Antibodies against Staphylococcus aureus leukotoxin

Abstract

The present disclosure relates to leukotoxin binding antibodies and antigen-binding fragments thereof. The antibodies and fragments can be used, for example, to detect leukotoxins and / or in methods for treating and preventing Staphylococcus aureus infections.

Description

Background Art

[0001] Infections caused by antimicrobial-resistant (AMR) bacterial pathogens pose an increasing threat to public health. Empirical broad-spectrum antibiotic therapy has in part stimulated the continued prevalence of AMR. This has led to the exploration of specific pathogen approaches including monoclonal antibodies (mAbs) to prevent or treat serious bacterial infections. Some monoclonal antibodies are currently being developed for the prevention or treatment of antibiotic-resistant bacterial infections (see, for example, A. DiGiandomenico, A. and BR Sellman, Curr. Opin. Microbiol., 27: 78-85 (2015)). Such passive immunization strategies provide immediate and effective immunoglobulin responses against target pathogens.

[0002] Staphylococcus aureus is a bacterial pathogen that causes a variety of diseases, including skin and soft tissue infections, endocarditis, osteomyelitis, pneumonia, and bacteremia (Lowy, FD, N. Engl. J. Med., 339(8):520-32 (1998)). Preclinical studies indicate that monoclonal antibody-based approaches hold promise for prophylaxis and adjunctive therapy against S. aureus infections (see, e.g., Hazenbos et al., PLoS Pathog., 9(10):e1003653.doi:10.1371 / journal.ppat.10036532013 (2013); Rouha, H., MAbs, 7(1):243-254 (2015); Foletti et al., J. Mol. Biol., 425(10):1641-1654 (2013); Karauzum et al., J Biol. Chem., 287(30):25203-15 (2012); and Hua et al., Antimicrob Agents Chemother., 58(2):1108-17 (2014). However, there is still a need for antibodies useful for treating S. aureus infections, especially infections that are resistant to currently commercially available antibiotics. The present disclosure provides such antibodies. Summary of the invention

[0003] The present invention provides antibodies and antigen-binding fragments thereof that bind to Staphylococcus aureus (S. aureus) leukotoxin.

[0004] In some cases, the antibody or antigen-binding fragment thereof that specifically binds to at least one Staphylococcus aureus leukotoxin comprises a variable heavy chain (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3, variable light chain (VL) CDR1, VL CDR2 and VL CDR3, wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 comprise a sequence selected from the group consisting of: (a) SEQ ID NOs: 1, 2, 3, 12, 5 and 6, respectively; (b) SEQ ID NOs: 1-6, respectively; (c) SEQ ID NOs: 1, 2, 17, 4, 5 and 6, respectively; (d) SEQ ID NOs: 1, 2, 17, 12, 5 and 6, respectively, and (e) SEQ ID NOs: 1, 20, 3, 4, 5 and 6, respectively.

[0005] In some cases, the antibody or antigen-binding fragment thereof that specifically binds to at least one Staphylococcus aureus leukotoxin comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VLCDR3 of SAN481-SYT-YTE. In some cases, the CDRs are Kabat-defined CDRs, Chothia-defined CDRs or AbM-defined CDRs.

[0006] In some cases, the antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises an amino acid sequence of SEQ ID NO: 7, 15, 18, 21 or 23. In some cases, the antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VL comprises an amino acid sequence of SEQ ID NO: 8 or 13. In some cases, the antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH and VL comprise a sequence selected from the group consisting of: (a) SEQ ID NO: 15 and 13, respectively; (b) SEQ ID NO: 7 and 8, respectively; (c) SEQ ID NO: 7 and 13, respectively; (d) SEQ ID NO: 15 and 8, respectively; (e) SEQ ID NO: 18 and 8, respectively; (f) SEQ ID NO: 18 and 13, respectively; (g) SEQ ID NO: 21 and 8, respectively; (h) SEQ ID NO: 23 and 13, respectively. In some cases, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence of SEQ ID NO: 15 and a VL comprising the sequence of SEQ ID NO: 13. In some cases, the antibody or antigen-binding fragment comprises a heavy chain comprising the sequence of SEQ ID NO: 16, 9, 11, 22, or 24. In some cases, the antibody or antigen-binding fragment comprises a light chain comprising the sequence of SEQ ID NO: 14 or 10. In some cases, the antibody comprises a heavy chain and a light chain, wherein the heavy chain and the light chain comprise a sequence selected from the group consisting of: (a) SEQ ID NO: 16 and 14, respectively; (b) SEQ ID NO: 9 and 10, respectively; (c) SEQ ID NO: 11 and 10, respectively; (d) SEQ ID NO: 11 and 14, respectively; (e) SEQ ID NO: 16 and 10, respectively; (f) SEQ ID NO: 19 and 10, respectively; (g) SEQ ID NO: 19 and 14, respectively; (h) SEQ ID NO: 22 and 10, respectively; and (i) SEQ ID NO: 24 and 14, respectively. In some cases, the antibody comprises a heavy chain comprising the sequence of SEQ ID NO: 16 and a light chain comprising the sequence of SEQ ID NO: 14.

[0007] In some cases, the antibodies or antigen-binding fragments thereof provided herein bind to the same S. aureus leukotoxin antigenic determinant as an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 13.

[0008] In certain instances, the antibodies or antigen-binding fragments thereof provided herein competitively inhibit binding of an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 13 to Staphylococcus aureus leukotoxin.

[0009] In some cases, the antibody or antigen binding fragment binds to LukF, LukD or HlgB and / or the antibody or antigen binding fragment neutralizes LukF, LukD or HlgB. In some cases, the antibody or antigen binding fragment (a) binds to LukF, LukD and HlgB and / or (b) neutralizes LukF, LukD and HlgB.

[0010] In some cases, the antibody or antigen-binding fragment further comprises a heavy chain constant region. In some cases, the heavy chain constant region is selected from the group consisting of: human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 heavy chain constant regions. In some cases, the heavy chain constant region is a human IgG1 constant region.

[0011] In some cases, the antibody or antigen-binding fragment further comprises a light chain constant region. In some cases, the light chain constant region is selected from the group consisting of: human immunoglobulin IgGκ and IgGλ light chain constant regions. In some cases, the light chain constant region is a human IgGκ light chain constant region.

[0012] In certain instances, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

[0013] In some cases, the antibody or antigen-binding fragment comprises an Fc region that has been engineered to improve half-life. In some cases, the antibody or antigen-binding fragment thereof comprises an Fc region having a YTE mutation.

[0014] In certain instances, the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment.

[0015] In some cases, the antibody or antigen-binding fragment is a full-length antibody. In some cases, the antibody or antigen-binding fragment is an antigen-binding fragment. In some cases, the antigen-binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, DVD-Ig, Fcab, mAb 2 , (scFv)2 or scFv-Fc.

[0016] In some cases, the antibody or antigen-binding fragment thereof has an affinity for S. aureus LukF, LukD and HlgB of less than 75 pM. In some cases, the antibody or antigen-binding fragment thereof has similar binding affinities for LukF, LukD and HlgB.

[0017] In some cases, the antibody or antigen-binding fragment thereof further comprises a detectable label.

[0018] Also provided herein are compositions comprising an antibody or antigen-binding fragment thereof provided herein and an optional pharmaceutically acceptable carrier.

[0019] Also provided herein are methods for using antibodies provided herein. In some cases, the method for treating or preventing Staphylococcus aureus (S.aureus) infection in an individual comprises administering to an individual an antibody or antigen binding fragment provided herein or a composition provided herein. In some cases, Staphylococcus aureus infection is sepsis. In some cases, Staphylococcus aureus infection is bacteremia. In some cases, Staphylococcus aureus infection is pneumonia. In some cases, Staphylococcus aureus infection is ICU pneumonia. In some cases, Staphylococcus aureus infection is skin or soft tissue infection (SSTI). In some cases, Staphylococcus aureus infection is diabetic lower limb infection. In some cases, Staphylococcus aureus infection is diabetic foot ulcer (DFU). In some cases, DFU is not infected. In some cases, DFU is infected. In some cases, DFU is grade 1, grade 2 or grade 3 DFU. In some cases, Staphylococcus aureus infection is bone or joint infection. In some cases, Staphylococcus aureus infection is joint infection, device infection, wound infection, surgical site infection or osteomyelitis.

[0020] In some cases, the subject is a surgical subject.

[0021] In some cases, the Staphylococcus aureus infection involves antibiotic-resistant Staphylococcus aureus.

[0022] In some cases, the individual has diabetes.

[0023] In some cases, the individual is a person.

[0024] In certain instances, treating or preventing S. aureus infection comprises toxin neutralization, inhibition of cell lysis, inhibition of multi-organ dysfunction, inhibition of S. aureus-associated sepsis, or any combination of the foregoing.

