Compositions and methods for treating and preventing Staphylococcus aureus infections

Abstract

Pharmaceutical compositions for treating a Staphylococcus aureus infection or reducing the risk of developing a Staphylococcus aureus infection in a subject are provided. The present invention provides a method for the preparation of a 1×10 Fab fragment containing a 1×10 Fab fragment, comprising administering the 1×10 Fab fragment to a pharmaceutically acceptable carrier via a paratope in the Fab region. -10 and a purified monoclonal antibody that specifically binds to Staphylococcus aureus protein A (SpA) with a KD of less than M, wherein the monoclonal antibody is capable of displacing human IgG immunoglobulins bound to SpA on Staphylococcus aureus bacteria via their Fc region.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Patent Application Nos. 62 / 007,242, filed June 3, 2014, 62 / 041,423, filed August 25, 2014, and 62 / 115,665, filed February 13, 2015.

[0002] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The above ASCII copy, created on May 28, 2015, is designated 5407-0234_SL.txt and is 83,036 bytes in size.

[0003] The present invention relates generally to methods of medical therapy, immunology and microbiology. More particularly, the present invention relates to compositions and methods for treating and preventing Staphylococcus aureus infections. [Background technology]

[0004] Staphylococcus aureus (SA) is a substantial cause of illness and death in both humans and animals. Infections caused by these gram-positive cocci often result in the development of superficial abscesses. Other cases of SA infection can be much more severe. For example, invasion of the lymphatics and blood by SA can lead to systemic infection, which in turn can cause complications such as endocarditis, arthritis, osteomyelitis, pneumonia, septic shock, and even death. Nosocomial SA infections are common and particularly challenging to resolve, as SA is the most frequent cause of nosocomial surgical site infections and pneumonia and the second most common cause of cardiovascular and bloodstream infections. Antibiotic administration has been, and remains, the standard of care for SA infections. Unfortunately, antibiotic use has also accelerated the development of antibiotic resistance in SA. Notably, methicillin-resistant SA (MRSA) has evolved the ability to resist β-lactam antibiotics, e.g., penicillin and cephalosporin. More concerningly, in recent years, SA resistance to mainstay antibiotics, e.g., vancomycin and linezolid, has emerged. Therefore, novel approaches to prevent and treat SA infections are needed. Summary of the Invention

[0005] It has been found that certain antibodies (Abs) bearing Fab region paratopes that specifically bind SA protein A (SpA) can mediate opsinization of SA bacteria despite the expression of antibody-neutralizing SpA. Previous Ab-based strategies for treating or preventing SA infections have shown promise in preclinical and early clinical trials but have failed to meet endpoints in phase III trials. Perhaps explaining these results is that previous strategies did not address the Ab-neutralizing properties of SpA. SpA is a highly expressed cell wall-associated protein that binds most immunoglobulins (Igs) via their Fc (effector) region. SpA binds human antibodies of the subclasses IgG1, IgG2, and IgG4 via their Fc region at approximately 1 × 10 -9 K of M D The Fc region acts as an anchor, directing the effector moiety of immunoglobulin (Ig) away from Fc-interacting immune effectors, such as complement, and Fc receptor (FcR)-bearing phagocytes. Thus, the majority of Abs specific for SA antigens are "sequestered" from immune effectors in this manner. Furthermore, because SpA is highly expressed on the SA cell wall (an estimated 7% of the cell wall), SpA mediates the formation of an Ig shield that coats the cell wall. This shield sterically prevents Abs specific for cell wall antigens from binding to their targets and mediating bacterial oponophagocytosis. The formation of the Ig shield has not previously been recognized as a virulence factor. Thus, despite the Fc-neutralizing ability of SpA and the formation of an Ig shield, SA-binding Abs with Fab regions that specifically bind to SpA while still allowing their Fc regions to interact with FcRs on immune effector cells and / or activate complement by binding Clq would be a significant step forward compared to anti-SA Ab-based approaches. Preferred versions of such Abs would be able to displace Ig already bound to SpA by their Fc regions.

