Reduction of Staphylococcus aureus Infection in Colony-Forming Patients

Abstract

The present disclosure is directed to a method of treating a subject colonized with S. aureus with anti-alpha toxin antibodies or antigen-binding fragments thereof, which can reduce the incidence of infections associated with the presence of S. aureus in the subject.

Description

Background Art

[0001] Bacterial pneumonia occurring within the hospitalized patient population or the intensive care unit (ICU) patient population is a clinically significant and serious disease that greatly contributes to morbidity and mortality. This disease constitutes the second major type of nosocomial infection and the leading cause of death due to nosocomial infection in the United States (Non-Patent Document 1). Staphylococcus aureus is a major cause of hospital-acquired pneumonia. Recent studies in European ICUs have reported that 23% of ICU patients on mechanical ventilation developed pneumonia caused by Staphylococcus aureus (S. aureus), and more than half of them were caused by methicillin-resistant Staphylococcus aureus (MRSA) (Non-Patent Document 2).

[0002] Staphylococcus aureus further causes a wide range of additional diseases including skin and soft tissue infections, endocarditis, osteomyelitis, pneumonia, and bacteremia (Non-Patent Document 3). During infection, S. aureus releases a number of toxins including α-toxin (AT) as the most dominant virulence factor that causes tissue invasion and necrosis (Non-Patent Document 4). The extremely important role played by AT in the pathogenesis of S. aureus is supported by animal models (skin necrosis, pneumonia, sepsis, endocarditis, and mastitis) and by analytical studies in humans in which the presence of anti-AT antibodies during severe infection was associated with improved outcomes.

[0003] Preclinical trials have shown that monoclonal antibody-based approaches are promising for the prevention and adjunctive therapy of Staphylococcus aureus (S. aureus) infections (see, for example, Non-Patent Document 5; Non-Patent Document 6; Non-Patent Document 7; and Non-Patent Document 8). Anti-AT antibodies have shown promising results in their ability to treat and prevent S. aureus infections. MEDI4893, or sprtoxumab, is a human monoclonal antibody with a long half-life that binds to AT with high affinity and effectively blocks AT pore formation within the target cell membrane. The results of preclinical trials have demonstrated that a prophylactic method using an anti-AT antibody containing the MEDI4893 binding region reduced disease severity in murine infection models of skin necrosis, pneumonia, and lethal bacteremia / sepsis (see, for example, Patent Document 1 and Patent Document 2, each of which is incorporated herein by reference in its entirety).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

[0006] However, Staphylococcus aureus (S. aureus) infections such as pneumonia can develop extremely rapidly in patients colonized with S. aureus. Therefore, a method is needed to identify patients at risk who would achieve the greatest benefit from anti-AT antibodies. [Means for Solving the Problems]

[0007] Staphylococcus aureus (S. aureus) pneumonia is a life-threatening complication that occurs early in intensive care unit (ICU) patients on mechanical ventilation, despite infection management and antibiotic use. As demonstrated herein, anti-alpha toxin (AT) antibodies have been evaluated for the prevention of S. aureus pneumonia and have been shown to be associated with clinically meaningful efficacy (≥25% reduction in relative risk) and acceptable safety. In particular, a 32% reduction in S. aureus pneumonia was observed in patients taking anti-AT antibodies, with no concerns about safety. Furthermore, even greater efficacy was observed in a subset of patients. Accordingly, provided herein is a method for identifying patients at high risk of developing S. aureus infections who would benefit from taking anti-AT antibodies.

[0008] Provided herein is a method of treating a subject colonized with Staphylococcus aureus (S. aureus) comprising the step of administering to the subject an antibody or antigen-binding fragment thereof that binds to S. aureus alpha toxin (AT), wherein polymerase chain reaction (PCR) is used to detect the level of S. aureus in a sample obtained from the subject. In certain examples, the sample obtained from the subject has a level of S. aureus that does not exceed the level of S. aureus that correlates with the polymerase chain reaction (PCR) cycle threshold (Ct) value. In certain examples, the method reduces the incidence of infections associated with the presence of S. aureus in the subject.

[0009] A method for preventing Staphylococcus aureus (S. aureus) infection in a subject, comprising the step of administering an antibody or an antigen-binding fragment thereof that binds to S. aureus AT to the subject, wherein a sample obtained from the subject has a level of S. aureus that does not exceed the level of S. aureus correlated with the PCR Ct value, is provided.

[0010] A method comprising the step of administering sublatoxumab to a subject to reduce the incidence of S. aureus pneumonia in the subject, wherein the reduction is determined by clinical, microbiological, and radiographic means, and optionally the incidence is reduced by about 30%, is provided.

[0011] A method comprising the step of administering sublatoxumab to a subject to reduce the incidence of pneumonia of any cause in the subject, wherein the reduction is determined by clinical, microbiological, and radiographic means, and optionally the incidence is reduced by about 30%, is provided.

[0012] In certain examples, the infection is determined by clinical, microbiological, and radiographic means.

[0013] In certain examples, the clinical means include abnormal body temperature, abnormal white blood cell count, cough, purulent sputum, bronchial breath sounds, dyspnea, tachypnea (respiratory rate > 30 breaths / min), hypoxemia, or any combination thereof.

[0014] In certain examples, the microbiological means include a respiratory specimen, blood culture, pleural fluid aspirate, or lung tissue culture that is positive for S. aureus.

[0015] In certain examples, the radiographic means include new or worsening infiltrates on a chest radiograph.

[0016] In a given example, PCR has been used to detect the level of Staphylococcus aureus (S. aureus) in a sample obtained from a subject. In a given example, the sample obtained from the subject has a level of S. aureus that does not exceed the level of S. aureus that correlates with the polymerase chain reaction (PCR) cycle threshold (Ct) value.

[0017] In a given example, the method provided herein further includes the step of detecting the level of S. aureus in a sample obtained from a subject.

[0018] In a given example, the sample obtained from the subject has a level of S. aureus that does not exceed the level of S. aureus that correlates with a PCR Ct value of 29 or more. In a given example, a PCR Ct value of 29 corresponds to a concentration of S. aureus of about 1,600 to about 1,700 colony forming units (CFU) / mL.

[0019] Provided herein is a method of treating a subject in whom S. aureus infection has colonized, the method including the step of administering to the subject an antibody that binds to S. aureus AT or an antigen-binding fragment thereof, wherein the sample obtained from the subject has a concentration of S. aureus that does not exceed 1,700 CFU / mL. In a given example, the method reduces the incidence of an infectious disease associated with the presence of S. aureus in the subject.

[0020] Provided herein is a method of preventing S. aureus infection in a subject, the method including the step of administering to the subject an antibody that binds to S. aureus AT or an antigen-binding fragment thereof, wherein the sample obtained from the subject has a concentration of S. aureus that does not exceed 1,700 CFU / mL.

[0021] In a given example, the concentration of Staphylococcus aureus (S. aureus) AT was measured using PCR.

[0022] In a given example, S. aureus has formed colonies in the subject.

[0023] In a given example, the sample obtained from the subject has S. aureus at a level correlated with at least the Ct value of PCR. In a given example, the sample obtained from the subject has S. aureus at a level correlated with at least a PCR Ct value of 3.

[0024] In a given example, the level of S. aureus is detected within 3 hours, optionally within 2 hours.

[0025] In a given example, PCR detects S. aureus protein A.

[0026] In a given example, the subject is mechanically ventilated, and optionally, a ventilator is attached to the subject. In a given example, the subject is taking antibiotics.

[0027] In a given example, the sample is a skin or soft tissue sample. In a given example, the sample is obtained from the lower respiratory tract of the subject. In a given example, the sample is an endotracheal aspirate. In a given example, the sample is a tracheal sample. In a given example, the sample is a bronchial sample.

[0028] In a given example, the sample contains bacteria that will not grow in culture for identifying S. aureus. In a given example, the sample contains bacteria that are not Staphylococcus. In a given example, S. aureus is antibiotic-resistant. In a given example, the method provided herein further includes a step of determining whether S. aureus is antibiotic-resistant.

[0029] In a given example, Staphylococcus aureus (S. aureus) is methicillin-resistant. In a given example, the method provided herein further includes the step of determining whether Staphylococcus aureus (S. aureus) is methicillin-resistant.

[0030] In a given example, resistance is determined using PCR.

[0031] In a given example, the infectious disease is pneumonia. In a given example, the infectious disease is ICU (intensive care unit) pneumonia.

[0032] In a given example, the subject is human.

[0033] In a given example, an antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT binds to the same Staphylococcus aureus (S. aureus) AT epitope as an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ ID NO: 8. In a given example, an antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT competitively inhibits the binding of an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ ID NO: 8 to Staphylococcus aureus (S. aureus) AT.

[0034] In a given example, an antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of MEDI4893. In a given example, an antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT comprises a variable heavy chain (VH) complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a variable light chain (VL) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0035] In a predetermined example, the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT comprises a VH comprising the amino acid sequence of SEQ ID NO: 7. In a predetermined example, the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT comprises a VL comprising the amino acid sequence of SEQ ID NO: 8.

[0036] In a predetermined example, the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9. In a predetermined example, the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT comprises a light chain comprising the amino acid sequence of SEQ ID NO: 10.

[0037] In a predetermined example, the antibody or antigen-binding fragment that binds to Staphylococcus aureus (S. aureus) AT further comprises a heavy chain constant region. In a predetermined example, the heavy chain constant region is human immunoglobulin IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 and IgA 2 selected from the group consisting of heavy chain constant regions. In a predetermined example, the heavy chain constant region is the human IgG 1 constant region.

[0038] In a predetermined example, the antibody or antigen-binding fragment that binds to Staphylococcus aureus (S. aureus) AT further comprises a light chain constant region. In a predetermined example, the light chain constant region is selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions. In a predetermined example, the light chain constant region is the human IgGκ light chain constant region.

[0039] In a predetermined example, the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT is an IgG antibody or an antigen-binding fragment thereof.