[0025] Polynucleotides are also provided herein. In some cases, the isolated polynucleotides comprise nucleic acid molecules encoding the VH or heavy chains of antibodies or their antigen-binding fragments provided herein. In some cases, the isolated polynucleotides comprise nucleic acid molecules encoding the VL or light chains of antibodies or their antigen-binding fragments provided herein.

[0026] Vectors are also provided herein.In some cases, polynucleotides are provided herein.

[0027] Host cells are also provided herein. In some cases, host cells comprise polynucleotides provided herein, carriers provided herein, or first carriers containing polynucleotides provided herein and second carriers containing polynucleotides provided herein. In some cases, host cells are selected from the group consisting of: CHO, NS0, PER-C6, HEK-293 and HeLa cells. In some cases, host cells are separated.

[0028] Also provided herein are methods of producing antibodies or antigen-binding fragments. In some cases, the method of producing an antibody or antigen-binding fragment thereof comprises culturing a host cell provided herein such that the antibody or antigen-binding fragment thereof is produced.

[0029] Also provided herein are methods for detecting S. aureus or S. aureus leukotoxins. In some cases, the method of detecting S. aureus or S. aureus leukotoxins in a sample comprises contacting the sample with an antibody or antigen-binding fragment thereof provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A graph is provided showing the in vitro activity of multiple SAN481 variants compared to SAN481. QD1 = SAN481-YTE; QD2 = SAN481VL26S32Y-YTE; QD3 = SAN481VH28T-YTE; QD4 = SAN481-VH28T100F-YTE; QD5 = SAN481-SY-T-YTE; QD6 = SAN481-SY-TF-YTE; QD11 = SAN481-EG-YTE; and QD12 = SAN481-SY-QFS-YTE. (See Example 2)

[0031] Figure 2 Provides a diagram demonstrating that SAN481-SYT-YTE has in vitro leukotoxin neutralization activity similar to SAN481. (See Example 3)

[0032] Figure 3 A sequence alignment of HIgB (SEQ ID NO: 27), LukF (SEQ ID NO: 25), and LukD (SEQ ID NO: 26) is provided. DETAILED DESCRIPTION

[0033] The present disclosure provides antibodies and antigen-binding fragments thereof (e.g., monoclonal antibodies and antigen-binding fragments thereof) that bind to Staphylococcus aureus leukotoxins. The present disclosure also provides methods of using such antibodies and antigen-binding fragments, such as in detecting Staphylococcus aureus leukotoxins and in treating or preventing Staphylococcus aureus infections.

[0034] I. Definitions

[0035] As used herein, the term "leukotoxin" refers to a bacterial leukotoxin polypeptide, including, but not limited to, a natural leukotoxin polypeptide and an isoform of a leukotoxin polypeptide. "Leukotoxin" encompasses unprocessed full-length leukotoxin polypeptides and leukotoxin polypeptide forms produced by intracellular processing. Leukotoxins include LukSF, leukotoxin ED (LukED), HlgAB, HlgCB) and leukotoxin AB (LukAB, also known as LukGH). As used herein, the term "Staphylococcus aureus LukF" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO: 25. As used herein, the term "Staphylococcus aureus LukD" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO: 26. As used herein, the term "Staphylococcus aureus HIgB" refers to a polypeptide comprising the amino acid sequence of SEQ ID NO: 27. (See Figure 3 ) "Leukotoxin polynucleotide", "leukotoxin nucleotide" or "leukotoxin nucleic acid" refers to a polynucleotide encoding a leukotoxin.

[0036] 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 of each of the above, through at least one antigen recognition site in the variable region of the immunoglobulin molecule. As used herein, as long as the antibody exhibits the desired biological activity, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising antibodies, and any other modified immunoglobulin molecules. Antibodies can have any of the five main categories of immunoglobulins based on the consistency of their heavy chain constant domains (referred to as α, δ, ε, γ, and μ, respectively): IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked antibodies or conjugated to other molecules such as toxins and radioisotopes.

[0037] The term "monoclonal antibody" as used herein refers to an antibody produced by a single clone of a B cell and binds to the same antigenic determinant. In contrast, the term "polyclonal antibody" refers to an antibody population produced by different B cells and binds to different antigenic determinants of the same antigen.

[0038] The term "antibody fragment" refers to a portion of an intact antibody. "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 may contain the antigenic determining region (e.g., complementary determining region (CDR)) of an intact antibody. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies may be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or may be artificially produced.

[0039] A complete antibody is usually composed of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains an N-terminal variable (VH) region and three C-terminal constant (CHI, CH2 and CH3) regions, and each light chain contains an N-terminal variable (VL) region and a 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 region and the VL region have the same general structure, wherein each region comprises four framework regions, and their sequences are relatively conservative. As used herein, the term "framework region" refers to a relatively conservative amino acid sequence within a variable region between a hypervariable region or a complementary determining region (CDR). There are four framework regions in each variable domain, referred to as FR1, FR2, FR3 and FR4. The framework regions form a beta-sheet structure that provides the structural framework for the variable regions (see, e.g., CA Janeway et al. (Eds.), Immunobiology, 5th ed., Garland Publishing, New York, NY (2001)). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of the antibody, which is responsible for antigen binding.

[0040] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0041] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0042] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding fragment thereof. In certain aspects, the CDRs may be determined according to the Kabat numbering system (see, e.g., Kabat EA and Wu TT (1971) Ann NY Acad Sci 190:382-391 and EA Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication 91-3242). Using the Kabat numbering system, the CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which may optionally include one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1); amino acid positions 50 to 65 (CDR2); and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRs of an antibody described herein are determined according to the Kabat numbering scheme.

[0043] Chothia refers to the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbering using the Kabat numbering convention, the Chothia CDR-H1 loop terminus varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if neither 35A nor 35B is present, the loop terminus is at 32; if only 35A is present, the loop terminus is at 33; if both 35A and 35B are present, the loop terminus is at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used in the AbM antibody modeling software from Oxford Molecular.

[0044]

[0045] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meaning in the art. The constant region is the portion of an antibody, such as the carboxyl terminal portion of the light and / or heavy chain, that is not directly involved in binding of the antibody to an antigen but can exhibit various effector functions (such as interaction with Fc receptors). The amino acid sequence of the constant region of an immunoglobulin molecule is generally more conserved than the immunoglobulin variable domain.

[0046] As used herein, the term "heavy chain" when used in relation to an antibody, based on the amino acid sequence of the constant domain, can refer to any of the different types, such as alpha (alpha, α), delta (delta, δ), epsilon (epsilon, ε), gamma (gamma, γ) and mu (mu, μ), which respectively produce the IgA, IgD, IgE, IgG and IgM classes of antibodies, including subclasses of IgG, such as IgG1, IgG2, IgG3 and IgG4. The amino acid sequences of heavy chains are well known in the art. In a specific embodiment, the heavy chain is a human heavy chain.

[0047] As used herein, the term "light chain" when used in relation to an antibody can refer to any of the different types, such as kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0048] A "chimeric" antibody refers to an antibody or fragment thereof that comprises both human and non-human domains. A "humanized" antibody is an antibody that comprises a human antibody backbone and at least one CDR obtained or derived from a non-human antibody. Non-human antibodies include antibodies isolated from any non-human animal, such as a rodent (e.g., mouse or rat). A humanized antibody may comprise one, two, or three CDRs obtained or derived from a non-human antibody. A fully human antibody does not contain any amino acid residues obtained or derived from a non-human animal. It should be understood that fully human and humanized antibodies have a lower risk of inducing an immune response in humans than mouse or chimeric antibodies (see, e.g., Harding et al., Monoclonal Antibodies, 2(3):256-26 (2010)).

[0049] As used herein, "antigenic determinant" is a term in the art and refers to a local region of an antigen to which an antibody or its antigen-binding fragment can specifically bind. An antigenic determinant may be, for example, an adjacent amino acid of a polypeptide (linear or adjacent antigenic determinant), or an antigenic determinant may be, for example, obtained by bringing together two or more non-adjacent regions of one or more polypeptides (conformation, nonlinearity, non-continuous or non-adjacent antigenic determinants). In certain embodiments, the antigenic determinant to which an antibody or its antigen-binding fragment binds may be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA analysis, hydrogen / deuterium exchange and mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry) coupling, array-based oligopeptide scanning analysis, and / or mutation-induced positioning (e.g., site-directed mutation-induced positioning). For X-ray crystallography, crystals 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(Part 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). Antibodies / antigen-binding fragments thereof: Antigen crystals can be studied using well-known X-ray diffraction techniques and computer software such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) Vols. 114 and 115, edited by HW Wyckoff et al.; US2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallographica Sin. D: Biological Crystallography 49(Part 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, edited by CW Carter CW; Roversi et al.; et al.; US2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallographica Sin. D: Biological Crystallography 49(Part 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, edited by CW Carter CW; Roversi et al.; P) et al., (2000) Acta Crystallographica Sinica D: Biological Crystallography 56(Part 10): 1316-1323). Mutagenesis localization studies can be performed using any method known to those skilled in the art.See, for example, Champe M et al. (1995) J. Biol. Chem. 270: 1388-1394 and Cunningham BC and Wells JA (1989) Science 244: 1081-1085 for a description of mutagenesis techniques, including alanine scanning mutagenesis.