[0006] As examples of the above, we describe herein isolated or purified antibodies (particularly human IgG3 antibodies with Fc regions with low or no affinity for SpA, e.g., allotypes with arginine at amino acid 435; Stapleton et al., Nature Communications 2, Article number: 599, 2011) whose Fab regions can specifically bind to target epitopes of SpA on SA bacteria, while their Fc regions remain capable of interacting with one (e.g., soluble recombinant or native) FcR on immune cells, despite the Fc-binding properties of SpA and steric hindrance of the target epitope by Ig bound to SpA via their Fc regions. Also provided herein are pharmaceutical compositions containing at least one of these antibodies and a pharmaceutically acceptable carrier (e.g., a non-naturally occurring pharmaceutically acceptable carrier). Further provided are methods for treating a subject having an SA infection or reducing the risk of developing an SA infection in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of any of the pharmaceutical compositions described herein, or any of the antibodies or antigen-binding fragments described herein.

[0007] As used herein, the terms "a" or "an" before a noun refers to one or more of that particular noun. For example, the phrase "an antibody" refers to "one or more antibodies."

[0008] The term "antibody" or "Ab" refers to any immunoglobulin (e.g., a human, cartilaginous, or camelid antibody) or conjugate thereof that specifically binds to an antigen (e.g., an SpA antigen such as SEQ ID NO: 1 or an antigen fragment of SEQ ID NO: 1). A wide variety of Abs are known to those skilled in the art. Non-limiting examples of Abs include monoclonal Abs (e.g., including full-length Abs), polyclonal Abs, multispecific Abs (e.g., bispecific Abs), dual variable domain Abs, single-chain Abs (e.g., single-domain Abs, camelid Abs, and cartilaginous antibodies), chimeric (e.g., humanized, e.g., humanized IgG3) Abs, and human Abs (e.g., human IgG3 Abs). The term "antibody" further includes Ab conjugates (e.g., Abs conjugated to a stabilizing protein, label, or therapeutic agent (e.g., any of the therapeutic agents described herein or known in the art)).

[0009] The term "antigen-binding fragment" refers to any portion of a full-length Ab that contains at least one variable domain (e.g., a mammalian (e.g., human, mouse, rat, rabbit, or sheep) heavy or light chain immunoglobulin), a camelid variable antigen-binding domain (VHH), or a cartilaginous fish immunoglobulin novel antigen receptor (Ig-NAR) domain capable of specifically binding to an antigen. For example, the antigen-binding fragments described herein can be sufficient to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) in a mammal (e.g., a human) and / or can include at least a portion of the Ab Fc region conjugated to a therapeutic agent (e.g., any of the therapeutic agents described herein or known in the art). Non-limiting examples of Ab fragments include Fab, Fab', F(ab'), Fv fragments, diabodies, linear antibodies, and multispecific Abs formed from Ab fragments. Additional Ab fragments containing at least one camelid VHH domain or at least one cartilaginous fish Ig-NAR domain include minibodies, microbodies, subnanobodies, and nanobodies, as well as any of the other forms of Abs described in U.S. Patent Application Publication No. 2010 / 0092470. The antigen-binding fragment may be, for example, an antigen-binding fragment of one of human or humanized IgG1, IgG2, IgG3 IgG4, IgD, IgA, IgE, or IgM.

[0010] The term "human antibody" refers to an Ab encoded by nucleic acid present in the human genome (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, human Abs are produced in mammalian (e.g., human) cell culture. In some embodiments, human Abs are produced in non-human cells (e.g., a Chinese hamster ovary cell line or a mouse or hamster cell line). In some embodiments, human Abs are produced in bacterial or yeast cells. Human Abs can include a conjugated therapeutic agent (e.g., any of the therapeutic agents described herein or known in the art). Human Abs can be human IgG1, IgG2, IgG4, IgD, IgA, IgE, or IgM, and are preferably human IgG3. The term "fully human antibody" refers to an Ab comprising heavy and light chain variable regions naturally present in human serum.

[0011] The term "humanized antibody" refers to an Ab that contains the majority of the sequence of a human Ab, but also includes minimal sequence derived from a non-human (e.g., mouse, rat, rabbit, or goat) Ig. In a non-limiting example, a humanized Ab is a human Ab (recipient Ab) in which hypervariable region residues of the recipient Ab are replaced with hypervariable region residues from a non-human species Ab (donor Ab), e.g., mouse, rabbit, or goat Ab, with the desired specificity, affinity, and capacity. In some embodiments, Fv framework residues of the human Ig are replaced with corresponding non-human residues. In some embodiments, a humanized Ab may contain residues not found in the recipient Ab or donor Ab. These modifications can be made to further refine Ab performance.