[0040] In certain examples, an antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT comprises an Fc region that has been genetically engineered to improve the half-life. In certain examples, an antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT comprises an Fc region that contains the YTE mutation.

[0041] In certain examples, the antibody or antigen-binding fragment that binds to Staphylococcus aureus (S. aureus) AT is a monoclonal antibody or antigen-binding fragment.

[0042] In certain examples, the antibody or antigen-binding fragment that binds to Staphylococcus aureus (S. aureus) AT is a full-length antibody. In certain examples, the antibody or antigen-binding fragment that binds to Staphylococcus aureus (S. aureus) AT is an antigen-binding fragment. In certain examples, the antigen-binding fragment is Fab, Fab’, F(ab’) 2 , single-chain Fv (scFv), disulfide-linked Fv, intrabodies, IgGΔCH2, minibodies, F(ab’) 3 , tetrabody, tribody, diabody, DVD-Ig, Fcab, mAb 2 , (scFv) 2 or scFv-Fc.

[0043] In certain examples, the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT has an affinity for Staphylococcus aureus (S. aureus) AT of 80 - 100 pM.

[0044] In certain examples, the antibody or antigen-binding fragment is sublatoxumab.

[0045] In certain examples, 2,000 mg of the antibody or antigen-binding fragment is administered. In certain examples, 5,000 mg of the antibody or antigen-binding fragment is administered.

[0046] In certain examples, preventing Staphylococcus aureus (S. aureus) infection includes neutralizing toxins, inducing opsonophagocytosis, inhibiting thromboembolic lesion formation, inhibiting S. aureus - related sepsis, or any combination of the above.

[0047] Provided herein is an antibody or antigen - binding fragment that binds to S. aureus AT for use in treating a subject colonized with Staphylococcus aureus (S. aureus), wherein a sample obtained from the subject has a level of S. aureus that does not exceed the level of S. aureus that correlates with the polymerase chain reaction (PCR) cycle threshold (Ct) value. In certain examples, the treating step reduces the incidence of infections associated with the presence of S. aureus in the subject.

[0048] Provided herein is an antibody or antigen - binding fragment thereof that binds to S. aureus AT for use in preventing S. aureus infection in a subject colonized with S. aureus, wherein a sample obtained from the subject has a level of S. aureus that does not exceed the level of S. aureus that correlates with the PCR Ct value.

[0049] In certain examples, a sample obtained from the subject has a level of S. aureus that does not exceed the level of S. aureus that correlates with 29 PCR Ct values. In certain examples, the antibody or antigen - binding fragment thereof is not administered to the subject in an instance where the level of S. aureus in a sample obtained from the subject is detected not to exceed the level of S. aureus that correlates with the PCR Ct value.

[0050] An in vitro method for identifying a subject in which Staphylococcus aureus (S. aureus) that is responsive to an antibody or an antigen-binding fragment thereof that binds to S. aureus AT has formed colonies, the method including a step of detecting the level of S. aureus in a sample obtained from the subject, is provided, wherein the level of S. aureus that does not exceed the level of S. aureus correlated with the PCR Ct value is responsive to the antibody or the antigen-binding fragment thereof. In a given example, the PCR Ct value is 29. BRIEF DESCRIPTION OF THE DRAWINGS

[0051]

Figure 1

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DETAILED DESCRIPTION OF THE INVENTION

[0052] The present disclosure is directed to methods for preventing Staphylococcus aureus infections in patients with low levels of Staphylococcus aureus colony formation and methods for identifying patients in whom Staphylococcus aureus is colonizing and who would benefit from α-toxin antibodies.

[0053] I. DEFINITIONS The use of the terms "a", "the", "at least one", and similar referents in the context of the present invention (particularly in the context of the following claims) should be construed to include both the singular and the plural unless otherwise indicated herein or clearly contradicted by the context.

[0054] The use of the term "at least one" following a listing of one or more items (e.g., "at least one of A and B") should be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B) unless otherwise indicated herein or clearly contradicted by the context.

[0055] The terms "comprising", "having", "including", and "containing" should be construed as non-limiting terms (i.e., meaning "including but not limited to") unless otherwise noted.

[0056] As used herein, the term "alpha toxin" or "AT" refers to a bacterial alpha toxin polypeptide including but not limited to native alpha toxin polypeptides and isoforms of alpha toxin polypeptides. "Alpha toxin" includes the full-length, unprocessed alpha toxin polypeptide as well as forms of the alpha toxin polypeptide resulting from intracellular processing. As used herein, the term "Staphylococcus aureus (S. aureus) alpha toxin":

Chem.

[0057] The Staphylococcus aureus (S. aureus) alpha toxin H35L mutant has the sequence

Chem.

[0058] "Alpha toxin polynucleotide", "alpha toxin nucleotide", or "alpha toxin nucleic acid" refers to a polynucleotide encoding alpha toxin.

[0059] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing antibodies, and any other modified immunoglobulin molecules, so long as the antibody exhibits the desired biological activity. Antibodies can be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, which are called α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. Different classes of immunoglobulins have different well-known subunit structures and three-dimensional conformations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[0060] As used in the present invention, the term "monoclonal antibody" refers to an antibody produced by a single clone of B cells that binds to the same epitope. In contrast, the term "polyclonal antibody" refers to a population of antibodies produced by different B cells that bind to different epitopes of the same antigen.

[0061] The term "antibody fragment" refers to a portion of an intact antibody. An "antigen-binding fragment", "antigen-binding domain", or "antigen-binding region" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment can include the antigen-determining regions (e.g., complementarity-determining regions (CDRs)) of an intact antibody. Examples of antigen-binding fragments of an antibody include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of an antibody can be obtained from any animal species such as rodents (e.g., mice, rats, or hamsters) and humans, or can be artificially produced.

[0062] A full antibody typically consists of the following four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have the same general structure, and each region contains four framework regions whose sequences are relatively conserved. As used herein, the term "framework region" refers to the relatively conserved amino acid sequences within the variable region located between the hypervariable regions or complementarity-determining regions (CDRs). Each variable domain has four framework regions designated FR1, FR2, FR3, and FR4. These framework regions form β-sheets that provide the structural framework of the variable region (see, for example, C.A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001)). Three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable regions" of the antibody that are responsible for antigen binding.

[0063] The terms "VL" and "VL domain" are used interchangeably to mean the variable region of the light chain of an antibody.

[0064] The terms "VH" and "VH domain" are used interchangeably to mean the variable region of the heavy chain of an antibody.

[0065] The terms "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering the amino acid residues of the heavy chain variable region and the light chain variable region of an antibody or antigen-binding fragments thereof. In certain embodiments, the CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs within an antibody heavy chain molecule are typically located at amino acid positions 31-35 (CDR1), amino acid positions 50-65 (CDR2), and amino acid positions 95-102 (CDR3), which may optionally include one or two additional amino acids (referred to as 35A and 35B in the Kabat numbering scheme) following 35. Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3). In certain embodiments, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.

[0066] Chothia, instead, refers to the positions of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The ends of the Chothia CDR-H1 loop when numbered using the Kabat numbering scheme vary from H32 to H34 depending on the length of this loop (this is because the Kabat numbering scheme places insertions at H35A and H35B, and if neither 35A nor 35B is present, this loop ends at 32, if only 35A is present, this loop ends at 33, and if both 35A and 35B are present, this loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0067]

Table 1

[0068] As used herein, the terms "constant region" or "constant domain" are interchangeable and have the meaning generally ascribed in the art. The constant region is the carboxyl-terminal portion of the light and / or heavy chains that is not directly involved in binding of the antibody portion, e.g., the antibody to an antigen, but can exhibit various effector functions such as interaction with Fc receptors. The constant regions of immunoglobulin molecules generally have more conserved amino acid sequences compared to the immunoglobulin variable domains.

[0069] As used herein, the term "heavy chain" when used in reference to an antibody refers to any distinct type, e.g., based on the amino acid sequence of the constant domain, IgG, e.g., IgG 1 , IgG 2 , IgG 3 and IgG 4Refers to α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses thereof. The heavy chain amino acid sequences are well known in the art. In certain embodiments, the heavy chain is a human heavy chain.

[0070] As used herein, the term "light chain," when used in reference to an antibody, can refer to any distinct type, such as κ (kappa) or λ (lambda), based on the amino acid sequence of the constant domain. The light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain.

[0071] A "chimeric" antibody refers to an antibody or fragment thereof that includes both human and non-human regions. A "humanized" antibody is an antibody that includes a human antibody framework and at least one CDR obtained from or derived from a non-human antibody. Examples of non-human antibodies include antibodies isolated from any non-human animal, such as rodents (e.g., mice or rats). A humanized antibody can include one, two, or three CDRs obtained from or derived from a non-human antibody. A fully human antibody does not include any amino acid residues obtained from or derived from a non-human animal. It will be understood that fully human antibodies and humanized antibodies have a lower risk of inducing an immune response in humans compared to mouse antibodies or chimeric antibodies (see, e.g., Harding et al., mAbs, 2(3):256-26(2010)).

[0072] As used herein, "epitope" is a term in the art and refers to a local region of an antigen to which an antibody or an antigen-binding fragment thereof can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear epitope or continuous epitope), or an epitope can be formed integrally from two or more non-contiguous regions of one or more polypeptides (conformational epitope, non-linear epitope, discontinuous epitope or non-contiguous epitope). In certain embodiments, the epitope to which an antibody or an antigen-binding fragment thereof binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallographic examination, ELISA assay, hydrogen / deuterium exchange with mass spectrometry (e.g., liquid chromatography liquid spray mass spectrometry), array-based oligopeptide scanning assay and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallographic structure analysis, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303).Antibody / its antigen-binding fragment: The crystals of the antigen can be studied using known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; for example, Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.,; U.S. Patent Application Publication No. 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316-1323). Mutagenesis mapping studies can be achieved using any method known to those skilled in the art. For example, for descriptions of mutagenesis techniques such as alanine scanning mutagenesis, see Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085.

