals were surveyed for survival 3 times daily for 7 days post-infection. Survival was recorded using GraphPad Prism and results were analyzed. Statistical significance was analyzed using Kaplan-Meir survival analysis and Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests.

[0130] Lagomorph pneumonia model

[0131] A group of 3–9 male New Zealand rabbits was treated with different doses of SYN100 24 hours before infection. The inoculation dose was maintained at 2.9–5.2 × 10⁻⁶. 7 CFU / rabbit or less. If linezolid is used, administer subcutaneously at a dose of 50 mg / kg / 8h 4 hours post-infection. Monitor animal survival twice daily for 7 days post-infection. Record survival rates and analyze results using GraphPad Prism. Statistical significance was analyzed using Kaplan-Meir survival analysis with Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests.

[0132] Example 1: Isolation of anti-α-toxin antibodies

[0133] BABL / c mice were immunized with recombinant α-toxin specifically inactivated by introducing an H35L mutation. We then constructed a single-chain variable fragment (scFv) phage library and panned it with α-toxin purified from Staphylococcus aureus. After three rounds of panning, 29 unique binders were identified, produced, and their neutralizing activity was further tested using an α-toxin-induced A549 cell lysis assay. The results showed that only 10 binders (25A1, 25A10, 25E4, 25E12, 25H3, 25B7, 25G1, 25G4, 5H9, and N2F6) could inhibit A549 cell lysis when the antibody was used at 40 μg / mL. Figure 1 Table 3 summarizes the neutralizing activity of the 10 antibodies. The antibodies were then converted to full-length antibodies, and their binding and neutralizing activities were further identified. The CDR sequences of the 10 clones are shown in Table 2 (as shown above). CDR sequence comparison revealed that 9 amino acid sequences were almost identical among the 10 inhibitory antibodies. Five of these (25A10, 25A1, 25E12, 25H3, and 25E4) exhibited very similar functional activities in neutralizing AT-induced A549 cell lysis. Clone 25A1 was screened based on binding affinity and functional activity.

[0134] Table 3:

[0135]

[0136]

[0137] Example 2: High affinity binding of 25A1 to recombinant α-toxin

[0138] The affinity of 25A1 for recombinant AT was evaluated using surface plasmon resonance (SPR). Figure 2 As shown, 25A1 binds to α-toxin, K D It is 8.346×10-10 M, with an association constant of 7.608 x 10 5 M -1 s -1 , with a dissociation constant of 6.349 x 10 -4 s -1 . This data indicates that 25A1 has a high affinity for AT.

[0139] Engineering and characterization of humanized 25A1

[0140] To reduce the immunogenicity introduced by the mouse antibody, we chose to graft the CDRs of the mouse 25A1 antibody onto the human frameworks IGVK3-11*01F (light chain) and IGHV1-2*02F (heavy chain) because they have high sequence and conformational similarity to mouse 25A1. Different combinations of back mutations were generated and subjected to antigen binding assays. Based on the binding affinity and the number of back mutations, two variants of heavy chain 25A1-VHB2 and 25A1-VHB5 and 2 variants of light chain 25A1-VLB4 and 25A1-VLB6 were selected to construct variants 25A1-HuB2B4, 25A1-HuB5B4, 25A1-HuB2B6 and 25A1-HuB5B6. The sequence changes of the antibodies after CDR grafting and back mutation are shown in Figure 3(A). The binding kinetics (KD) of humanized antibodies 25A1-HuB2B4, 25A1-HuB5B4, 25A1-HuB2B6 and 25A1-HuB5B6 to recombinant a-toxin were determined by ForteBio to be 1.1 x 10 D M, 1.5 x 10 -9 M, 1.1 x 10 -9 M, 1.1 x 10 - 9 M and 1.1 x 10 -9 M, respectively, which are highly similar to the KD of the parental mouse antibody 25A1, which is 1.5 x 10 -9 M (Figure 3(B)).

[0141] We then selected 25A1-B2B4 and 25A1-B5B6 for sequence reliability check. A potential glycosylation site was found at N61 in the CDR2 region of the heavy chain (Figure 3(C)). Therefore, N61 was mutated to alanine to avoid unnecessary complications caused by additional glycosylation; the N61A mutant clones were named 25A1-B2B4AQT and 25A1-B5B6AQT.