[0012] In some embodiments, a humanized Ab will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (complementarity-determining regions) correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. A humanized antibody may further contain at least a portion of an Ig constant region (Fc region), typically of a human Ig (e.g., human IgG3). Humanized Abs can be produced by molecular biology methods well known in the art. Non-limiting examples of methods for producing humanized antibodies are described herein. A humanized Ab can include a conjugated therapeutic agent (e.g., any of the therapeutic agents described herein or known in the art).

[0013] The term "single-chain antibody" refers to a single polypeptide containing at least one variable binding domain (e.g., a variable domain of a mammalian heavy or light chain Ig, a camelid variable antigen binding domain (VHH), or a cartilaginous fish (e.g., shark) immunoglobulin novel antigen receptor (Ig-NAR) domain) that can specifically bind to an antigen. Non-limiting examples of single-chain Abs are described herein and known in the art (e.g., antibodies described in U.S. Patent Application Publication No. 2010 / 0092470). Single-domain antibodies can include a conjugated therapeutic agent (e.g., any of the therapeutic agents described herein or known in the art).

[0014] An Ab or antigen-binding fragment thereof "specially binds" or "binds specially" to a particular antigen, e.g., SpA (e.g., an epitope comprising SEQ ID NO: 1 or an antigen-binding fragment of SEQ ID NO: 1), but when the Ab or antigen-binding fragment thereof binds to that antigen, it recognizes and binds to (e.g., does not recognize and bind to) other molecules in the sample only to a lesser extent. In some embodiments, the Ab or antigen-binding fragment thereof is present at a concentration of 1 x 10 (as determined by surface plasmon resonance) in phosphate buffered saline. -10 M or less (e.g., 1×10 -11 M or less or 1 x 10 -12 Affinity (K D The ability of an Ab or antigen-binding fragment to specifically bind to a protein epitope can be determined using any of the methods known in the art or described herein.

[0015] The term "complementarity-determining region" or "CDR" refers to a region within an Ig (heavy or light chain Ig) that forms part of the antigen-binding site (paratope) in an Ab or antigen-binding fragment thereof. As is known in the art, heavy chain Igs typically contain three CDRs: CDR1, CDR2, and CDR3, respectively, and light chain Igs typically contain three CDRs: CDR1, CDR2, and CDR3, respectively. Within any Ab or antigen-binding fragment thereof, the three CDRs from the heavy chain Ig and the three CDRs from the light chain Ig together form an antigen-binding site within the Ab or antigen-binding fragment thereof. The Kabat database is one system used in the art for numbering CDR sequences present within a light or heavy chain Ig.

[0016] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. This specification describes methods and materials for use in the present invention. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are intended to be illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions of terms, will control. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1 is a schematic diagram of SpA showing the different domains and locations of each of the five antigenic peptides. The sequence of antigenic peptide number 5 (SEQ ID NO: 1) is shown. [Figure 2] Figure 2 is a set of two graphs showing histograms of fluorescence of SA clinical isolate 00X (top) and SA strain ATCC#25923 (bottom) incubated with biotinylated PA8-G3 Ab (light line) or control biotinylated anti-interleukin 1α Ab (MABp1) (dark line) and then incubated with streptavidin-APC. [Figure 3] Figure 3 is a set of two graphs showing histograms of fluorescence of SA clinical isolate 00X (top) and SA strain ATCC#25923 (bottom) incubated with unlabeled PA8-G3 Ab (light line) or unlabeled MABp1 Ab (dark line), then incubated with biotinylated recombinant Fcγ receptor 1, and then incubated with streptavidin-APC. [Figure 4]Figure 4 is a graph of the mean fluorescence intensity (using fluorescence cell sorting) of differentiated HL60 cells after co-incubation with PA8-G3 Ab opsonized with pH-loaded green-labeled strain ATCC #25923 or clinical isolate 00X. Similar samples incubated with control Ab MABp1 instead of PA8-G3 Ab were used as negative controls. [Figure 5] Figure 5 is a set of two graphs showing the fluorescence intensity of clinical isolate 00X (top) or ATCC#25923 (bottom) preincubated with human serum for 15 minutes before addition of biotinylated PA8-G3 Ab or negative control MABP1 Ab, and then incubated with streptavidin APC. [Figure 6] Figure 6 is a graph showing the mean fluorescence intensity of differentiated or undifferentiated HL-60 cells after co-incubation with pH-loaded green-labeled SA and one of the following unlabeled Abs: PA7.2-G3, PA4-G3, PA8-G3, PA15-G3, PA21-G3, PA27-G3, PA32-G3, PA37-G3, or MABp1. The MABp1 Ab sample was used as a negative control. [Figure 7] 7A-D are graphs showing that administration of mAb PA8 enhances survival of mouse subjects infected with S. aureus. [Figure 8] 8A-C are graphs showing the synergy between PA8-G3 and vancomycin. DETAILED DESCRIPTION OF THE INVENTION