[0073] An antibody that "binds to the same epitope" as a reference antibody refers to an antibody that binds to the same amino acid residues as the reference antibody. The ability of an antibody to bind to the same epitope as a reference antibody can be determined by hydrogen / deuterium exchange assays (see Coales et al. Rapid Commun. Mass Spectrom. 2009; 23: 639-647) or X-ray crystallographic analysis.

[0074] As used herein, the terms "immunologically specifically bind", "immunologically specifically recognize", "specifically bind" and "specifically recognize" are similar terms in the context of an antibody or an antigen-binding fragment thereof. These terms mean that an antibody or an antigen-binding fragment thereof binds to an epitope via its antigen-binding domain and that the binding requires some complementarity between the antigen-binding domain and the epitope. Thus, an antibody that "specifically binds" to a first Staphylococcus aureus (S. aureus) leukotoxin may also bind to other S. aureus leukotoxins, but the degree of binding to unrelated, non-leukotoxin proteins is, for example, less than about 10% of the binding of the antibody to the first S. aureus leukotoxin as measured by, for example, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), BiaCore or octet binding assay.

[0075] An antibody is said to "competitively inhibit" the binding of a reference antibody to an epitope if it preferentially binds to that epitope or an overlapping epitope to the extent that it blocks the binding of the reference antibody to the given epitope to some degree. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. An antibody can be said to competitively inhibit the binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60% or at least 50%.

[0076] The term "nucleic acid sequence" is intended to encompass polymers of DNA or RNA, i.e., polynucleotides that can be single-stranded or double-stranded and can contain non-natural or modified nucleotides. The terms "nucleic acid" and "polynucleotide" as used in the present invention refer to polymeric forms of nucleotides of either ribonucleotide (RNA) or deoxyribonucleotide (DNA), of any length. These terms refer to the primary structure of the molecule and thus include double-stranded and single-stranded DNA as well as double-stranded and single-stranded RNA. These terms include, by way of equivalents, nucleotide analogs and modified polynucleotides, e.g., but not limited to, any analogs of RNA or DNA made from methylated and / or capped polynucleotides. Nucleic acids are typically linked via phosphodiester bonds to form a nucleic acid sequence or polynucleotide, although many other linkages are known in the art (e.g., phosphorothioate, boranophosphate, etc.).

[0077] As used herein, "transfection," "transformation," or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (e.g., Murray E.J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (see Brash et al, Mol. Cell Biol., 7:2031-2034 (1987)). Phage or viral vectors can be introduced into host cells after growth of infectious particles in suitable packaging cells, many of which are commercially available.

[0078] As used herein, the terms "treatment", "treating", etc. refer to means (e.g., administration of an antibody or an antigen-binding fragment thereof provided herein to a subject) for curing, alleviating, reducing, and / or halting the progression of the symptoms of a diagnosed pathological condition or disorder. Accordingly, subjects in need of treatment include those that have already been diagnosed as having a disorder or are suspected of having a disorder. A "therapeutically effective amount" refers to an amount effective over the required dosage and period to achieve a desired therapeutic result (e.g., treatment of a Staphylococcus aureus (S. aureus) infection).

[0079] Prophylactic or preventative means refer to means (e.g., administration of an antibody or an antigen-binding fragment thereof provided herein to a subject) for preventing and / or alleviating the occurrence of a targeted pathological condition or disorder. Accordingly, subjects in need of prophylactic or preventative means include those that are susceptible to a disorder and those in which the disorder must be prevented. A "prophylactically effective amount" refers to an amount effective over the required dosage and period to achieve a desired prophylactic result (e.g., prevention of a Staphylococcus aureus (S. aureus) infection or onset of disease).

[0080] The subject in whom Staphylococcus aureus has "formed colonies" refers to a subject in whom Staphylococcus aureus is present in or on the body. Colony formation can be determined, for example, by a process of detecting Staphylococcus aureus in a sample obtained from the subject. Staphylococcus aureus can be detected, for example, by a process of culturing or by polymerase chain reaction (PCR). Infectious diseases resulting from or associated with the presence of Staphylococcus aureus in or on the subject's body exhibit radiographic signs and / or clinical signs of the bacterium. Staphylococcus aureus infections can occur, for example, as skin or soft tissue infections (SSTIs) or bacteremia. Staphylococcus aureus bacteria can move through the bloodstream, infect sites within the body, and as a result cause pneumonia, ICU pneumonia, bone or joint infections, device infections, wound infections, surgical site infections, or osteomyelitis. Radiographic signs include, for example, an X-ray showing infiltrates. Clinical signs include, for example, abnormal body temperature, abnormal white blood cell count, cough, purulent sputum, bronchial breath sounds, dyspnea, tachypnea (respiratory rate > 30 breaths / min) and / or hypoxemia.

[0081] As used herein, the terms "administer," "the process of administering," "administration," etc. refer to methods (e.g., intravenous administration) that can be used to enable the delivery of a drug, such as a combination of an anti-Staphylococcus aureus antibody or an antigen-binding fragment thereof, to a desired biological site of action. Administration techniques that can be used in conjunction with the agents and methods described herein can be found, for example, in the following: Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington’s, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0082] Administration in "combination" with one or more additional therapeutic agents includes co-administration (simultaneous administration) and sequential administration in any order.

[0083] Unless otherwise specified or clear from the context, as used herein, the term "or" is understood to be inclusive. The term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", and both "A" and "B". Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0084] II. Anti-Staphylococcus aureus α-toxin antibody As provided herein, antibodies and antigen-binding fragments thereof (e.g., monoclonal antibodies and fragments) that bind to Staphylococcus aureus (S. aureus) α-toxin can be used to avoid S. aureus in subjects colonized with low levels of S. aureus.

[0085] Alpha toxin (AT) is an important pathogenic factor in several Staphylococcus aureus (S. aureus) syndromes, including pneumonia, skin and soft tissue infections (SSTIs), bacteremia, etc. (Bubeck Wardenburg, J. and O. Schneewind, J. Exp. Med., 205:287-294 (2008); Inoshima et al., J. Invest. Dermatol., 132:1513-1516 (2012); and Foletti et al., see above). Passive immunization with anti-AT monoclonal antibodies reduces the severity of disease in pneumonia and skin necrosis models (Hua et al., Antimicrob. Agents Chemother., 58:1108-1117 (2014); Tkaczyk et al., Clin. Vaccine Immunol., 19:377-385 (2012); and Ragle, B.E. and J. Wardenburg Bubeck, Infect. Immun., 77:2712-2718 (2009)), and vaccination with an AT toxoid containing the H35L mutation (ATH35L) protected against death in murine lethal bacteremia and pneumonia models (Bubeck Wardenburg, supra, Foletti et al., supra, Hua et al., supra, Ragle, supra, Menzies, B.E. and D.S Kernodle, Infect. Immun., 77:2712-2718 (2009); and Adhikari et al., PLoS One, 7:e38567 (2012)). AT contributes to multiple aspects of the pathogenesis of S. aureus during bacteremia and sepsis, including stimulation of the hyperinflammatory response characteristic of sepsis, which leads to loss of vascular integrity, and activation of the cleavage of ADAM10-mediated endothelial adherens junctions (Powers et al., J Infect. Dis., 206:352-356 (2012); Wilke, G.A. and J. Bubeck Wardenburg, Proc. Natl. Acad. Sci. USA, 107:13473-13478 (2010); and Becker et al., J Innate Immun., 6:619-631 (2014)).AT has also been demonstrated to target platelets, which impairs the repair of the damaged endothelial barrier and promotes organ dysfunction through the formation of platelet-neutrophil aggregates (Powers et al., Cell Host Microbe, 17:775-787 (2015)). The structure and function of alpha-toxin are described in detail, for example, in Bhakdi, S. and J. Tranum-Jensen, Microbiol. Mol. Biol. Rev., 55(4):733-751 (1991).

[0086] Monoclonal and polyclonal antibodies that bind to AT are also known in the art (see, for example, Hua et al., Antimicrob. Agents Chemother., 58(2):1108-1117 (2014); and Oganesyan et al., J. Biol. Chem., 289:29874-29880 (2014)) and are commercially available from suppliers such as Sigma Aldrich (St. Louis, MO) and AbCam (Cambridge, MA). Representative antibodies that bind to AT are disclosed in International Publication Nos. WO 2012 / 109285 and WO 2014 / 074540, both of which are incorporated herein by reference in their entireties.

[0087] In one example, an antibody or antigen-binding fragment (e.g., monoclonal antibody or fragment) that specifically binds to Staphylococcus aureus (S. aureus) α-toxin (AT) consists essentially of or consists of: (i) a heavy chain polypeptide comprising the CDR1 amino acid sequence of SEQ ID NO: 1, the CDR2 amino acid sequence of SEQ ID NO: 2, and the CDR3 amino acid sequence of SEQ ID NO: 3; and (ii) a light chain polypeptide comprising the CDR1 amino acid sequence of SEQ ID NO: 4, the CDR2 amino acid sequence of SEQ ID NO: 5, and the CDR3 amino acid sequence of SEQ ID NO: 6. In another example, the heavy chain polypeptide of an antibody or antigen-binding fragment that specifically binds to S. aureus AT (e.g., monoclonal antibody or fragment) consists essentially of or consists of the variable region amino acid sequence of SEQ ID NO: 7. In another example, the light chain polypeptide of an antibody or antigen-binding fragment that specifically binds to S. aureus AT (e.g., monoclonal antibody or fragment) consists essentially of or consists of the variable region amino acid sequence of SEQ ID NO: 8. In another example, an antibody or antigen-binding fragment that specifically binds to S. aureus AT (e.g., monoclonal antibody or fragment) consists essentially of or consists of a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, which consists essentially of or consists of it. In another example, an antibody or antigen-binding fragment that specifically binds to S. aureus AT (e.g., monoclonal antibody or fragment) consists essentially of or consists of a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10, which consists essentially of or consists of it.