[0050] An antibody that "binds to the same antigenic determinant" as a reference antibody is an antibody that binds to the same amino acid residue as the reference antibody. The ability of an antibody to bind to the same antigenic determinant as the reference antibody can be determined by hydrogen / deuterium exchange analysis (see Coales et al. Rapid Commun. Mass Spectrom. 2009; 23: 639-647) or x-ray crystallography.

[0051] As used herein, the terms "immunospecific binding", "immunospecific recognition", "specific binding" and "specific recognition" are similar terms in the context of an antibody or antigen-binding fragment thereof. These terms indicate that the antibody or antigen-binding fragment thereof binds to an antigenic determinant through its antigen-binding domain, and that the binding requires some complementarity between the antigen-binding domain and the antigenic determinant. Thus, for example, an antibody that "specifically binds" to a first Staphylococcus aureus leukotoxin may also bind to other Staphylococcus aureus leukotoxins, as measured by, for example, a radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), a BiaCore or an octet binding assay, but the extent of binding to unrelated non-leukotoxin proteins is less than about 10% of the binding of the antibody to the first Staphylococcus aureus leukotoxin.

[0052] An antibody is said to "competitively inhibit" binding of a reference antibody to a given antigenic determinant if the antibody preferentially binds to the antigenic determinant or overlapping antigenic determinant to an extent that causes the antibody to block to some extent the binding of the reference antibody to the antigenic determinant. Competitive inhibition can be determined by any method known in the art, such as a competition ELISA assay. An antibody may be said to competitively inhibit binding of a reference antibody to a given antigenic determinant by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0053] The term "nucleic acid sequence" is intended to encompass polymers of DNA or RNA, i.e., polynucleotides, which may be single-stranded or double-stranded and may contain non-natural or altered nucleotides. As used herein, the terms "nucleic acid" and "polynucleotide" refer to polymeric forms of nucleotides, ribonucleotides (RNA) or deoxyribonucleotides (DNA) of any length. These terms refer to the primary structure of the molecule, and therefore include double-stranded and single-stranded DNA and double-stranded and single-stranded RNA. The term includes RNA or DNA analogs made from nucleotide analogs and modified polynucleotides (such as, but not limited to, methylated and / or capped polynucleotides) as equivalents. Although many other bonds (e.g., phosphorothioates, boranophosphates, and analogs thereof) are known in the art, nucleic acids are typically linked by phosphate bonds to form nucleic acid sequences or polynucleotides.

[0054] S. aureus infections can occur, for example, as skin or soft tissue infections (SSTIs) or bacteremia. S. aureus bacteria can travel through the bloodstream and infect sites within the body, causing pneumonia, ICU pneumonia, diabetic leg infections, diabetic foot ulcers (DFUs), bone or joint infections, device infections, wound infections, surgical site infections, or osteomyelitis.

[0055] 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 coprecipitation (see, e.g., EJ Murray (EJ) (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-polydextrose; electroporation; cationic liposome-mediated transfection; tungsten particle-promoted microprojectile bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors can be introduced into host cells following growth of the infectious particles in appropriate packaging cells, many of which are commercially available.

[0056] As used herein, the term "treatment" or "treating" and its analogous terms refer to obtaining a desired pharmacological and / or physiological effect. In one embodiment, the effect is therapeutic, i.e., the effect partially or completely cures the disease and / or the adverse symptoms attributable to the disease.

[0057] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result (e.g., treatment of Staphylococcus aureus infection) at the dosage and time period required. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antigen-binding fragment to elicit a desired response in the individual.

[0058] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (eg, preventing S. aureus infection or disease onset).

[0059] As used herein, the term "administer / administering / admini-stration" and its analogous terms refer to methods that can be used to enable a drug (e.g., leukotoxin-binding antibody or antigen-binding fragment thereof) to be delivered to a desired biological site of action (e.g., intravenous administration). Administration techniques that can be used with the agents and methods described herein are found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0060] As used in the disclosure and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0061] Unless the context specifically dictates or is obvious, as used herein, the term "or" is to be understood as inclusive. The term "and / or" as used in the phrase "A and / or B" herein is intended to include both "A and B", "A or B", "A" and "B". Similarly, the term "and / or" as used in the phrase "A, B and / or C" is intended to encompass 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).

[0062] II. Anti-leukotoxin antibodies

[0063] The present invention provides antibodies and antigen-binding fragments thereof that bind to at least one S. aureus leukotoxin.

[0064] Leukotoxin is a toxic factor of Staphylococcus aureus. Leukotoxin targets a wide range of immune cells for destruction. Leukotoxins include Panton-Valentine leukocidin (LukSF-PV, also referred to as LukSF), leukotoxin ED (LukED), gamma hemolysin (which exists in two toxin forms: HlgAB and HlgCB) and leukotoxin AB (LukAB, also referred to as LukGH). In some cases, an antibody or its antigen binding fragment that is bound to at least one leukotoxin is bound to LukF, LukD and / or HIgB. In some cases, an antibody or its antigen binding fragment that is bound to at least one leukotoxin is bound to LukF, LukD and HIgB.

[0065] In one instance, an antibody or antigen-binding fragment (e.g., a monoclonal antibody or fragment) that specifically binds to at least one Staphylococcus aureus leukotoxin comprises, consists essentially of, or consists of (i) a heavy chain polypeptide comprising a CDR1 amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQ ID NO: 2, and a CDR3 amino acid sequence of SEQ ID NO: 3, and (ii) a light chain polypeptide comprising a CDR1 amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 5, and a CDR3 amino acid sequence of SEQ ID NO: 6. In another instance, a heavy chain polypeptide of an antibody or antigen-binding fragment (e.g., a monoclonal antibody or fragment) that specifically binds to at least one Staphylococcus aureus leukotoxin comprises, consists essentially of, or consists of a variable region amino acid sequence of SEQ ID NO: 15. In another instance, the light chain polypeptide of an antibody or antigen-binding fragment (e.g., a monoclonal antibody or fragment) that specifically binds to at least one Staphylococcus aureus leukotoxin comprises, consists essentially of, or consists of the variable region amino acid sequence of SEQ ID NO: 13. In another instance, an antibody or antigen-binding fragment (e.g., a monoclonal antibody or fragment) that specifically binds to at least one Staphylococcus aureus leukotoxin comprises, consists essentially of, or consists of a variable heavy chain comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 15, and a variable light chain comprising, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 13. In another instance, an antibody or antigen-binding fragment (e.g., a monoclonal antibody or fragment) that specifically binds to at least one Staphylococcus aureus leukotoxin comprises, consists essentially of, or consists of a variable heavy chain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 16, and / or a variable light chain comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 14.

[0066] Sequences of exemplary anti-leukotoxin antibodies are provided below.In certain instances, an antibody or antigen-binding fragment thereof described herein binds to at least one leukotoxin and comprises the six CDRs of Tables 1 and 2 below (i.e., VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3).

[0067] The SAN481-SYT-YTE antibody comprises the VH CDRs of SEQ ID NOs: 1-3 and the VLCDRs of SEQ ID NOs: 12, 5 and 6.

[0068] Table 1: VH CDR amino acid sequences

[0069]

[0070] Table 2: VL CDR amino acid sequences

[0071]

[0072] In certain instances, an antibody or antigen-binding fragment thereof described herein binds to at least one leukotoxin and comprises an antibody VH listed in the table below, for example, in combination with a VL.

[0073] Table 3: Variable heavy chain (VH) amino acid sequence

[0074]

[0075] In certain instances, an antibody or antigen-binding fragment thereof described herein binds to at least one leukotoxin and comprises an antibody VL listed in the table below, for example, in combination with a VH, optionally in combination with a VH listed in the preceding table.

[0076] Table 4: Variable light chain (VL) amino acid sequence

[0077]

[0078] In certain instances, an antibody or antigen-binding fragment thereof described herein binds to at least one leukotoxin and comprises an antibody heavy chain, for example, in combination with a light chain as listed in the table below.

[0079] Table 5: Full length heavy chain amino acid sequence

[0080]

[0081]

[0082]

[0083] In certain instances, the antibodies or antigen-binding fragments thereof described herein bind to at least one leukotoxin and comprise an antibody light chain listed in the table below, for example, in combination with a heavy chain, optionally in combination with a heavy chain listed in the preceding table.