[0142] Inhibition of a-toxin-induced rabbit red blood cell hemolysis in Example 4

[0143] Humanized 25A1, 25A1-B2B4AQT and 25A1-B5B6AQT were then prepared and tested for their ability to inhibit rabbit red blood cell hemolysis induced by native alpha toxin. Briefly, stationary phase (overnight culture) bacterial supernatants were collected from five S. aureus clinical strains (BAA-1717, BAA-1756, ATCC33592, BAA-42 and Wood46) and added to rabbit red blood cells along with various anti-alpha toxin antibodies at concentrations ranging from 0.195 to 25 pg / mL. The percent inhibition of red blood cell hemolysis induced by recombinant and native alpha toxin for the five test strains is shown in Figure 4 Table 1. Antibodies 25A1, 25A1-B2B4AQT and 25A1-B5B6AQT all bound to native alpha toxin from the test strains and produced about 50-90% inhibition of red blood cell lysis mediated by the various native alpha toxins. The IC 50 values for 25A1, 25A1-B2B4AQT and 25A1-B5B6AQT inhibition of recombinant alpha toxin induced hemolysis were 462.3, 442.9 and 304.2 pg / mL, respectively; the IC 50 values for ATCC33592 were 1518, 1801 and 1830 ng / mL, respectively; the IC 50 values for BAA-1756 were 6913, 7956 and 7322 ng / mL, respectively; the IC values for Wood46 were 1299, 1707 and 1537 ng / mL, respectively; and the IC values for BAA-42 were 860.6, 910.8 and 996.3 ng / mL, respectively, showing that both 25A1-B2B4AQT and 25A1-B5B6AQT retained comparable inhibitory capacity to 25A1.

[0144] Example 5 SYN100 improves survival in mouse bacteremia and pneumonia models

[0145] Staphylococcus aureus is a common cause of sepsis, a systemic inflammatory condition accompanied by multi-organ dysfunction. Alpha toxin plays an important role in sepsis models, as S. aureus hla mutants exhibit delayed time to death and increased survival in sepsis models. Therefore, we tested the ability of 25A1 to protect mice from S. aureus infection. As shown in Figure 5 Figure 6, deaths were observed in the control group between days 2 and 5. In contrast, survival was improved in the 25A1 treatment groups; the overall survival rates for the 50, 25, 10 and 5 mg / kg dose groups were 83%, 67%, 33% and 50%, respectively Figure 5), indicating that prophylactic treatment with 25A1 protected against septicemia infection. In another mouse sepsis model established with a methicillin-sensitive S. aureus strain, ATCC 29213, SYN100 also demonstrated protective effects as a prophylactic treatment at doses of 100, 50, and 10 mg / kg Figure 6 ). While there are some differences between strains, these observed effects support the prophylactic use of SYN100 in S. aureus sepsis and septicemia.

[0146] Since S. aureus is a common cause of respiratory tract-associated pneumonia in patients, the protective effects of SYN100 were evaluated in a mouse pneumonia model induced by S. aureus. Twenty-four hours after SYN100 administration, mice were challenged intranasally with three S. aureus clinical isolates, BAA1556 (USA300), SF8300 (USA300), or NRS261 (USA200) to induce infection. In the acute pneumonia model, the vehicle control group died within 18-20 hours post-challenge. In contrast, prophylactic administration of SYN100 significantly prolonged survival in all three models, indicating that SYN100 can provide protection against different S. aureus clinical isolates Figure 6 ).

[0147] In a subsequent experiment, the relationship of SYN100 to antibiotic treatment was tested in the mouse NRS261 pneumonia model. Vancomycin is a commonly prescribed treatment for MRSA infections in the clinic, so we tested the efficacy of SYN100 in combination with vancomycin as a standard treatment. As shown in Figure 8 , neither SYN100 at 10 mg / kg nor vancomycin at up to 30 mg / kg significantly prolonged survival in mice. Nonetheless, the three groups of mice that received SYN100 and vancomycin concurrently exhibited dose-dependent survival, highly suggesting a synergistic effect between SYN100 and vancomycin.