[0088] The sequences of representative anti-AT antibodies are provided below. Additional anti-AT antibodies are provided, for example, in U.S. Patent No. 9,527,905, which is incorporated herein by reference. In certain examples, the antibodies or antigen-binding fragments thereof described herein bind to AT and have six CDRs of the antibodies listed in the following two tables (i.e., three VH CDRs of the antibodies listed in the first table and three VL CDRs of the same antibodies listed in the second table).

[0089] The anti-AT antibody MEDI4893 (also known as sublatticexumab) is a human monoclonal antibody that binds to AT with high affinity and has a long half-life that effectively blocks AT pore formation within the target cell membrane. The extended half-life was achieved by introducing three amino acid substitutions (M252Y / S254T / T256E; called YTE) within the Fc domain to increase binding to the neonatal Fc receptor (FcRn), resulting in an increased serum half-life. The YTE mutation was used to demonstrate efficacy in preclinical test models previously described in International Publication Nos. 2012 / 109285 and 2014 / 074540 (both of which are incorporated herein by reference in their entirety), except for the YTE modification called "MEDI4893 * " or "LC10". MEDI4893 (or sublatticexumab) contains a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10. The identical CDR sequences, VH sequences, and VL sequences of MEDI4893 (sublatticexumab) and MEDI4893 * are provided in the table below.

[0090]

Table 2

[0091] In certain examples, the antibodies or antigen-binding fragments thereof described herein bind to AT and include, for example, the VH of the antibodies listed in the following table in combination with VL.

[0092]

Table 3

[0093] In a given example, the antibody or antigen-binding fragment thereof described herein binds to AT and includes the VL of the antibodies listed in the following table, optionally in combination with VH, for example, and optionally in combination with the VH of the same antibody listed in the previous table.

[0094]

Table 4

[0095] In a given example, the antibody or antigen-binding fragment thereof described herein binds to AT and includes the heavy chain of the antibodies listed in the following table, for example, in combination with a light chain.

[0096]

Table 5

[0097] In a given example, the antibody or antigen-binding fragment thereof described herein binds to AT and includes the light chain of the antibodies listed in the following table, for example, in combination with a heavy chain, and optionally includes the heavy chain of the same antibody listed in the previous table.

[0098]

Table 6

[0099] In certain embodiments, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the Chothia numbering scheme that refers to the positions of immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Patent No. 7,709,226). Typically, when using the Kabat numbering rules, the Chothia CDR-H1 loop is present at heavy chain amino acids 26-32, 33 or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52-56, the Chothia CDR-H3 loop is present at heavy chain amino acids 95-102, while the Chothia CDR-L1 loop is present at light chain amino acids 24-34, the Chothia CDR-L2 loop is present at light chain amino acids 50-56, and the Chothia CDR-L3 loop is present at light chain amino acids 89-97. The termini of the Chothia CDR-H1 loop when numbered using the Kabat numbering rules vary as H32-H34 depending on the length of this loop (this is because the Kabat numbering scheme places insertions at H35A and H35B, and when neither 35A nor 35B is present, this loop ends at 32, when only 35A is present, this loop ends at 33, and when both 35A and 35B are present, this loop ends at 34).

[0100] In certain embodiments, provided herein are combinations of antibodies and antigen-binding fragments thereof that include the Chothia VH CDRs and VL CDRs of the MEDI4893 antibody. In certain embodiments, the antibody or antigen-binding fragment thereof includes one or more CDRs, wherein the Chothia CDRs and Kabat CDRs have the same amino acid sequence. In certain embodiments, provided herein are antibodies and antigen-binding fragments thereof that include a combination of Kabat CDRs and Chothia CDRs.

[0101] In certain embodiments, the CDRs of the antibody or antigen-binding fragment thereof can be determined according to the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7:132-136 and Lefranc M-P et al., (1999) Nucleic Acids Res 27:209-212. According to the IMGT numbering scheme, VH-CDR1 is positions 26-35, VH-CDR2 is positions 51-57, VH-CDR3 is positions 93-102, VL-CDR1 is positions 27-32, VL-CDR2 is positions 50-52, and VL-CDR3 is positions 89-97. In certain embodiments, provided herein are combinations of antibodies and antigen-binding fragments thereof that include the IMGT VH CDRs and VL CDRs of MEDI4893, as described, for example, in Lefranc M-P (1999) supra and Lefranc M-P et al., (1999) supra.

[0102] In certain embodiments, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to MacCallum RM et al., (1996) J Mol Biol 262:732-745. See also, for example, Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Duebel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, provided herein are combinations of antibodies or antigen-binding fragments thereof that include the VH CDR and VL CDR of the MEDI4893 antibody as determined by the method in MacCallum RM et al.

[0103] In certain embodiments, the CDRs of an antibody or antigen-binding fragment thereof represent a compromise between Kabat CDRs and Chothia structural loops and can be determined according to the AbM numbering scheme that refers to the AbM hypervariable regions used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In certain embodiments, provided herein are combinations of antibodies or antigen-binding fragments that include the VH CDR and VL CDR of the MEDI4893 antibody as determined by the AbM numbering scheme.

[0104] In another embodiment, the antibodies or antigen-binding fragments thereof described herein (e.g., monoclonal antibodies or fragments) can include a constant region (Fc) of any suitable class (e.g., IgG, IgA, IgD, IgM, and IgE) that has been modified to improve the half-life of the antibody or antigen-binding fragment (e.g., monoclonal antibody or fragment). For example, the antibodies or antigen-binding fragments thereof described herein (e.g., monoclonal antibodies or fragments) can include an Fc that contains mutations that extend the half-life compared to the same antibody without the mutations.

[0105] Genetic manipulation of the Fc region is widely used in the art to extend the half-life of therapeutic antibodies and to protect them from in vivo degradation. In some embodiments, the Fc region of an IgG antibody or antigen-binding fragment can be modified to enhance the affinity of the IgG molecule for the neonatal Fc receptor (FcRn), which mediates IgG catabolism and protects IgG molecules from degradation. Suitable Fc region amino acid substitutions or modifications are known in the art and include, for example, the triple substitution M252Y / S254T / T256E (referred to as "YTE") (see, e.g., U.S. Patent No. 7,658,921; U.S. Patent Application Publication No. 2014 / 0302058; and Yu et al., Antimicrob. Agents Chemother., 61(1):e01020-16 (2017)). In certain embodiments, an antibody or antigen-binding fragment (e.g., a monoclonal antibody or fragment) that binds to Staphylococcus aureus AT contains an Fc region that includes the YTE mutation.

[0106] The antibodies or antigen-binding fragments (e.g., monoclonal antibodies or fragments) described herein can be or can be obtained from human antibodies, humanized antibodies, non-human antibodies, or chimeric antibodies. In one embodiment, the antibodies or antigen-binding fragments thereof described herein are fully human antibodies.

[0107] Human antibodies, non-human antibodies, chimeric antibodies or humanized antibodies can be obtained by any means including in vitro sources (e.g., hybridomas or cell lines that recombinantly produce antibodies) and in vivo sources (e.g., rodents, human tonsils). Methods for generating antibodies are known in the art and are described, for example, in Koehler and Milstein, Eur. J. Immunol., 5:511-519 (1976); Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988); and Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001). In certain embodiments, human or chimeric antibodies can be generated using transgenic animals (e.g., mice) in which one or more endogenous immunoglobulin genes have been replaced with one or more human immunoglobulin genes. Examples of transgenic mice in which the endogenous antibody genes have been effectively replaced with human antibody genes include, but are not limited to, Medarex HUMAB-MOUSE™, Kirin TC MOUSE™, and Kyowa Kirin KM-MOUSE™ (see, e.g., Lonberg, Nat. Biotechnol., 23(9):1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181:69-97 (2008)). Humanized antibodies can be generated using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley & Sons, Inc., Hoboken, N.J. (2009)), including, for example, transplantation of non-human CDRs onto a human antibody framework (see, e.g., Kashmiri et al., Methods, 36(1):25-34 (2005); and Hou et al., J. Biochem., 144(1):115-120 (2008)).In one embodiment, the humanized antibody can be produced using, for example, the method described in U.S. Patent Application Publication No. 2011 / 0287485A1.

[0108] III. Nucleic Acids, Vectors, and Host Cells Also provided herein are one or more isolated nucleic acid sequences encoding an antibody that binds to AT or an antigen-binding fragment thereof (optionally, the antibody or antigen-binding fragment thereof is a monoclonal antibody or fragment).

[0109] The present disclosure further provides one or more vectors comprising one or more nucleic acid sequences encoding an antibody that binds to AT or an antigen-binding fragment thereof (optionally, the antibody or antigen-binding fragment thereof is a monoclonal antibody or fragment). The vector can be, for example, a plasmid, episome, cosmid, viral vector (e.g., retrovirus or adenovirus), or phage.

[0110] A vector comprising a nucleic acid sequence encoding an antibody that binds to AT or an antigen-binding fragment thereof (optionally, the antibody or antigen-binding fragment thereof is a monoclonal antibody or fragment) can be introduced into a host cell capable of expressing the encoded polypeptide, including any suitable prokaryotic or eukaryotic cell. Accordingly, the present disclosure provides an isolated cell comprising this vector. Host cells that can be used include cells that can grow easily and reliably, have a moderately fast growth rate, have a well-characterized expression system, and can be easily and efficiently transformed or transfected.

[0111] A nucleic acid sequence encoding any amino acid of the antibodies or antigen-binding fragments (optionally, monoclonal antibodies or fragments) described herein can be introduced into a cell by "transfection," "transformation," or "transduction."

[0112] IV. Pharmaceutical Compositions and Methods of Administering Anti-AT Antibodies The present disclosure provides a composition comprising an effective amount of any of the AT antibodies or antigen-binding fragments thereof described herein and a pharmaceutically acceptable carrier, the amount being effective to reduce the risk of infection in a subject colonized with Staphylococcus aureus (S. aureus).