[0084] Table 6: Full length light chain amino acid sequence

[0085]

[0086] The sequences of the antibodies used in the following examples are summarized in Table 7.

[0087] Table 7: SEQ ID NOs of antibody CDRs, variable regions, heavy chains and light chains

[0088] Antibody H CDRs L CDRs VH V L H L SAN481 1-3 4-6 7 8 9 10 SAN481-YTE 1-3 4-6 7 8 11 10 SAN481-SY-YTE 1-3 12,5,6 7 13 11 14 SAN481-T-YTE 1-3 4-6 15 8 16 10 SAN481-TF-YTE 1,2,17 4-6 18 8 19 10 SAN481-SYT-YTE 1-3 12,5,6 15 13 16 14 SAN481-SY-TF-YTE 1,2,17 12,5,6 18 13 19 14 SAN481-EG-YTE 1,20,3 4-6 21 8 22 10 SAN481-SY-QFS-YTE 1-3 12,5,6 23 13 24 14 SAN481-SYT* 1-3 12,5,6 15 13 28 14

[0089] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be identified according to the Chothia numbering scheme, which refers to the positions of immunoglobulin structural loops (see, e.g., C Chothia and AM Leske, (1987), J. Mol. Biol. 196:901-917; Al-Lazikani B et al., (1997) J. Mol. Biol. 273:927-948; C Chothia et al., (1992) J. Mol. Biol. 227:799-817; Tramontano A et al., (1990) J. Mol. Biol. 215(1):175-82; and U.S. Pat. No. 7,709,226). Generally, when using the Kabat numbering convention, the Chothia CDR-H1 loop occurs at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop occurs at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop occurs at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop occurs at light chain amino acids 24 to 34, the Chothia CDR-L2 loop occurs at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop occurs at light chain amino acids 89 to 97. When numbered using the Kabat numbering convention, the Chothia CDR-H1 loop end varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if neither 35A nor 35B is present, the loop end is at 32; if only 35A is present, the loop end is at 33; if both 35A and 35B are present, the loop end is at 34).

[0090] In certain aspects, provided herein are antibodies and antigen-binding fragments thereof comprising the Chothia VH and VL CDRs of the SAN481 or SAN481-SYT antibodies. In certain embodiments, the antibodies or antigen-binding fragments thereof comprise one or more CDRs wherein the Chothia and Kabat CDRs have the same amino acid sequence. In certain embodiments, provided herein are antibodies and antigen-binding fragments thereof comprising a combination of Kabat CDRs and Chothia CDRs.

[0091] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof may be determined according to the IMGT numbering system as described in: MP Lefranc MP, (1999) The Immunologist 7:132-136 and MP Lefranc et al., (1999) Nucleic Acids Res 27:209-212. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97. In a specific embodiment, provided herein are antibodies and antigen-binding fragments thereof comprising the IMGT VH and VLCDR of the SAN481 or SAN481-SYT-YTE antibody, eg, as described in MP Lefranc (1999) supra and MP Lefranc et al., (1999) supra.

[0092] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof may be determined according to RM MacCallum RM et al., (1996) Journal of Molecular Biology 262:732-745. See also, for example, A. Martin, "Protein Sequence and Structure Analysis of Antibody Variable Domains", in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In a specific embodiment, an antibody or antigen-binding fragment thereof is provided herein, comprising the VH and VL CDRs of SAN481 or SAN481-SYT-YTE antibodies determined by the method in RM MacCallum et al.

[0093] In certain aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable regions that represent a compromise between Kabat CDRs and Chothia structural loops and is used by the Oxford Molecular AbM antibody modeling software (Oxford Molecular Group, Inc.). In a specific embodiment, provided herein is an antibody or antigen-binding fragment comprising the VH and VL CDRs of the SAN481 or SAN481-SYT-YTE antibody as determined by the AbM numbering scheme.

[0094] In another aspect, the antibodies or antigen-binding fragments thereof (e.g., monoclonal antibodies or fragments) described herein may comprise a constant region (Fc) of any suitable class (e.g., IgG, IgA, IgD, IgM, and IgE) that is modified so as 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 (e.g., monoclonal antibodies or fragments) described herein may comprise an Fc that comprises a mutation that extends half-life relative to the same antibody without the mutation.

[0095] Fc region engineering is widely used in the art to extend the half-life of therapeutic antibodies and protect them from in vivo decomposition. In some embodiments, the Fc region of an IgG antibody or antigen-binding fragment may be modified to increase the affinity of IgG molecules to Fc neoreceptors (FcRn), which mediate IgG metabolism and protect IgG molecules from decomposition. Suitable Fc region amino acid substitutions or modifications are known in the art and include, for example, triple substitutions M252Y / S254T / T256E (referred to as "YTE") (see, for example, U.S. Patent No. 7,658,921; U.S. Patent Application Publication No. 2014 / 0302058; and Yu et al., Antimicrobial Agents and Chemotherapy, 61 (1): e01020-16 (2017)). In certain aspects, an antibody or antigen-binding fragment (e.g., a monoclonal antibody or fragment) that is bound to at least one Staphylococcus aureus leukotoxin comprises an Fc region containing a YTE mutation.

[0096] The antibodies or antigen-binding fragments described herein (eg, monoclonal antibodies or fragments) may be or may be obtained from human antibodies, humanized antibodies, non-human antibodies, or chimeric antibodies. In one aspect, the antibodies or antigen-binding fragments thereof described herein are fully human antibodies.

[0097] Human, non-human, chimeric 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 producing antibodies are known in the art and described, for example, in Kohler et al. 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 Press, New York, NY (2001). In certain embodiments, human antibodies or chimeric antibodies can be produced using transgenic animals (e.g., mice) in which one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes. Examples of transgenic mice that effectively replace endogenous antibody genes with human antibody genes include, but are not limited to, the Medarex HUMAB-MOUSE TM 、Kirin TC MOUSE TM Kyowa Kirin KM-MOUSE TM (See, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005) and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). Humanized antibodies can be produced 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, NJ (2009)), including, e.g., grafting non-human CDRs onto human antibody frameworks (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, humanized antibodies can be produced using methods such as those described in U.S. Patent Application Publication No. 2011 / 0287485A1.

[0098] In certain aspects, the antibodies or antigen-binding fragments provided herein have similar binding affinities for LukF, LukD, and HIgB.

[0099] III. Nucleic Acids, Vectors and Host Cells

[0100] Also provided herein is one or more isolated nucleic acid sequences encoding an antibody or antigen-binding fragment thereof that binds to at least one leukotoxin (wherein optionally the antibody or antigen-binding fragment thereof is a monoclonal antibody or fragment).

[0101] The present disclosure further provides one or more vectors comprising one or more nucleic acid sequences encoding antibodies or antigen-binding fragments thereof that bind to at least one leukotoxin (wherein optionally one or more of the antibodies or antigen-binding fragments thereof are monoclonal antibodies or fragments). The vector may be, for example, a plasmid, an episome, a cosmid, a viral vector (e.g., a retrovirus or adenovirus), or a phage. Suitable vectors and vector preparation methods are well known in the art (see, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994)).

[0102] In addition to the nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that binds to at least one leukotoxin (wherein optionally the antibody or antigen-binding fragment thereof is a monoclonal antibody or fragment), the vector preferably comprises expression control sequences, such as a promoter, enhancer, polyadenylation signal, transcription terminator, internal ribosome entry site (IRES), and the like, which provide for expression of the coding sequence in a host cell. Exemplary expression control sequences are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0103] A vector comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof that binds to at least one leukotoxin (optionally wherein one or more of the antibodies or antigen-binding fragments thereof are monoclonal antibodies or fragments) can be introduced into a host cell, including any suitable prokaryotic or eukaryotic cell, capable of expressing the polypeptide encoded thereby. Therefore, the present disclosure provides a separated cell comprising a vector. Useful host cells include host cells that can be easily and reliably grown, have a reasonably fast growth rate, have a well-characterized expression system, and can be easily and efficiently transformed or transfected. Examples of suitable prokaryotic cells include, but are not limited to, cells from the genus Bacillus (such as Bacillus subtilis and Bacillus brevis), the genus Escherichia (such as Escherichia coli), the genus Pseudomonas (Pseudomonas), the genus Streptomyces (Streptomyces), the genus Salmonella (Salmonella) and the genus Erwinia (Erwinia). Particularly useful prokaryotic cells include various strains of E. coli (e.g., K12, HB101 (ATCC No. 33694), DH5a, DH10, MC1061 (ATCC No. 53338), and CC102). Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. In one embodiment, the vector is expressed in a mammalian cell. Many suitable mammalian host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, VA). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573) and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651) and CV-1 cell line (ATCC No. CCL70). The mammalian cell is preferably a human cell. For example, the mammalian cell may be a human lymphoid or lymphoid-derived cell line, such as a cell line derived from pre-B lymphocytes, Cell lines (Crucell Holland BV, The Netherlands) or human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573).