[0148] Example 6 SYN100 improves survival in a rabbit pneumonia model

[0149] In many ways, rabbits are a more suitable model organism for S. aureus infection than mice. Therefore, we tested the efficacy of SYN100 in a rabbit pneumonia model established with a hospital-acquired MRSA strain, ST20120426. ST20120406 is a highly virulent strain that secretes relatively high amounts of alpha-toxin, causing control animals to collapse within 24 h. As shown in Figure 9 , all doses of SYN100 tested in this model, from 25 to 125 mg / kg, significantly prolonged survival, thus providing yet another demonstration of the practicality of SYN100 in S. aureus pneumonia.

[0150] The combination of SYN100 and antibiotics was further explored in the rabbit pneumonia model on ST20120426. The data in Figure 10(A) show that a single use of 30 mg / kg of SYN100 and 50 mg / kg / 8h of linezolid (LZD) treatment, the overall survival rates were 56% and 33%, respectively, while the group receiving both SYN100 and LZD had a survival rate of 89%. Further examination of lung tissue found that only the combination therapy group had significantly reduced lung swelling, bacterial load, and a more normal macroscopic appearance. LZD inhibits the initiation of protein synthesis in bacteria and has been shown to be equally effective as vancomycin, a cell wall synthesis blocker. Taken together, these results indicate that SYN100 can complement the effects of two antibiotics and further protect against MRSA infection in an additive or synergistic manner.

[0151] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. All such alternatives, modifications, and variations are intended to fall within the scope of the disclosure. SEQUENCE LISTING <110> Unicarion Biopharm (Suzhou) Co., Ltd. <120> Staphylococcus aureus alpha-toxin specific antibodies and uses thereof <130> None <160> 31 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Mouse <400> 1 Gly Tyr Ser Phe Thr Asp Tyr Asn Met Asn 1 5 10 <210> 2 <211> 10 <212> PRT <213> Mouse <400> 2 Gly Tyr Ser Phe Thr Gly Tyr Phe Met Asn 1 5 10 <210> 3 <211> 17 <212> PRT <213> Mouse <400> 3 Ser Ile Asn Pro Tyr Tyr Gly Ile Thr Ser Tyr Asn Gln Thr Phe Lys 1 5 10 15 Gly <210> 4 <211> 17 <212> PRT <​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <210> 8 <211> 11 <212> PRT <213> Mouse <400> 8 Asp Gly Asp Gly Tyr Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 9 <211> 11 <212> PRT <213> Mouse <400> 9 Val Tyr Tyr Gly Asp Ser Leu Gly Leu Asp Tyr 1 5 10 <210> 10 <211> 10 <212> PRT <213> Mouse <400> 10 Ser Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 <210> 11 <211> 10 <212> PRT <213> Mouse <400> 11 Ser Ala Ser Ser Ser Ile Ser Tyr Met His 1 5 10 <210> 12 <211> 10 <212> PRT <213> Mouse <400> 12 Ser Ala Ser Ser Ser Lys Ser Tyr Ile His 1 5 10 <210> 13 <211> 10 <212> PRT <213> Mouse <400> 13 Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr 1 5 10 <210> 14 <211> 7 <212> PRT <213> Mouse <400> 14 Asp Thr Ser Lys Leu Ala Ser 1 5 <210> 15 <211> 7 <212> PRT <213> Mouse <400> 15 Asp Thr Ser Asn Leu Ala Ser 1 5 <210> 16 <211> 9 <212> PRT <213> Mouse <400> 16 Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5 <210> 17 <211> 9 <212> PRT <213> Mouse <400> 17 Gln Gln Trp Ser Ser Asn Pro Pro Thr 1 5 <210> 18 <211> 9 <212> PRT <213> Mouse <400> 18 His Gln Arg Ser Ser Tyr Pro Trp Thr 1 5 <210> 19 <211> 120 <212> PRT <213> Mouse <400> 19 Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Ser Ile Asn Pro Tyr Tyr Gly Ile Thr Ser Tyr Asn Gln Thr Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asp Ser Leu Gly Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 20 <211> 107 <212> PRT <213> Mouse <400> 20 Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 21 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody <400> 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Asn Pro Tyr Tyr Gly Ile Thr Ser Tyr Asn Gln Thr Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asp Ser Leu Gly Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 22 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody <400> 22 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Asn Pro Tyr Tyr Gly Ile Thr Ser Tyr Asn Gln Thr Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asp Ser Leu Gly Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 23 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ser Ile Asn Pro Tyr Tyr Gly Ile Thr Ser Tyr Asn Gln Thr Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Tyr Gly Asp Ser Leu Gly Leu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 24 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody <400> 24 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 25 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody <400> 25 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 26 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody <400> 26 Glu Ile Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gin Gin Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gin Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 27 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Humanized antibody <400> 27 Gln Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Asn Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Ser He Asn Pro Tyr Tyr Gly He Thr Ser Tyr Ala Gin Thr Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Lys Ser He Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg He Tyr Tyr Gly Asp Ser Leu Gly Leu Asp Tyr Trp Gly Gin 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 28 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody <400> 28 Glu He Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Arg Trp lie Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly lie Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gin Gin Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gin Gly Thr Lys Val Glu lie Lys 100 105 <210> 29 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody <400> 29 Gln Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Asn Met Asn Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Ser lie Asn Pro Tyr Tyr Gly lie Thr Ser Tyr Ala Gin Thr Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Thr Ser lie Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg lie Tyr Tyr Gly Asp Ser Leu Gly Leu Asp Tyr Trp Gly Gin 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 30 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Humanized antibody <400> 30 Glu lie Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Arg Leu lie Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly lie Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 31 <211> 17 <212> PRT <213> Mouse <400> 31 Ser Ile Asn Pro Tyr Tyr Gly Ile Thr Ser Tyr Asn Gln Thr Phe Arg 1 5 10 15 Gly