[0113] In another aspect, the composition may comprise a nucleic acid sequence encoding an AT-binding antibody or antigen-binding fragment. The nucleic acid sequence may be present within a vector or a combination of vectors.

[0114] In one aspect, the composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition comprising a carrier, such as a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and an AT-binding antibody or antigen-binding fragment nucleic acid sequence or vector.

[0115] Any suitable carrier can be used within the scope of the context of the present disclosure, and such carriers are well known in the art. The choice of carrier will be determined, at least in part, by the particular site to which the composition can be administered and the particular method used to administer the composition. Optionally, the composition can be sterile. The composition can be frozen or lyophilized for storage and reconstituted with a suitable sterile carrier prior to use. The composition can be produced, for example, according to conventional techniques described in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0116] The composition preferably comprises an AT-binding antibody or antigen-binding fragment in an amount effective to reduce the risk of S. aureus infection in a patient colonized with S. aureus. For this purpose, the methods of the present disclosure include administering a therapeutically effective amount or a prophylactically effective amount of an AT-binding antibody or antigen-binding fragment thereof or a composition of the above-described antibodies or fragments (including monoclonal antibodies or fragments).

[0117] In the case of repeated administration for several days or more according to the condition, the treatment can be repeated until the desired suppression of the disease symptoms occurs. However, other dosing regimens may be useful and are included within the scope of the present disclosure. The desired dosage can be delivered by single bolus administration of the composition, multiple bolus administrations of the composition, or continuous infusion administration of the composition.

[0118] An effective amount of an anti-AT antibody or an antigen-binding fragment thereof can be administered to a subject such as a human using standard administration techniques including intravenous, intraperitoneal, subcutaneous, and intramuscular administration routes. The anti-AT antibody or an antigen-binding fragment thereof may be suitable for parenteral administration. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In some embodiments, the anti-AT antibody or an antigen-binding fragment thereof is administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0119] A composition comprising an AT-binding antibody or antigen-binding fragment or the like can be administered alone or in combination with other drugs (e.g., adjuvants) conventionally used for treating Staphylococcus aureus infections. A composition comprising an AT-binding antibody or antigen-binding fragment can be used in combination with one or more antibiotics such as penicillinase-resistant β-lactam antibiotics (e.g., oxacillin or flucloxacillin). Gentamicin can be used to treat serious infections such as endocarditis. However, most strains of Staphylococcus aureus are currently resistant to penicillin, and 2 out of 100 people carry methicillin-resistant Staphylococcus aureus (MRSA). MRSA infections are typically treated with vancomycin, and mild skin infections can be treated with a triple antibiotic ointment.

[0120] A composition comprising an AT-binding antibody or antigen-binding fragment can be used in combination with, for example, one or more anti-Staphylococcus aureus antibiotics.

[0121] V. Method for Identifying Patients Who Would Benefit from Anti-AT Antibodies As demonstrated herein, administration of an anti-AT antibody or an antigen-binding fragment thereof to a subject in which low levels of Staphylococcus aureus (S. aureus), e.g., levels of S. aureus that do not exceed a threshold level of S. aureus, are colonizing can reduce the incidence of infectious diseases associated with the presence of S. aureus in that subject.

[0122] The amount of S. aureus in a subject can be determined based on the amount of S. aureus in a sample obtained from the subject. The sample can be, for example, a skin or soft tissue sample. The sample can be obtained, for example, from the lower respiratory tract of the subject. The sample can be, for example, an endotracheal aspirate, a tracheal sample, or a bronchial sample.

[0123] In certain embodiments, the anti-AT antibody or an antigen-binding fragment thereof is administered to a subject, where the sample obtained from the subject has a concentration of S. aureus that does not exceed a predetermined threshold that is estimated to be 3.2 log10 CFU / mL (about 1,600 - 1,700 CFU / mL).

[0124] The amount of S. aureus in a sample obtained from a subject can be quantified using polymerase chain reaction (PCR). For example, the amount of S. aureus in a sample obtained from a subject can be a number that can be quantified based on the number of PCR cycles required to reach a threshold signal, herein referred to as the "cycle threshold" or "Ct value". A high Ct value (i.e., a large number of PCR cycles required to reach the threshold signal) indicates low levels of S. aureus, while a low Ct value (i.e., a small number of PCR cycles required to reach the threshold signal) indicates high levels of S. aureus.

[0125] PCR is a particularly useful method for quantifying the amount of Staphylococcus aureus (S. aureus) in a sample because it can be performed quickly and evenly. For example, PCR can detect the amount of S. aureus in a sample within two hours. Laboratories are extremely diverse in their methods regarding bacterial culture, but laboratories will perform PCR using the same type of equipment that produces a uniform PCR yield across various laboratories.

[0126] PCR can be used to determine the amount of S. aureus in a sample based on the amplification of a single S. aureus gene or a combination of S. aureus genes. For example, PCR can be used to detect the S. aureus protein A gene. Detection of S. aureus protein A detects all S. aureus regardless of their methicillin susceptibility. However, PCR can also be used to further determine whether S. aureus is antibiotic resistant, for example methicillin resistant. For example, PCR can be used to detect the presence of the methicillin resistance determinant (mecA) and the staphylococcal cassette chromosome (SCCmec). Detection of both mecA and SCCmec indicates that S. aureus is methicillin resistant.

[0127] In certain embodiments, the anti-AT antibody or antigen-binding fragment thereof is administered to a subject, wherein a sample obtained from the subject has a level of Staphylococcus aureus (S. aureus) that does not exceed the highest level of S. aureus correlated with the PCR Ct value. In certain embodiments, the anti-AT antibody or antigen-binding fragment thereof is administered to a subject, wherein a sample obtained from the subject has a level of S. aureus that does not exceed the level of S. aureus correlated with a PCR Ct value of about 29. In certain embodiments, the anti-AT antibody or antigen-binding fragment thereof is administered to a subject, wherein a sample obtained from the subject has a level of S. aureus that does not exceed the level of S. aureus correlated with a PCR Ct value of 29 - 36.

[0128] In certain embodiments, the anti-AT antibody or antigen-binding fragment thereof is administered to a subject, wherein a sample obtained from the subject has a level of S. aureus that is at least at the lowest level of S. aureus correlated with the PCR Ct value. In certain embodiments, the anti-AT antibody or antigen-binding fragment thereof is administered to a subject, wherein a sample obtained from the subject has a level of S. aureus correlated with a PCR Ct value of about 3 to about 29. In certain embodiments, the anti-AT antibody or antigen-binding fragment thereof is administered to a subject, wherein a sample obtained from the subject has a level of S. aureus correlated with a PCR Ct value of 3 - 29.

[0129] A PCR Ct value of 29 is estimated to correspond to a concentration of about 1,600 - 1,700 colony forming units (CFU) / mL of S. aureus.

[0130] The methods provided herein for identifying subjects who would benefit from ingesting an anti-AT antibody or antigen-binding fragment thereof may be particularly useful in subjects wearing a ventilator. Thus, the subject may be a subject wearing a ventilator. The overall disease incidence rate is quite low, about 1-2%, but in patients colonized with Staphylococcus aureus, the incidence of Staphylococcus aureus pneumonia is much higher (greater than 20%). In subjects wearing a ventilator, Staphylococcus aureus pneumonia is generally an early event that occurs within the first week after the initiation of mechanical ventilation, so earlier administration of an anti-AT antibody or antigen-binding fragment thereof may be extremely important in avoiding pneumonia. PCR-based techniques for assessing the level of Staphylococcus aureus in a patient can be completed within about 2 hours, while culture techniques, on the other hand, are much slower (and lack the accurate quantitative nature of PCR-based techniques).

[0131] PCR-based techniques are further beneficial compared to culture-based techniques for Staphylococcus aureus colony formation because PCR-based techniques may be more sensitive in detecting Staphylococcus aureus, particularly in subjects taking antibiotics. Thus, the subject may be able to take antibiotics. Further, in certain aspects of the methods provided herein, PCR analysis of a sample obtained from a subject indicates that the subject would benefit from ingesting an anti-AT antibody or antigen-binding fragment thereof, and at the same time the sample does not contain bacteria that would grow in a culture assay used to determine the presence of Staphylococcus aureus.

[0132] The following examples further illustrate the invention, but of course should not be construed as limiting the scope of the invention.

Examples

[0133] Example 1 Staphylococcus aureus (S. aureus) pneumonia in patients wearing a ventilator is generally an early event that occurs within the first week after mechanical ventilation. Therefore, the rapid identification of patients at risk of developing this infectious disease can potentially save lives. Although the overall disease incidence rate is relatively low at about 1 - 2%, in patients colonized with S. aureus, the incidence rate is much higher (over 20%).

[0134] Rapid polymerase chain reaction (PCR) was analyzed as an assay for identifying patients at risk of S. aureus colonization in the lower respiratory tract (LRT) in a phase 2 clinical trial of ventilated patients in the intensive care unit (ICU). The Xpert® MRSA / SA SSTI assay (Cepheid, Sunnyvale, CA) was used according to the product protocol for performing PCR. This assay is a high - speed fully automated DNA test for directly and simultaneously detecting methicillin - resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA) from skin and soft - tissue specimens. Specimens are collected on a double swab that is placed inside a tube containing elution reagent. After briefly vortexing, the elution material provided with the assay and two single - use reagents (reagent 1 and reagent 2) are transferred to various uniquely marked chambers of a disposable fluid cartridge (Xpert MRSA / SA cartridge). This cartridge is placed on the GeneExpert® DX system instrument platform, which performs automated real - time multiplex PCR for detecting DNA. In this platform, additional sample preparation, amplification, and real - time detection are fully automated and fully integrated.