[0104] A nucleic acid sequence encoding the amino acids of any of the antibodies or antigen-binding fragments described herein (optionally a monoclonal antibody or fragment) can be introduced into a cell by transfection, transformation or transduction.

[0105] IV. Pharmaceutical Compositions and Methods Using Anti-Staphylococcus aureus Leukotoxin Antibodies

[0106] The present disclosure provides compositions comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0107] The present disclosure also provides compositions comprising one or more nucleic acid sequences encoding the antibodies or antigen-binding fragments thereof provided herein, or one or more vectors comprising these nucleic acid sequences.

[0108] The compositions provided herein (e.g., comprising an antibody or antigen-binding fragment thereof, one or more nucleic acid sequences, or one or more vectors) can be pharmaceutically acceptable (e.g., physiologically acceptable) compositions comprising a carrier, such as a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and an antibody or antigen-binding fragment, nucleic acid sequence, or vector.

[0109] Any suitable carrier can be used in the present disclosure, and these carriers are well known in the art. The choice of carrier will be determined in part by the specific site where the composition can be administered and the specific method for administering the composition. The composition can optionally be sterile. The composition can be stored frozen or lyophilized and reconstituted in a suitable sterile carrier before use. The composition can be produced according to the conventional techniques described below, such as Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, Philadelphia, Pennsylvania (Philadelphia, PA) (2001).

[0110] The composition ideally comprises an amount of the antibody or antigen-binding fragment that is effective for treating and / or preventing Staphylococcus aureus infection. To this end, the disclosed method comprises administering a therapeutically effective amount or a prophylactically effective amount of a leukotoxin-binding antibody or antigen-binding fragment thereof or a composition comprising the aforementioned antibody or antigen-binding fragment thereof (including a monoclonal antibody or fragment).

[0111] The present disclosure provides a method for treating or preventing Staphylococcus aureus infection in an individual (e.g., a human), comprising administering to an individual in need thereof a leukotoxin-binding antibody or antigen-binding fragment as described herein, followed by treating or preventing the Staphylococcus aureus infection in the individual. The present disclosure also provides the use of the leukotoxin-binding antibody or antigen-binding fragment as described herein, or a composition containing the antibody or fragment thereof as described herein, for the manufacture of a medicament for treating or preventing Staphylococcus aureus infection.

[0112] As discussed herein, Staphylococcus aureus is the main human pathogen causing widespread clinical infections. Staphylococcus aureus is the main cause of bacteremia and infective endocarditis as well as bone and joint, skin and soft tissue, pleural and lung and device-related infections. About 30% of the human population is colonized by Staphylococcus aureus (Wertheim et al., Lancet Infect. Dis., 5:751-762 (2005)). Symptoms of Staphylococcus aureus skin infection include, for example, boils, cellulitis and pustules. Staphylococcus aureus can also cause food poisoning, blood poisoning (also known as bacteremia), toxic shock syndrome and septic arthritis. The epidemiology, pathophysiology and clinical manifestations of S. aureus infection are described in detail in, for example, Tong et al., Clin. Microbiol. Rev., 28(3): 603-661 (2015), and the genomes of several different S. aureus strains have been sequenced (see, for example, GenBank / EMBL deposit numbers BX571856, BX571857, BX571858, FN433596, FN433597, FN433598, HE681097, FR821777, FR821778, FR821779 and FR821780). As discussed herein, an individual (e.g., a human individual) may suffer from diabetes.

[0113] In certain instances, a therapeutically effective amount of a leukotoxin binding antibody or antigen-binding fragment is an amount that inhibits S. aureus-associated sepsis (e.g., in humans), neutralizes toxins, inhibits cell lysis, inhibits multi-organ dysfunction, or any combination of the foregoing.

[0114] Alternatively, the pharmacological and / or physiological effect may be prophylactic, i.e., the effect completely or partially prevents a disease or its symptoms. In this regard, the disclosed methods comprise administering a "prophylactically effective amount" of a leukotoxin binding antibody or antigen-binding fragment (including monoclonal antibodies or fragments).

[0115] The therapeutic or preventive efficacy can be monitored by regularly evaluating the treated patient. For repeated administration over several days or longer, depending on the condition, the treatment can be repeated until the desired disease symptom suppression occurs. However, other dosing regimens may be applicable and within the scope of the present disclosure. The desired dose can be delivered by a single rapid administration of the composition, by multiple rapid administrations of the composition, or by continuous infusion of the composition.

[0116] Compositions comprising an effective amount of an antibody or antigen-binding fragment thereof as described herein, a nucleic acid sequence encoding any of the foregoing, or a vector containing a nucleic acid sequence can be administered to an individual, such as a human, using standard administration techniques, including intravenous, intraperitoneal, subcutaneous, and intramuscular administration routes. The composition may be suitable for parenteral administration. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In some embodiments, peripheral systemic delivery is used to administer the composition to an individual by intravenous, intraperitoneal, or subcutaneous injection.

[0117] The leukotoxin binding antibody or antigen binding fragment or a composition comprising the same can be administered alone or in combination with other drugs (e.g., as an adjuvant) that are routinely used to treat S. aureus infections. Compositions comprising the leukotoxin binding antibody or antigen binding fragment can be used in combination with, for example, one or more antibiotics such as penicillinase-resistant β-lactam antibiotics (e.g., oxacillin or flucloxacillin). Gentamycin can be used to treat serious infections such as endocarditis. However, most strains of S. aureus are now resistant to penicillin, and two out of every 100 people carry methicillin-resistant S. aureus strains (MRSA). MRSA infections are usually treated with vancomycin, and minor skin infections can be treated with triple antibiotic ointment.

[0118] In addition to therapeutic and prophylactic uses, any of the antibodies or antigen-binding fragments thereof described herein may be used in diagnostic or research applications. In this regard, the leukotoxin-binding antibodies or antigen-binding fragments may be used in assays to monitor an individual for S. aureus infection. Research applications include, for example, methods of detecting S. aureus in a sample (e.g., in human body fluids or in cell or tissue extracts) using the leukotoxin-binding antibodies or antigen-binding fragments and a label. The leukotoxin-binding antibodies or antigen-binding fragments may be used with or without modification, such as covalent or non-covalent labeling of the detectable moiety. For example, the detectable moiety may be a radioactive isotope (e.g., 3 H. 14 C. 32 P. 35 S or 125I), fluorescent or chemiluminescent compounds (such as fluorescent isothiocyanate, rhodamine or luciferin), enzymes (such as alkaline phosphatase, β-galactosidase or horseradish peroxidase), or prosthetic groups. Any method known in the art for conjugating antibodies or antigen-binding fragments thereof, respectively, to a detectable moiety may be used in the context of the present disclosure (see, e.g., Hunter et al., Nature, 194:495-496 (1962); David et al., Biochemistry, 13:1014-1021 (1974); Pain et al., J. Immunol. Meth., 40:219-230 (1981); and Nygren, J., Histochem. And Cytochem., 30:407-412 (1982)).

[0119] Any of the antibodies or antigen-binding fragments thereof (e.g., monoclonal antibodies or fragments) described herein, nucleic acid sequences encoding any of the foregoing, vectors comprising nucleic acid sequences, or compositions comprising any of the foregoing can be provided in a kit (i.e., a packaged combination of reagents) in predetermined amounts along with instructions for performing a diagnostic assay. If the leukotoxin-binding antibody or antigen-binding fragment is labeled with an enzyme, the kit preferably includes substrates and cofactors required for the enzyme (e.g., substrate precursors that provide a detectable chromophore or fluorophore). In addition, other additives may be included in the kit, such as stabilizers, buffers (e.g., blocking buffers or dissolution buffers), and the like. The relative amounts of the various reagents can be varied to provide concentrations in the reagent solution that generally optimize the sensitivity of the assay. The reagents can be provided in the form of a dry powder (usually lyophilized), including excipients that will provide a reagent solution having an appropriate concentration when dissolved.

[0120] The following examples further illustrate the invention but, of course, should not be construed as limiting its scope in any way.

[0121] Example 1

[0122] The anti-leukotoxin antibody SAN481 comprises a heavy chain having an amino acid sequence of SEQ ID NO: 9 and a light chain having an amino acid sequence of SEQ ID NO: 10. Several sequence disadvantages were identified in these sequences. For example, oxidation of the heavy chain W100a (VH-CDR3) and M256 (in the Fc domain) was observed. In addition, the glycosylation site (NFS) in the variable heavy chain was 70% glycosylated. Two NS deamidation sites were identified in VL-CDR1, and a DG / DS isomerization site was identified in VH-CDR2. In addition, at 1 week CWLa-2kLux, photosensitivity caused an increase of 3.5% in the aggregation of SAN481.