Claims

1. An antibody or antigen-binding fragment that specifically binds to a Staphylococcus aureus alpha-toxin epitope or fragment thereof, wherein the antibody or antigen-binding fragment comprises: a complementarity determining region of a heavy chain variable region and a complementarity determining region of a light chain variable region, wherein, The complementarity determining regions of the heavy chain variable region include CDRH1, CDRH2, and CDRH3 regions, and the complementarity determining regions of the light chain variable region include CDRL1, CDRL2, and CDRL3 regions, wherein: the CDRH1 region consists of the amino acid sequence of SEQ ID NO: 1; the CDRH2 region consists of the amino acid sequence of SEQ ID NO: 3; the CDRH3 region consists of the amino acid sequence of SEQ ID NO: 7; and the CDRL1 region consists of the amino acid sequence of SEQ ID NO: 10; the CDRL2 region consists of the amino acid sequence of SEQ ID NO: 14; and the CDRL3 region consists of the amino acid sequence of SEQ ID NO:

16.

2. The antibody or antigen-binding fragment of claim 1, which is a mammalian antibody.

3. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 19; and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:

20.

4. The antibody or antigen-binding fragment of claim 1, wherein the antibody is a monoclonal antibody.

5. The antibody or antigen-binding fragment of claim 1, wherein the antibody is a chimeric antibody.

6. The antibody or antigen-binding fragment of claim 1, wherein the antibody is a humanized antibody.

7. The antibody or antigen-binding fragment of claim 1, wherein the antibody is a human antibody.

8. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

9. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 7 and a pharmaceutically acceptable excipient.

10. Use of the antibody or antigen-binding fragment of any one of claims 1 to 7 for the manufacture of a medicament for treating or preventing bacteremia or pneumonia in a subject in need thereof.

11. Use of the antibody or antigen-binding fragment of any one of claims 1 to 7 for the manufacture of a medicament for prophylactic treatment of bacteremia or pneumonia in a subject in need thereof.

12. The use according to claim 10 or 11, wherein the antibody binds to the α-toxin with a KD in the range of 1 x 10 -7 to 1 x 10 -10 M.

13. The use of claim 10 or 11, wherein the medicament provides passive immunotherapy for Staphylococcus aureus infection.

14. Use of the antibody or antigen-binding fragment of any one of claims 1 to 7 for the manufacture of a reagent for detecting Staphylococcus aureus alpha-toxin in a sample.

15. A kit for detecting Staphylococcus aureus alpha-toxin in a sample, comprising the antibody or antigen-binding fragment of any one of claims 1 to 7.

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