[0135] The primers and probes in the Xpert MRSA / SA assay detect the nucleic acid sequences of Staphylococcus aureus protein A (spa), the gene for MecA-mediated oxacillin resistance (mecA), and the staphylococcal cassette chromosome (SCCmec) inserted into the SA chromosomal attB site. This assay includes sample processing controls to manage proper processing of the target bacteria and to monitor for the presence of inhibitors in the PCR assay. The probe check control device verifies reagent rehydration, filling of the PCR tubes within the cartridge, probe integrity, and dose stability.

[0136] The assay was depicted in Figure 1: Endotracheal aspirate (ETA) samples were obtained from patients and the swabs of the samples were inserted into the elution reagent. The mixture was vortexed and dispensed into the cartridge. The cartridge was then inserted into the PCR instrument to initiate the test. The total time for the test to be carried out was less than 2 minutes and the entire test was completed within 75 minutes.

[0137] The PCR test generates a cycle threshold (Ct) value that represents the number of PCR cycles required to reach the threshold signal. The Ct value is inversely proportional to the bacterial load: the more Staphylococcus aureus (S. aureus) there are in the sample, the fewer cycles are required to reach the threshold level, thus having a low Ct value, while on the other hand, fewer numbers of Staphylococcus aureus (S. aureus) in the sample require more cycles to reach the threshold level, thus having a higher Ct value for this reason.

[0138] Example 2 Using the rapid PCR test considered in Example 1, the presence or absence of Staphylococcus aureus (S. aureus) was detected by looking at three molecular targets, and it was determined whether the Staphylococcus aureus (S. aureus) was methicillin-resistant Staphylococcus aureus (S. aureus (MRSA)). The first target detects Staphylococcus aureus protein A (SPA), which detects all Staphylococcus aureus (S. aureus) regardless of their methicillin susceptibility status. The second and third targets are the methicillin-resistance determinant mecA and the Staphylococcus aureus chromosomal cassette (SCCmec) used to detect MRSA.

[0139] The rapid PCR test was performed on 720 patients at the time of screening in the Phase 2 clinical trial. Among these patients, 299 (41.5%) had colonies of Staphylococcus aureus (S. aureus). This number is higher than the colony-forming rate of Staphylococcus aureus (S. aureus) in the general public (25 - 30%), but it is a reasonable number for patients wearing ventilators in the ICU. Of the 299 patients with colonies of Staphylococcus aureus (S. aureus), 277 (92.6%) had methicillin-sensitive Staphylococcus aureus (S. aureus) (MSSA), and 22 (7.4%) had MRSA.

[0140] The Ct values for 295 of the 299 patients with colonies of Staphylococcus aureus (S. aureus) are shown in Figure 2. (Patients with 4PCR + zero or Ct values that could not be obtained are not shown.) The average Ct value in these patients was 25.7, and a significant number of patients had low Ct values (high bacterial load) at the time of screening.

[0141] Screening was performed at various time points after the start of wearing the ventilator. Therefore, the Ct value was compared with the number of days of wearing the ventilator. The results shown in Figure 3 prove that the Ct value has a wide range of distribution independent of the number of days of wearing the ventilator. Therefore, the number of days of wearing the ventilator could not be used to predict the colony formation or Ct value of Staphylococcus aureus (S. aureus).

[0142] Example 3 In this example, it is demonstrated that rapid PCR is more sensitive than the culture assay in detecting Staphylococcus aureus (S. aureus) colony formation. The culture assay usually includes either the step of plating an undiluted sample or serial dilutions on agar containing an agar plate or the step of streak culture, and varies greatly among different laboratories. After incubating the plate for 24 to 48 hours, the scattered colonies of Staphylococcus aureus (S. aureus) were counted (quantitative culture method), or either the number of quadrants of Staphylococcus aureus (S. aureus) growth and the growth density (semi - quantitative culture method) were evaluated. Qualitative culture does not provide quantification and only evaluates the presence or absence of Staphylococcus aureus (S. aureus). (See Figure 4.) Therefore, using different culture methods, the culture status (positive or negative for Staphylococcus aureus (S. aureus) infection) may be different, and if inaccurate, it may disadvantageously bias the treatment regimen.

[0143] Culture was performed on a randomized subgroup of 299 patients (N = 209). Only 162 out of 209 (77.5%) of these patients produced positive culture results, while the other 47 (22.5%) produced negative culture results. Therefore, the culture assay would miss 22.5% of the subjects in whom Staphylococcus aureus (S. aureus) formed colonies. In other words, a concordance rate of 77.5% and a discordance rate of 22.5% were observed between the rapid PCR test and the culture assay. All of this discordance was due to samples where the test result was positive by rapid PCR and negative by culture. (See Figure 6.)

[0144] Further scrutiny of the results demonstrated that a significantly higher percentage of patients with samples that were PCR positive and culture negative had been concomitantly using antibiotics compared to patients with samples that were positive by both PCR and culture. (See Figure 7.) These data suggest that antibiotic use has a negative impact on culture results. However, the history of antibiotic use had no impact on culture results. (See Figure 8.)

[0145] Furthermore, the sensitivity of the culture assay varied due to differences in plate culturing (e.g., sample dilution, plate culture volume, and number of plates) among various laboratories. The investigation of the laboratories is summarized in the following table.

[0146]

Table 7

[0147] Among these 9 laboratories, the detection limit varied between 3.3 and 10 5 CFU / mL. Therefore, the low culture sensitivity in some laboratories also contributed to the discrepancy between PCR and the culture assay.

[0148] The Ct cutoff value regarding whether the culture was classified as mild, moderate, or severe was examined. The majority of cultures classified as severe decreased to less than 29 PCR Ct values. However, a significant portion of cultures classified as moderate also decreased to less than 29 PCR Ct values. In fact, some cultures classified as mild also decreased to less than 29 PCR Ct values. Therefore, the evaluation of these cultures was inconsistent. (See Figure 5.)

[0149] The determination of methicillin susceptibility was consistent using either a rapid PCR or culture assay. Among 162 samples examined by both PCR and culture, regardless of the method, 9 cases (5.6%) were identified as MRSA and 153 cases (94.4%) were identified as MSSA.

[0150] These results demonstrate that both the culture and the PCR assay are equally effective in detecting methicillin susceptibility, but the PCR assay is more sensitive with respect to Staphylococcus aureus colony formation.

[0151] Example 4 Ct values were analyzed in culture-positive and culture-negative samples. The distribution of culture-positive and culture-negative samples according to Ct value is shown in FIG. 9, and the results are also summarized in the following table.

[0152] [Table 8]

[0153] Based on these results, 29 Ct values perform the best job of discriminating between culture-positive and culture-negative samples.

[0154] Ct values were also correlated with Staphylococcus aureus concentration. The results shown in FIG. 10 demonstrate that there is a statistically significant inverse linear correlation between Ct value and the number of CFU / mL. The majority of samples with Ct values greater than 29 have low Staphylococcus aureus loads (≦10 3 CFU / mL), and the majority of samples with Ct values less than 29 have high Staphylococcus aureus loads (>10 4 CFU / mL). A Ct value of 29 corresponded to approximately 3.2 log10 CFU / mL (approximately 1,600 - 1,700 CFU / mL).

[0155] A similar correlation between Ct value and Staphylococcus aureus concentration was detected in bronchial and tracheal cultures. (See FIG. 11.)

[0156] A similar correlation between Ct value and Staphylococcus aureus concentration was also detected with respect to whether or not non-Staphylococcus growth was observed. (See FIG. 12.)

[0157] These results demonstrate that PCR is a robust method for quantifying Staphylococcus aureus colony formation and that a Ct cutoff value of 29 effectively discriminates between low and high levels of Staphylococcus aureus colony formation.

[0158] Example 5 The efficacy of MEDI4893, an anti-Staphylococcus aureus antibody for preventing Staphylococcus aureus pneumonia in ICU patients on mechanical ventilation, was compared in patients with low and high levels of Staphylococcus aureus colony formation using a Ct cutoff value of 29.

[0159] Subjects were considered to be on mechanical ventilation if they had (i) an endotracheal or nasotracheal tube in place and were receiving positive pressure ventilation support, or (ii) no endotracheal or nasotracheal tube in place but had required ≥8 hours of positive pressure ventilation (e.g., patients who had received tracheotomy, continuous positive airway pressure [CPAP], etc.) within the previous 24 hours.

[0160] Staphylococcus aureus pneumonia was diagnosed in mechanically ventilated patients at the time of diagnosis when they met the following radiographic, clinical, and microbiological criteria and did not have any obvious non-infectious cause.

[0161] Radiographic criteria: New or worsening infiltrates consistent with pneumonia on chest X-ray obtained within 24 hours of the event (diagnosed by a qualified radiologist), and

[0162] Clinical criteria: At least two of the following minor or one major respiratory sign or presenting symptom · Minor criteria: Systemic signs of infection (one or more of the following): Abnormal body temperature (hypothermia defined by oral temperature > 38°C or tympanic temperature, or core temperature ≥ 38.3°C, or core temperature < 35°C), and / or abnormal white blood cell count (> WBC count of 10,000 cells / mm3, < WBC count of 4,500 cells / mm3, or > 15% band neutrophils) Production of purulent tracheal secretions New physical examination findings consistent with pneumonia / pulmonary fibrosis (e.g., crackles, ronchi, bronchial breath sounds), dullness on percussion · Major criteria: Rapid changes occurring in a ventilation support system for improving oxygen supply as determined below PaO maintained for at least 4 hours < 240 mmHg 2 / FiO 2 Ratio, or A reduction of ≥ 50 mmHg in PaO maintained for at least 4 hours 2 / FiO 2 And

[0163] Microbiological confirmation (obtained within 24 hours after the start of the event) of at least one of the following · Respiratory specimens are positive for Staphylococcus aureus (S. aureus) by culture. Examples include specimens of respiratory secretions obtained by endotracheal aspiration in intubated subjects, or by bronchoscopy using bronchoalveolar lavage (BAL) or protected specimen brush (PSB) sampling. Sputum specimens were acceptable in non-intubated subjects who met the ventilator protocol definition. · Blood cultures positive for Staphylococcus aureus (S. aureus) (and no obvious primary extra-pulmonary source of infection) · Pleural fluid aspirates or lung tissue cultures positive for Staphylococcus aureus (S. aureus) during an episode of pneumonia

[0164] The results are shown in the following table.