[0123] In order to achieve improved SAN481 antibody variants, a series of sequence variants were designed and tested. The variants were designed to remove these disadvantages and increase half-life without affecting the leukotoxin neutralizing activity of the antibody. (Preliminary attempts to remove sequence disadvantages resulted in loss of LukSF binding and neutralizing activity. These preliminary attempts included mutating W102 in constructs containing the N28T mutation to F, Y, A, L, I, G, and V.) The YTE mutation was used for half-life extension, and this same mutation also removed the methionine oxidation site in the Fc region (residue M256).

[0124] These variants were then tested for their binding and neutralization properties to LukSF, their photostability, and their imageability. The results are summarized in Table 8 below, and additional information is found in the Examples below.

[0125] Table 8: SAN481 and SAN481 variants

[0126]

[0127] "mon" = monomer

[0128] "agg." = Aggregate

[0129] SAN481-SYT-YTE was chosen as a particularly advantageous variant in view of the fact that SAN481-YTE has similar IC50 to LukSF, LukED and HIgAB, minimal increase in aggregation upon light exposure, no significant CDR deamidation and isomerization detected (1.1%), no stability issues, no self-association and no nonspecific binding.

[0130] Example 2

[0131] This example demonstrates that, unlike other SAN481 variants, the SAN481-SYT-YTE antibody maintains the in vitro activity of SAN481.

[0132] In vitro assays were performed to evaluate the activity of SAN481 variants. Figure 1 In these assays, differentiated HL60 human monocytes (2.5e4 wells / 25 μl) were incubated with a mixture of 50 μl LukSF (100 ng / ml each) or HIgAB (400 ng / ml each) and serial dilutions of each mAb mutant (25 μl) for 2 hours at 37°C as indicated.

[0133] The percentage of cell viability was measured using the Cell Glo assay and calculated as follows: 100*[(OD450 cells+toxin+mAb) / (OD450 cells alone)]. The percentage of cell viability required to achieve 50% inhibition of viability (IC 50 ) and are reported in Table 9.

[0134] Table 9: LukSF and HIgAB activities of SAN481 variants

[0135]

[0136] Compared to SAN481, the SAN481-TF-YTE and SAN481-SYTF-YTE antibodies lost 5.07 and 8.56 fold of performance against LukSF, respectively. However, the SAN481-SYT-YTE antibody did not lose performance.

[0137] Example 3

[0138] This example demonstrates that the SAN481-SYT-YTE antibody has similar in vitro leukotoxin neutralization as the SAN481 antibody.

[0139] In vitro leukotoxin neutralization activity was tested by an assay that measures cell viability. More specifically, Figure 2 Indicated, differentiated HL60 human monocytes (2.5e4 wells / 25 μl) were incubated with a mixture of 50 μl LukSF (100 ng / ml each), LukED (2000 ng / ml each), HlgCB (200 ng / ml each) or HIgAB (400 ng / ml each) and serial dilutions of SAN481 or SAN481_SYT-YTE (25 μl) for 2 hours at 37° C. The percentage of cell viability was measured using the Cell Glo assay and calculated as follows: 100*[(OD450 cells+toxin+mAb) / (OD450 cells alone)], and is shown graphically in Figure 2 superior.

[0140] The results are shown in Figure 2 In the present study, it was confirmed that SAN481-SYT-YTE and SAN481 had similar in vitro neutralizing activities against all LukSFs, HIgABs, HIgBCs and LukEDs.

[0141] Example 4

[0142] This example demonstrates that SAN481-SYT-YTE exhibits excellent photostability.

[0143] The photostability of SAN481 variants was tested. In these analyses, the binding affinity of mAb variants to recombinant antigens was measured by biolayer interferometry on an Octet384 instrument (ForteBio, Menlo Park, CA). To determine intrinsic binding affinity, 2 μg / mL antibody in PBS pH 7.2, 3 mg / mL BSA, 0.05% (v / v) Tween 20 (1× kinetic buffer, ForteBio) was captured by an anti-human IgG Fc biosensor (ForteBio). After washing, association and dissociation measurements were performed using serial dilutions of the antigen protein. The dissociation constant (KD) was derived as the ratio of two rate constants (koff / kon) from the nonlinear fit of the data using Octet384 software v.7.2.

[0144] The results are shown in Tables 10 and 11 below.

[0145] Table 10: One-week photostability of SAN481 variants

[0146]

[0147] Table 11: Octet binding activity of SAN481, SAN481-YTE and SAN481-SYT-YTE under light stress

[0148]

[0149]

[0150] The results confirmed the excellent photostability of SAN481-SYT-YTE and the absence of binding loss for the light stressed samples of SAN481-SYT-YTE.

[0151] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0152] Unless otherwise indicated herein or clearly contradictory to the context, the use of the terms "a / an" and "the" and "at least one" and similar references in the context of describing the present invention (especially in the context of the following claims) should be interpreted as covering both the singular and the plural. Unless otherwise indicated herein or clearly contradictory to the context, the use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") should be understood to mean one item (A or B) selected from the listed items or any combination of two or more of the listed items (A and B). Unless otherwise indicated, the terms "comprising", "having", "including" and "containing" should be understood as opening terms (i.e., meaning "including (but not limited to)"). Unless otherwise indicated, the enumeration of value ranges herein is intended only to serve as a shorthand method of individually referring to each independent value within the range, and each independent value is incorporated into the present specification as if individually listed herein. Unless otherwise indicated herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. Unless otherwise required, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and does not limit the scope of the present invention. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the present invention.