[0018] Described herein are methods and compositions for treating a subject with an SA infection or reducing the risk of developing an SA infection in a subject.

[0019] Antibodies and their antigen-binding fragments Described herein are purified or isolated (e.g., at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% pure by weight) Abs (e.g., preferably fully human, human, or humanized IgG3) that bind to SpA and are capable of mediating opsonization of SA bacteria despite expression of SA antibody (Ab)-neutralizing SpA. Preferred such Abs bind to the peptide of SEQ ID NO: 1 with sufficient binding affinity to displace human IgG immunoglobulins (e.g., one or more of IgG1, IgG2, and IgG4) bound to SpA via their Fc region. Preferred Abs bind to the peptide of SEQ ID NO: 1 with sufficient binding affinity to displace human IgG immunoglobulins (e.g., one or more of IgG1, IgG2, and IgG4) bound to SpA via their Fab region paratopes ... -10 Less than M (e.g., 1 × 10 -11 Less than M, 1 x 10 -12 Less than M, 0.5 x 10 -12 Less than M or 1 x 10 -13 K (less than M) D and can bind to SpA under physiological conditions (e.g., in phosphate buffered saline) (e.g., as determined using surface plasmon resonance or Bio-Layer Interferometry using recombinant SpA). For example, 1×10 Fab fragments described herein can bind to SpA via their Fab regions (e.g., under physiological conditions, e.g., in phosphate buffered saline, as measured using surface plasmon resonance, e.g., using recombinant SpA). -10 M~0.5×10 -12 M, 1 x 10 -11 M~0.5×10 -12 M, 1 x 10 -11 M~0.2×10 -12 K of M D Preferred are Abs that bind at 1 × 10 to Staphylococcus aureus protein A (SpA) via their Fab region paratopes. Those Abs or antigen-binding fragments described herein are preferably capable of displacing human Abs (e.g., one or more of IgG1, IgG2, and IgG4) that bind to SpA in the cell wall of SA bacteria via their Fc region. Further, herein, Abs or antigen-binding fragments that bind to Staphylococcus aureus protein A (SpA) via their Fab region paratopes are preferably capable of displacing human Abs (e.g., one or more of IgG1, IgG2, and IgG4) that bind to SpA in the cell wall of SA bacteria via their Fc region paratopes. -10 K less than M DProvided are purified or isolated (e.g., at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% pure by weight) mAbs (e.g., preferably fully human, human, or humanized IgG3) that specifically bind to SpA, wherein the mAbs are capable of mediating opsonization of SpA-expressing Staphylococcus aureus bacteria in the presence of at least 1 mg / mL (e.g., at least 1, 2, 3, 4, 5, 10, 25, 50, or 100 mg / mL, or the amount normally contained in human serum) IgG immunoglobulins that bind SpA via their Fc region.