[0165]

Table 9

[0166] Reduction in relative risk (5,000 mg of MEDI4893 vs placebo; p-value based on Poisson regression with 90% confidence interval (CI) and robust variance.

[0167] An 82.6% reduction in relative risk of Staphylococcus aureus (S. aureus) monomicrobial pneumonia (90% CI: -1.0%, 97.0%), a 30.6% reduction in relative risk of pneumonia of any cause (90% CI: -4.9%, 54.0%) and a 23.1% reduction in relative risk of pneumonia or death of any cause (90% CI: 23.1% - 4.9%, 43.6%) were also observed.

[0168]

Table 10

[0169] The p-value for interaction was obtained from a Poisson regression analysis with robust variance, including treatment group, subgroup under examination and treatment by subgroup interaction. The reduction in relative risk (5,000 mg of MEDI4893 vs placebo) and 90% confidence interval (CI) were based on unconditional confidence intervals for ratios.

[0170] Patients with at least 29 Ct values (low Staphylococcus aureus (S. aureus)) had an incidence of 33.3% and MEDI4893 produced a statistically significant relative risk reduction (RRR) of approximately 67%. In contrast, the overall population had an incidence of 26% and MEDI4893 produced a non-statistically significant 32% RRR. Patients with less than 29 Ct values (high Staphylococcus aureus (S. aureus)) had an incidence of 22.2% and a non-statistically significant RRR of approximately 2.5%.

[0171] These results demonstrate that MEDI4893, an anti-α-toxin antibody, showed increased efficacy in patients with low Staphylococcus aureus (S. aureus) colony formation.

[0172] Example 6 The effect of the anti-Staphylococcus aureus (S. aureus) antibody on the conservation of medical resources was also analyzed. The main results are summarized in the following table.

[0173]

Table 11

[0174] These results prove that MEDI4893 reduced the length of hospital stay, the length of stay in the intensive care unit (ICU), and the duration of mechanical ventilation.

[0175] Aspects of the present invention are further described in the following clauses: [Clause 1] A method for treating a subject colonized with Staphylococcus aureus (S. aureus), said method comprising administering to said subject an antibody or antigen-binding fragment thereof that binds to S. aureus alpha toxin (AT), wherein polymerase chain reaction (PCR) is used to detect the level of S. aureus in a sample obtained from said subject. [Clause 2] The method according to clause 1 above, wherein the sample obtained from the subject has a level of S. aureus that does not exceed the level of S. aureus correlated with the polymerase chain reaction (PCR) cycle threshold (Ct) value. [Clause 3] The method according to clause 1 or 2 above, wherein the method reduces the incidence of an infectious disease associated with the presence of S. aureus in the subject. [Clause 4] A method for preventing S. aureus infection in a subject, said method comprising administering to said subject an antibody or antigen-binding fragment thereof that binds to S. aureus AT, wherein the sample obtained from the subject has a level of S. aureus that does not exceed the level of S. aureus correlated with the PCR Ct value. [Clause 5] A method for reducing the incidence of S. aureus pneumonia in a subject, said method comprising administering sublatoxumab to said subject, wherein the reduction is determined by clinical, microbiological and radiological means, and optionally the incidence is reduced by about 30%. [Clause 6] A method for reducing the incidence of pneumonia of any cause in a subject, said method comprising administering sublatoxumab to said subject, wherein the reduction is determined by clinical, microbiological and radiological means, and optionally the incidence is reduced by about 30%. [Clause 7] The method according to any one of clauses 3 and 4 above, wherein the infectious disease is determined by clinical, microbiological and radiological means. [Clause 8] The clinical means is the method according to any one of items 5, 6, and 7, including abnormal body temperature, abnormal white blood cell count, cough, purulent sputum, bronchial breath sounds, dyspnea, tachypnea (respiratory rate > 30 breaths / min), hypoxemia, or any combination thereof. [Item 9] The microbiological means is the method according to any one of items 5, 6, 7, and 8, including a respiratory specimen, blood culture, pleural fluid aspirate, or lung tissue culture that is positive for Staphylococcus aureus (S. aureus). [Item 10] The radiograph means is the method according to any one of items 5, 6, and 7 to 9, including a new or worsening infiltrate on a chest radiograph. [Item 11] PCR is the method according to any one of items 5 to 10, which is used to detect the level of Staphylococcus aureus (S. aureus) in a sample obtained from the subject. [Item 12] The sample obtained from the subject has a level of Staphylococcus aureus (S. aureus) that does not exceed the level of Staphylococcus aureus (S. aureus) correlated with the polymerase chain reaction (PCR) cycle threshold (Ct) value, according to the method described in item 11. [Item 13] The method according to any one of items 1 to 12 further includes a step of detecting the level of Staphylococcus aureus (S. aureus) in a sample obtained from the subject. [Item 14] The sample obtained from the subject has a level of Staphylococcus aureus (S. aureus) that does not exceed the level of Staphylococcus aureus (S. aureus) correlated with 29 or more of the PCR Ct values, according to the method described in any one of items 1 to 4 and 11 to 13. [Item 15] The 29 PCR Ct values correspond to a concentration of approximately 1,600 to approximately 1,700 colony forming units (CFU) / mL of Staphylococcus aureus (S. aureus), according to the method described in item 16. [Item 16] A method for treating a subject in whom Staphylococcus aureus (S. aureus) infection has colonized, the method including administering to the subject an antibody that binds to Staphylococcus aureus (S. aureus) AT or an antigen-binding fragment thereof, and the sample obtained from the subject having a concentration of Staphylococcus aureus (S. aureus) that does not exceed 1,700 CFU / mL. [Item 17] The method according to item 18 reduces the incidence of the infectious disease associated with the presence of Staphylococcus aureus (S. aureus) in the subject. [Item 18] A method for preventing Staphylococcus aureus (S. aureus) infection in a subject, the method comprising administering to the subject an antibody or an antigen-binding fragment thereof that binds to S. aureus AT, wherein the sample obtained from the subject has a concentration of S. aureus not exceeding 1,700 CFU / mL. [Item 19] The concentration of S. aureus AT is measured by PCR, and the method according to any one of Items 18 to 20 above. [Item 20] The method according to any one of Items 4 to 17 and 20 to 21 above, wherein S. aureus forms colonies in the subject. [Item 21] The method according to any one of Items 1 to 22 above, wherein the sample obtained from the subject has a level of S. aureus that correlates with at least the Ct value of PCR. [Item 22] The method according to Item 23 above, wherein the sample obtained from the subject has a level of S. aureus that correlates with at least 3 PCR Ct values. [Item 23] The method according to any one of Items 1 to 24 above, wherein the level of S. aureus is detected within 3 hours, optionally within 2 hours. [Item 24] The method according to any one of Items 1 to 4, 11 to 15, and 19 to 23 above, wherein the PCR detects S. aureus protein A. [Item 25] The method according to any one of Items 1 to 24 above, wherein the subject is mechanically ventilated, and optionally, a ventilator is attached to the subject. [Item 26] The method according to any one of Items 1 to 25 above, wherein the subject is taking an antibiotic. [Item 27] The method according to any one of Items 1 to 4 and 11 to 26 above, wherein the sample is a skin or soft tissue sample. [Item 28] The method according to any one of Items 1 to 4 and 11 to 26 above, wherein the sample is obtained from the lower respiratory tract of the subject. [Item 29] The method according to any one of Items 1 to 4 and 11 to 26 above, wherein the sample is an endotracheal aspirate. [Item 30] The method according to any one of Items 1 to 4 and 11 to 26 above, wherein the sample is a respiratory tract sample. [Item 31] The method according to any one of Items 1 to 4 and 11 to 26 above, wherein the sample is a bronchial sample. [Item 32] The sample contains bacteria that will not grow during culturing for identifying Staphylococcus aureus (S. aureus), and the method according to any one of items 1 to 4 and 11 to 31 above. [Item 33] The sample contains bacteria that are not Staphylococcus, and the method according to any one of items 1 to 4 and 11 to 32, 13 to 34 above. [Item 34] The Staphylococcus aureus (S. aureus) is antibiotic-resistant, and the method according to any one of items 1 to 5 and 7 to 33 above. [Item 35] The method according to any one of items 1 to 5 and 7 to 34 above, further comprising the step of determining whether the Staphylococcus aureus (S. aureus) is antibiotic-resistant. [Item 36] The Staphylococcus aureus (S. aureus) is methicillin-resistant, and the method according to any one of items 1 to 5 and 7 to 35 above. [Item 37] The method according to any one of items 1 to 5 and 7 to 36 above, further comprising the step of determining whether the Staphylococcus aureus (S. aureus) is methicillin-resistant. [Item 38] The resistance is determined using PCR, and the method according to any one of items 34 to 37 above. [Item 39] The infectious disease is pneumonia, and the method according to any one of items 3, 4 and 7 to 37 above. [Item 40] The infectious disease is intensive care unit (ICU) pneumonia, and the method according to any one of items 3, 4 and 7 to 37 above. [Item 41] The subject is a human, and the method according to any one of items 1 to 40 above. [Item 42] The antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT binds to the same Staphylococcus aureus (S. aureus) AT epitope as an antibody comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 8, and the method according to any one of items 1 to 4 and 7 to 41 above. [Item 43] The antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT competitively inhibits the binding of an antibody comprising VH containing the amino acid sequence of SEQ ID NO: 7 and VL containing the amino acid sequence of SEQ ID NO: 8 to Staphylococcus aureus (S. aureus) AT, and the method according to any one of items 1 to 4 and 7 to 42 above. [Item 44] The antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT is the method according to any one of items 1 to 4 and 7 to 43 above, comprising VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of MEDI4893. [Item 45] The antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT is the method according to any one of items 1 to 4 and 7 to 44 above, comprising a variable heavy chain (VH) complementarity-determining region (CDR) 1 containing the amino acid sequence of SEQ ID NO: 1, a VH CDR2 containing the amino acid sequence of SEQ ID NO: 2, a VH CDR3 containing the amino acid sequence of SEQ ID NO: 3, a variable light chain (VL) CDR1 containing the amino acid sequence of SEQ ID NO: 4, a VL CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 containing the amino acid sequence of SEQ ID NO: 6. [Item 46] The antibody or antigen-binding fragment that binds to Staphylococcus aureus (S. aureus) AT is the method according to any one of items 1 to 4 and 7 to 45 above, comprising a VH containing the amino acid sequence of SEQ ID NO: 7. [Item 47] The antibody or antigen-binding fragment that binds to Staphylococcus aureus (S. aureus) AT is the method according to any one of items 1 to 4 and 7 to 46 above, comprising a VL containing the amino acid sequence of SEQ ID NO: 8. [Item 48] The antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT is the method according to any one of items 1 to 4 and 7 to 47 above, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 9. [Item 49] The antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT is the method according to any one of items 1 to 4 and 7 to 48 above, comprising a light chain containing the amino acid sequence of SEQ ID NO: 10. [Item 50] The antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT is the method according to any one of items 1 to 4, 7 to 47, and 49 above, further comprising a heavy chain constant region. [Item 51] The heavy chain constant region is human immunoglobulin IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 The method according to item 50 above, selected from the group consisting of heavy chain constant regions. [Item 52] The heavy chain constant region is a human IgG 1 constant region. The method according to item 51 above. [Item 53] The method according to any one of items 1 to 4, 7 to 48, and 51 to 52 above, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT further comprises a light chain constant region. [Item 54] The method according to item 53 above, wherein the light chain constant region is selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions. [Item 55] The method according to item 54 above, wherein the light chain constant region is a human IgGκ light chain constant region. [Item 56] The method according to any one of items 1 to 4 and 7 to 47 above, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT is an IgG antibody or antigen-binding fragment thereof. [Item 57] The method according to any one of items 1 to 4, 7 to 47, and 56 above, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT comprises an Fc region that has been genetically engineered to improve the half-life. [Item 58] The method according to any one of items 1 to 4, 7 to 47, and 56 above, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT comprises an Fc region with a YTE mutation. [Item 59] The method according to any one of items 1 to 4 and 7 to 58 above, wherein the antibody or antigen-binding fragment that binds to Staphylococcus aureus (S. aureus) AT is a monoclonal antibody or antigen-binding fragment. [Item 60] The method according to any one of items 1 to 4 and 7 to 59 above, wherein the antibody or antigen-binding fragment that binds to Staphylococcus aureus (S. aureus) AT is a full-length antibody. [Item 61] The method according to any one of items 1 to 4 and 7 to 59 above, wherein the antibody or antigen-binding fragment that binds to Staphylococcus aureus (S. aureus) AT is an antigen-binding fragment. [Item 62] The antibody or antigen-binding fragment that binds to Staphylococcus aureus (S. aureus) AT 2 is Fab, Fab’, F(ab’) 3 , single-chain Fv (scFv), disulfide-linked Fv, intrabody, IgGΔCH2, minibody, F(ab’) 2 , tetrabody, triabody, diabody, DVD-Ig, Fcab, mAb 2 , (scFv) [Item 63] The antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT has an affinity of 80 to 100 pM for Staphylococcus aureus (S. aureus) AT, and the method according to any one of items 1 to 4 and 7 to 62 above. [Item 64] The antibody or antigen-binding fragment thereof is sublatoxumab, and the method according to any one of items 1 to 4 and 7 to 63 above. [Item 65] The method according to any one of items 1 to 4 and 7 to 64 above, wherein 2,000 mg of the antibody or antigen-binding fragment thereof is administered. [Item 66] The method according to any one of items 1 to 4 and 7 to 64 above, wherein 5,000 mg of the antibody or antigen-binding fragment is administered. [Item 67] The step of preventing Staphylococcus aureus (S. aureus) infection includes neutralizing toxins, inhibiting cell lysis, inhibiting multiple organ failure, inhibiting Staphylococcus aureus (S. aureus)-related sepsis, or any combination of the foregoing, and the method according to any one of items 1 to 4, 7 to 15 and 18 to 66 above. [Item 68] An antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT for use in treating a subject colonized with Staphylococcus aureus (Staphylococcus aureus (S. aureus)), wherein the sample obtained from the subject has a level of Staphylococcus aureus (S. aureus) that does not exceed the level of Staphylococcus aureus (S. aureus) correlated with the polymerase chain reaction (PCR) cycle threshold (Ct) value. Antibody or antigen-binding fragment thereof. [Item 69] The step of treating reduces the incidence of the infection associated with the presence of Staphylococcus aureus (S. aureus) in the subject, and the antibody or antigen-binding fragment according to item 68 above. [Item 70] An antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT for use in preventing Staphylococcus aureus (S. aureus) infection in a subject colonized with Staphylococcus aureus (S. aureus), wherein the sample obtained from the subject has a level of Staphylococcus aureus (S. aureus) that does not exceed the level of Staphylococcus aureus (S. aureus) correlated with the PCR Ct value. Antibody or antigen-binding fragment thereof. [Item 71] The sample obtained from the subject has a level of Staphylococcus aureus (S. aureus) that does not exceed the level of S. aureus correlated with the 29 PCR Ct values, and is the antibody or antigen-binding fragment thereof according to any one of items 68 to 70 above. [Item 72] The antibody or antigen-binding fragment thereof is not administered to the subject in an example where the level of S. aureus in the sample obtained from the subject is detected to exceed the level of S. aureus correlated with the PCR Ct value, and is the antibody or antigen-binding fragment thereof according to any one of items 68 to 70 above. [Item 73] An in vitro method for identifying a subject in which S. aureus that is responsive to an antibody or antigen-binding fragment thereof that binds to S. aureus AT forms colonies, the method including a step of detecting the level of S. aureus in a sample obtained from the subject, and the level of S. aureus that does not exceed the level of S. aureus correlated with the PCR Ct value indicates that the subject is responsive to the antibody or antigen-binding fragment thereof. [Item 74] The PCR Ct value is 29, and is the method according to item 73 above. All references, including publications, patent applications, and patents cited in this specification, are hereby incorporated by reference in their entirety to the same extent as if each reference had been individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0176] The recitation of numerical ranges in this specification is merely intended to provide a convenient summary of the individual numerical values within the range and, unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described in this specification can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any examples, or exemplary language (e.g., "such as") provided herein is for illustrative purposes only and does not limit the scope of the invention unless otherwise claimed. Words in this specification should not be construed as indicating any non-claimed element essential to the practice of the invention.