[0153] Preferred embodiments of the present invention are described herein, including the best modes known to the inventors for implementing the present invention. After reading the foregoing description, variations of those preferred embodiments may become apparent to those of ordinary skill in the art. The inventors expect that those of ordinary skill in the art will adopt the variations as appropriate, and the inventors intend to implement the present invention in other ways different from those specifically described herein. Therefore, if applicable law permits, the present invention includes all modifications and equivalents of the subject matter recited in the claims attached hereto. In addition, unless otherwise indicated herein or otherwise clearly contradictory to the context, the present invention encompasses any combination of the above elements in all possible variations thereof. Sequence Listing <110> MedImmune, LLC Humabs BioMed SA <120> Antibodies against Staphylococcus aureus leukotoxin <130> 2943.103PC02 <150> US 62 / 743,501 <151> 2018-10-09 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> SAN481 VH CDR1 <400> 1 Thr Tyr Ala Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial sequence <220> <223> SAN481 VH CDR2 <400> 2 Val Thr Ser Phe Asp Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 12 <212> PRT <213> Artificial sequence <220> <223> SAN481 VH CDR3 <400> 3 Asp Glu Tyr Thr Gly Gly Trp Tyr Ser Val Gly Tyr 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial sequence <220> <223> SAN 481 VL CDR1 <400> 4 Ser Gly Asn Ser Tyr Asn Ile Gly Ser Asn Ser Val Tyr 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> SAN481 VL CDR2 <400> 5 Arg Ser Ile Gln Arg Pro Ser 1 5 <210> 6 <211> 11 <212> PRT <213> Artificial sequence <220> <223> SAN481 VL CDR3 <400> 6 Ala Ala Trp Asp Asp Ser Leu Arg Ala Trp Val 1 5 10 <210> 7 <211> 121 <212> PRT <213> Artificial sequence <220> <223> SAN481 VH <400> 7 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Ser Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ser Phe Asp Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Gly Leu Arg Val Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95 Ala Arg Asp Glu Tyr Thr Gly Gly Trp Tyr Ser Val Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 8 <211> 110 <212> PRT <213> artificial sequence <220> <223> SAN481 VL <400> 8 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Asn Ser Tyr Asn Ile Gly Ser Asn 20 25 30 Ser Val Tyr Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Ser Arg Ser Ile Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Val Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Arg Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 9 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> SAN481 Heavy Chain <400> 9 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Ser Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ser Phe Asp Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Gly Leu Arg Val Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95 Ala Arg Asp Glu Tyr Thr Gly Gly Trp Tyr Ser Val Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Light 450 <210> 10 <211> 216 <212> PRT <213> artificial sequence <220> <223> SAN481 <400> 10 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Asn Ser Tyr Asn Ile Gly Ser Asn 20 25 30 Ser Val Tyr Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Ser Arg Ser Ile Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Val Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Arg Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 11 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> SAN481-YTE heavy chain <400> 11 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Ser Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ser Phe Asp Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Gly Leu Arg Val Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95 Ala Arg Asp Glu Tyr Thr Gly Gly Trp Tyr Ser Val Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 245 250 255 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Light 450 <210> 12 <211> 13 <212> PRT <213> artificial sequence <220> <223> SAN481-SY VL CDR1 <400> 12 Ser Gly Ser Ser Tyr Asn Ile Gly Ser Asn Tyr Val Tyr 1 5 10 <210> 13 <211> 110 <212> PRT <213> artificial sequence <220> <223> SAN481-SY VL <400> 13 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Tyr Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Ser Arg Ser Ile Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Val Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Arg Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 14 <211> 216 <212> PRT <213> artificial sequence <220> <223> SAN481-SY chain link <400> 14 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Tyr Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Ser Arg Ser Ile Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Val Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Arg Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 15 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> SAN481-T VH <400> 15 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ser Phe Asp Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Gly Leu Arg Val Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95 Ala Arg Asp Glu Tyr Thr Gly Gly Trp Tyr Ser Val Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 16 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> SAN481-T-YTE Heavy Chain <400> 16 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ser Phe Asp Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Gly Leu Arg Val Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95 Ala Arg Asp Glu Tyr Thr Gly Gly Trp Tyr Ser Val Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 245 250 255 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 17 <211> 12 <212> PRT <213> Artificial sequence <220> <223> SAN481-TF VH CDR3 <400> 17 Asp Glu Tyr Thr Gly Gly Phe Tyr Ser Val Gly Tyr 1 5 10 <210> 18 <211> 121 <212> PRT <213> Artificial sequence <220> <223> SAN481-TF VH <400> 18 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ser Phe Asp Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Gly Leu Arg Val Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95 Ala Arg Asp Glu Tyr Thr Gly Gly Phe Tyr Ser Val Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> SAN481-TF-YTE Heavy Chain <400> 19 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ser Phe Asp Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Gly Leu Arg Val Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95 Ala Arg Asp Glu Tyr Thr Gly Gly Phe Tyr Ser Val Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 245 250 255 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leucine Threonine Cysteine Leucine Valine Lysine Glycine Phenylalanine Tyrosine Proline Serine Aspartic Acid Isoleucine Alanine Valine Glutamic Acid 370 375 380 Tryptophan Glutamic Acid Serine Asparagine Glycine Glutamine Proline Glutamic Acid Asparagine Asparagine Tyrosine Lysine Threonine Threonine Proline Proline 385 390 395 400 Valine Leucine Aspartic Acid Serine Aspartic Acid Glycine Serine Phenylalanine Phenylalanine Leucine Tyrosine Serine Lysine Leucine Threonine Valine 405 410 415 Aspartic Acid Lysine Serine Arginine Tryptophan Glutamine Glutamine Glycine Asparagine Valine Phenylalanine Serine Cysteine Serine Valine Methionine 420 425 430 Histidine Glutamic Acid Alanine Leucine Histidine Asparagine Histidine Tyrosine Threonine Glutamine Lysine Serine Leucine Serine Leucine Serine 435 440 445 Proline Glycine Lysine 450 <210> 20 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> SAN481-EG VH CDR2 <400> 20 Valine Threonine Serine Phenylalanine Glutamic Acid Glycine Serine Asparagine Glutamic Acid Tyrosine Tyrosine Isoleucine Aspartic Acid Serine Valine Lysine 1 5 10 15 Glycine <210> 21 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> SAN481-EG VH <400> 21 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Ser Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ser Phe Glu Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Gly Leu Arg Val Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95 Ala Arg Asp Glu Tyr Thr Gly Gly Trp Tyr Ser Val Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 22 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> SAN481-EG-YTE Heavy Chain <400> 22 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Ser Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ser Phe Glu Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Gly Leu Arg Val Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95 Ala Arg Asp Glu Tyr Thr Gly Gly Trp Tyr Ser Val Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 245 250 255 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 23 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> SAN481-QFS VH <400> 23 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Gln Phe Ser Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ser Phe Asp Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Gly Leu Arg Val Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95 Ala Arg Asp Glu Tyr Thr Gly Gly Trp Tyr Ser Val Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 24 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> SAN481 - QFS - YTE Heavy Chain <400> 24 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Gln Phe Ser Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ser Phe Asp Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Gly Leu Arg Val Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95 Ala Arg Asp Glu Tyr Thr Gly Gly Trp Tyr Ser Val Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 245 250 255 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 25 <211> 302 <212> PRT <213> Artificial Sequence <220> <223> LukF <400> 25 Gly Ala Gln His Ile Thr Pro Val Ser Glu Lys Lys Val Asp Asp Lys 1 5 10 15 Ile Thr Leu Tyr Lys Thr Thr Ala Thr Ser Asp Ser Asp Lys Leu Lys 20 25 30 Ile Ser Gln Ile Leu Thr Phe Asn Phe Ile Lys Asp Lys Ser Tyr Asp 35 40 45 Lys Asp Thr Leu Ile Leu Lys Ala Ala Gly Asn Ile Tyr Ser Gly Tyr 50 55 60 Thr Lys Pro Asn Pro Lys Asp Thr Ile Ser Ser Gln Phe Tyr Trp Gly 65 70 75 80 Ser Lys Tyr Asn Ile Ser Ile Asn Ser Asp Ser Asn Asp Ser Val Asn 85 90 95 Val Val Asp Tyr Ala Pro Lys Asn Gln Asn Glu Glu Phe Gln Val Gln 100 105 110 Gln Thr Val Gly Tyr Ser Tyr Gly Gly Asp Ile Asn Ile Ser Asn Gly 115 120 125 Leu Ser Gly Gly Gly Asn Gly Ser Lys Ser Phe Ser Glu Thr Ile Asn 130 135 140 Tyr Lys Gln Glu Ser Tyr Arg Thr Ser Leu Asp Lys Arg Thr Asn Phe 145 150 155 160 Lys Lys Ile Gly Trp Asp Val Glu Ala His Lys Ile Met Asn Asn Gly 165 170 175 Trp Gly Pro Tyr Gly Arg Asp Ser Tyr His Ser Thr Tyr Gly Asn Glu 180 185 190 Met Phe Leu Gly Ser Arg Gln Ser Asn Leu Asn Ala Gly Gln Asn Phe 195 200 205 Leu Glu Tyr His Lys Met Pro Val Leu Ser Arg Gly Asn Phe Asn Pro 210 215 220 Glu Phe Ile Gly Val Leu Ser Arg Lys Gln Asn Ala Ala Lys Lys Ser 225 230 235 240 Lys Ile Thr Val Thr Tyr Gln Arg Glu Met Asp Arg Tyr Thr Asn Phe 245 250 255 Trp Asn Gln Leu His Trp Ile Gly Asn Asn Tyr Lys Asp Glu Asn Arg 260 265 270 Ala Thr His Thr Ser Ile Tyr Glu Val Asp Trp Glu Asn His Thr Val 275 280 285 Lys Leu Ile Asp Thr Gln Ser Lys Glu Lys Asn Pro Met Ser 290 295 300 <210> 26 <211> 302 <212> PRT <213> Artificial Sequence <220> <223> LukD <400> 26 Gly Ala Gln His Ile Thr Pro Val Ser Glu Lys Lys Val Asp Asp Lys 1 5 10 15 Ile Thr Leu Tyr Lys Thr Thr Ala Thr Ser Asp Asn Asp Lys Leu Asn 20 25 30 Ile Ser Gln Ile Leu Thr Phe Asn Phe Ile Lys Asp Lys Ser Tyr Asp 35 40 45 Lys Asp Thr Leu Val Leu Lys Ala Ala Gly Asn Ile Asn Ser Gly Tyr 50 55 60 Lys Lys Pro Asn Pro Lys Asp Tyr Asn Tyr Ser Gln Phe Tyr Trp Gly 65 70 75 80 Gly Lys Tyr Asn Val Ser Val Ser Ser Glu Ser Asn Asp Ala Val Asn 85 90 95 Val Val Asp Tyr Ala Pro Lys Asn Gln Asn Glu Glu Phe Gln Val Gln 100 105 110 Gln Thr Leu Gly Tyr Ser Tyr Gly Gly Asp Ile Asn Ile Ser Asn Gly 115 120 125 Leu Ser Gly Gly Leu Asn Gly Ser Lys Ser Phe Ser Glu Thr Ile Asn 130 135 140 Tyr Lys Gln Glu Ser Tyr Arg Thr Thr Ile Asp Arg Lys Thr Asn His 145 150 155 160 Lys Ser Ile Gly Trp Gly Val Glu Ala His Lys Ile Met Asn Asn Gly 165 170 175 Trp Gly Pro Tyr Gly Arg Asp Ser Tyr Asp Pro Thr Tyr Gly Asn Glu 180 185 190 Leu Phe Leu Gly Gly Arg Gln Ser Ser Ser Asn Ala Gly Gln Asn Phe 195 200 205 Leu Pro Thr His Gln Met Pro Leu Leu Ala Arg Gly Asn Phe Asn Pro 210 215 220 Glu Phe Ile Ser Val Leu Ser His Lys Gln Asn Asp Thr Lys Lys Ser 225 230 235 240 Lys Ile Lys Val Thr Tyr Gln Arg Glu Met Asp Arg Tyr Thr Asn Gln 245 250 255 Trp Asn Arg Leu His Trp Val Gly Asn Asn Tyr Lys Asn Gln Asn Thr 260 265 270 Val Thr Phe Thr Ser Thr Tyr Glu Val Asp Trp Gln Asn His Thr Val 275 280 285 Lys Leu Ile Gly Thr Asp Ser Lys Glu Thr Asn Pro Gly Val 290 295 300 <210> 27 <211> 300 <212> PRT <213> Artificial Sequence <220> <223> HIgB <400> 27 Gly Glu Gly Lys Ile Thr Pro Val Ser Val Lys Lys Val Asp Asp Lys 1 5 10 15 Val Thr Leu Tyr Lys Thr Thr Ala Thr Ala Asp Ser Asp Lys Phe Lys 20 25 30 Ile Ser Gln Ile Leu Thr Phe Asn Phe Ile Lys Asp Lys Ser Tyr Asp 35 40 45 Lys Asp Thr Leu Val Leu Lys Ala Thr Gly Asn Ile Asn Ser Gly Phe 50 55 60 Val Lys Pro Asn Pro Asn Asp Tyr Asp Phe Ser Lys Leu Tyr Trp Gly 65 70 75 80 Ala Lys Tyr Asn Val Ser Ile Ser Ser Gln Ser Asn Asp Ser Val Asn 85 90 95 Val Val Asp Tyr Ala Pro Lys Asn Gln Asn Glu Glu Phe Gln Val Gln 100 105 110 Asn Thr Leu Gly Tyr Thr Phe Gly Gly Asp Ile Ser Ile Ser Asn Gly 115 120 125 Leu Ser Gly Gly Leu Asn Gly Asn Thr Ala Phe Ser Glu Thr Ile Asn 130 135 140 Tyr Lys Gln Glu Ser Tyr Arg Thr Thr Leu Ser Arg Asn Thr Asn Tyr 145 150 155 160 Lys Asn Val Gly Trp Gly Val Glu Ala His Lys Ile Met Asn Asn Gly 165 170 175 Trp Gly Pro Tyr Gly Arg Asp Ser Phe His Pro Thr Tyr Gly Asn Glu 180 185 190 Leu Phe Leu Ala Gly Arg Gln Ser Ser Ala Tyr Ala Gly Gln Asn Phe 195 200 205 Ile Ala Gln His Gln Met Pro Leu Leu Ser Arg Ser Asn Phe Asn Pro 210 215 220 Glu Phe Leu Ser Val Leu Ser His Arg Gln Asp Gly Ala Lys Lys Ser 225 230 235 240 Lys Ile Thr Val Thr Tyr Gln Arg Glu Met Asp Leu Tyr Gln Ile Arg 245 250 255 Trp Asn Gly Phe Tyr Trp Ala Gly Ala Asn Tyr Lys Asn Phe Lys Thr 260 265 270 Arg Thr Phe Lys Ser Thr Tyr Glu Ile Asp Trp Glu Asn His Lys Val 275 280 285 Lys Leu Leu Asp Thr Lys Glu Thr Glu Asn Asn Lys 290 295 300 <210> 28 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of SAN481-T <400> 28 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Ala Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ser Phe Asp Gly Ser Asn Glu Tyr Tyr Ile Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Thr Gly Leu Arg Val Glu Asp Thr Ala Leu Tyr Phe Cys 85 90 95 Ala Arg Asp Glu Tyr Thr Gly Gly Trp Tyr Ser Val Gly Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to at least one Staphylococcus aureus leukotoxin, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) complementarity determining region CDR1 sequence as shown in SEQ ID NO:1, a VH CDR2 sequence as shown in SEQ ID NO:2, a VH CDR3 sequence as shown in SEQ ID NO:3, a variable light chain (VL) CDR1 sequence as shown in SEQ ID NO:12, a VL CDR2 sequence as shown in SEQ ID NO:5, and a VL CDR3 sequence as shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID NO:

15.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises VH and VL, wherein the VL comprises the amino acid sequence of SEQ ID NO:

13.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1, wherein the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence of SEQ ID NO: 15 and a VL comprising the sequence of SEQ ID NO:

13.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising the sequence of SEQ ID NO:

16.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment comprises a light chain comprising the sequence of SEQ ID NO:

14.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody comprises a heavy chain comprising the sequence of SEQ ID NO: 16 and a light chain comprising the sequence of SEQ ID NO:

14.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein (a) the antibody or antigen-binding fragment binds to LukF, LukD or HlgB and / or (b) the antibody or antigen-binding fragment neutralizes LukF, LukD or HlgB.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment (a) binds to LukF, LukD and HlgB and / or (b) neutralizes LukF, LukD and HlgB.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment further comprises a heavy chain constant region.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the heavy chain constant region is selected from the group consisting of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 heavy chain constant regions. 12 . The antibody or antigen-binding fragment thereof according to claim 11 , wherein the heavy chain constant region is a human IgG1 constant region.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment further comprises a light chain constant region.

14. The antibody or antigen-binding fragment thereof according to claim 13, wherein the light chain constant region is selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions.

15. The antibody or antigen-binding fragment thereof according to claim 14, wherein the light chain constant region is a human IgGκ light chain constant region.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof comprises an Fc region that has been engineered to improve half-life.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof comprises an Fc region having a YTE mutation.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment is a full-length antibody.

21. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment is an antigen-binding fragment.

22. The antigen binding fragment of claim 21, wherein the antigen binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabodies, tribodies, bibodies, DVD-Ig, Fcab, mAb 2 , (scFv)2 or scFv-Fc.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof has an affinity for LukF, LukD and HlgB of Staphylococcus aureus of less than 75 pM.

24. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof has similar binding affinity to LukF, LukD and HIgB.

25. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, further comprising a detectable label.

26. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 25.

27. The composition of claim 26, wherein the composition further comprises a pharmaceutically acceptable carrier.

28. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 25 or a composition according to claim 26 or 27 in the preparation of a medicament for treating or preventing a Staphylococcus aureus (S. aureus) infection in a subject.

29. The use according to claim 28, wherein the Staphylococcus aureus infection is sepsis.

30. The use according to claim 28, wherein the Staphylococcus aureus infection is bacteremia.

31. The use according to claim 28, wherein the Staphylococcus aureus infection is pneumonia.

32. The use according to claim 28, wherein the Staphylococcus aureus infection is ICU pneumonia.

33. The use according to claim 28, wherein the Staphylococcus aureus infection is a skin or soft tissue infection (SSTI).

34. The use according to claim 28, wherein the Staphylococcus aureus infection is a diabetic lower limb infection.

35. The use according to claim 28, wherein the Staphylococcus aureus infection is diabetic foot ulcer DFU.

36. The use according to claim 35, wherein the DFU is not infected.

37. The use according to claim 35, wherein the DFU is infected.

38. The use according to claim 35, wherein the DFU is a grade 1, grade 2 or grade 3 DFU.

39. The use according to claim 28, wherein the S. aureus infection is a bone or joint infection.

40. The use according to claim 28, wherein the S. aureus infection is a joint infection, a device infection, a wound infection, a surgical site infection or osteomyelitis.

41. The use according to any one of claims 28 to 40, wherein the subject is a surgical subject.

42. The use of any one of claims 28 to 40, wherein the S. aureus infection comprises antibiotic-resistant S. aureus.

43. The use according to any one of claims 28 to 40, wherein the subject suffers from diabetes.

44. The use according to any one of claims 28 to 40, wherein the subject is a human.

45. The use according to any one of claims 28 to 40, wherein the treatment or prevention of Staphylococcus aureus infection comprises toxin neutralization, inhibition of cell lysis, inhibition of multiple organ dysfunction, inhibition of Staphylococcus aureus-associated sepsis, or any combination thereof.

46. ​​An isolated polynucleotide comprising a nucleic acid molecule encoding the heavy and light chains of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 24.

47. An isolated polynucleotide comprising a nucleic acid molecule encoding the VH and VL of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 24.

48. An isolated vector comprising the polynucleotide according to claim 46 or 47.

49. A host cell comprising the polynucleotide of claim 46 or 47, or the vector of claim 48.

50. The host cell of claim 49, wherein the host cell is selected from the group consisting of CHO, NS0, PER-C6, HEK-293 and HeLa cells.

51. The host cell of claim 49 or 50, wherein the host cell is isolated.

52. A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing the host cell according to any one of claims 49 to 51 to produce the antibody or the antigen-binding fragment thereof.

53. A method for detecting Staphylococcus aureus or Staphylococcus aureus leukotoxin in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 25, wherein the method is not for diagnostic purposes.

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