[0177] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to appropriately employ such variations, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Further, any combination of the above elements in all possible variations thereof is included in the present invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

**Claim 1** A pharmaceutical composition for treating or preventing Staphylococcus aureus (S. aureus) infection in a subject in whom S. aureus has formed colonies, the pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof that binds to S. aureus alpha toxin (AT), wherein in a sample obtained from the subject, the level of S. aureus has been detected by polymerase chain reaction (PCR), and the sample has a level of S. aureus that does not exceed the level of S. aureus correlated with 29 PCR cycle thresholds (Ct values); The antibody or antigen-binding fragment thereof that binds to S. aureus AT comprises a variable heavy chain (VH) complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a variable light chain (VL) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; The 29 PCR Ct values correspond to the S. aureus concentration in the sample of 1,600 to 1,700 colony-forming units (CFU) / mL of S. aureus, The sample obtained from the subject is an endotracheal aspirate, Pharmaceutical composition. **Claim 2** The pharmaceutical composition according to claim 1, wherein the PCR detects S. aureus protein A. **Claim 3** The pharmaceutical composition according to claim 1 or 2, wherein the S. aureus is antibiotic-resistant as determined using PCR. **Claim 4** The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof that binds to S. aureus AT comprises a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ ID NO:

8. **Claim 5** The antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus AT is human IgG １ The pharmaceutical composition according to any one of claims 1 to 4, comprising a heavy chain constant region. **Claim 6** The pharmaceutical composition according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof that binds to S. aureus AT comprises an Fc region with a YTE mutation. **Claim 7** The pharmaceutical composition according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment that binds to Staphylococcus aureus (S. aureus) AT is a monoclonal antibody or antigen-binding fragment.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT has an affinity of 80 to 100 pM for Staphylococcus aureus (S. aureus) AT.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof is Subratoxumab.

10. An in vitro method for identifying a subject in which Staphylococcus aureus (S. aureus) has formed colonies that is responsive to an antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) α-toxin (AT), the method comprising detecting the level of Staphylococcus aureus (S. aureus) in a sample obtained from the subject, wherein not exceeding the level of Staphylococcus aureus (S. aureus) that correlates with a PCR Ct value of 29 is the criterion for identifying that the subject is responsive to the antibody or antigen-binding fragment thereof; The antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT comprises a variable heavy chain (VH) complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a variable light chain (VL) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 6; The PCR Ct value of 29 corresponds to the Staphylococcus aureus (S. aureus) concentration in the sample of 1,600 to 1,700 colony-forming units (CFU) / mL of Staphylococcus aureus (S. aureus), The sample obtained from the subject is an endotracheal aspirate. Method.

11. The in vitro method according to claim 10, wherein the antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus (S. aureus) AT comprises a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ ID NO:

8.

12. The antibody or antigen-binding fragment thereof that binds to Staphylococcus aureus AT is human IgG １ The in vitro method according to claim 10 or 11, comprising a heavy chain constant region and an Fc region having a YTE mutation.

13. The in vitro method according to any one of claims 10 to 12, wherein the antibody or antigen-binding fragment thereof is sublatoxumab.

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