[0020] The purified or isolated Abs provided herein are present in the extracellular domain of SpA (e.g., X RIt is possible that the IgG-binding domains bind to epitopes present in the repeat region and one or more IgG-binding domains. Non-limiting examples of antigens that can be specifically recognized by any of the Abs (or antigen-binding fragments thereof) provided herein include 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQ ID NO: 1 (e.g., a fragment starting at amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of SEQ ID NO: 1); 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 contiguous amino acids of SEQ ID NO: 82 (e.g., a fragment starting at amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of SEQ ID NO: 82); 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of SEQ ID NO: 83; 2, 13, 14, 15, or 16 contiguous amino acids (e.g., fragments beginning at amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO: 83); 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 contiguous amino acids of SEQ ID NO: 84 (e.g., fragments beginning at amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of SEQ ID NO: 84); 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids of SEQ ID NO: 85 (e.g., fragments beginning at amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of SEQ ID NO: 85);or amino acids 1 to 20, 10 to 30, 20 to 40, 30 to 50, 40 to 60, 50 to 70, 60 to 80, 70 to 90, 80 to 100, 90 to 110, 100 to 120, 110 to 130, 120 to 140, 130 to 150, 140 to 160, 150 to 170, 160 to 180, 170 to 190, 180 to 200, 190 to 210, 200 to 220, 210 to 230, 220 to 240, 230 to 250, 240 to 260, 250 to 270 of SEQ ID NO: 86, The antigens include 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids from positions 260-280, 270-290, 280-300, 290-310, 300-320, 310-330, 320-340, 330-350, 340-360, 350-370, 360-380, 370-390, 380-400, 390-410, 400-420, 410-430, 420-440, or 430-450. Other exemplary antigens include similar fragments of SpA having an amino acid sequence that differs from that of SEQ ID NO: 86;

[0021] Methods for determining the ability of an Ab or its antigen-binding fragment to bind to a target protein (e.g., SpA or a portion thereof) can be performed using methods known in the art. Non-limiting examples of such methods include competitive binding assays using Abs known to bind to a target protein (e.g., SpA), enzyme-linked immunosorbent assays, BioCoRE®, affinity columns, immunoblotting, or protein array technologies. In some embodiments, the binding ability of an Ab or its antigen-binding fragment is determined by contacting SA bacteria with the Ab or its antigen-binding fragment. Exemplary methods for determining the ability of an Ab or its antigen-binding fragment to displace human Abs (e.g., one or more of IgG1, IgG2, and IgG4) bound to SpA in the cell wall of SA bacteria are described in the Examples section below. Additional methods for determining the ability of an Ab or its antigen-binding fragment to displace human Abs (e.g., one or more of IgG1, IgG2, and IgG4) bound to SpA in the cell wall of SA bacteria are known in the art.

[0022] The Ab can be, for example, a mAb, a multispecific Ab (e.g., a bispecific Ab), a chimeric Ab (e.g., a humanized Ab, e.g., a humanized IgG Ab), a human Ab, or a fragment of any of the above. For example, the Ab can be a human or humanized monoclonal IgG3 Ab. The Ab can also be a single-chain Ab (e.g., a single-domain Ab), such as a single-chain camelid or cartilaginous (e.g., shark) Ab or a single-chain Ab containing at least one camelid variable antigen-binding domain (VHH) or at least one cartilaginous (e.g., shark) immunoglobulin novel antigen receptor (Ig-NAR) domain (e.g., the Ab described in U.S. Patent Application Publication No. 2010 / 0092470). The Ab can be a whole Ab molecule or an Ab multimer.

[0023] The term "Ab" further includes Ab conjugates (e.g., Ab conjugated to a stabilizing protein, label, or therapeutic agent (e.g., any of the therapeutic agents described herein or known in the art)). For example, the Abs provided herein can include an Fc domain or portion of an Fc domain sufficient to mediate Ab-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) in a mammal (e.g., a human) and / or conjugated to a therapeutic agent (e.g., any of the therapeutic agents described herein or known in the art). The Ab can be, for example, human or humanized IgG1, IgG2, IgG4, IgD, IgA, IgE, or IgM, and is preferably human or humanized IgG3.

[0024] Antigen-binding fragments described herein can, for example, be sufficient to mediate Ab-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) in a mammal (e.g., a human) and / or can include at least a portion of an Fc domain conjugated to a therapeutic agent (e.g., any of the therapeutic agents described herein or known in the art). Non-limiting examples of Ab fragments include Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv fragments, fragments containing either the variable light or variable heavy chain domains, diabodies, linear Abs, and multispecific Abs formed from Ab fragments. Additional Ab fragments containing at least one camelid VHH domain or at least one cartilaginous fish Ig-NAR domain include minibodies, microAbs, subnano-Abs, and nano-Abs, as well as any of the other forms of Abs described in U.S. Patent Application Publication No. 2010 / 0092470.

[0025] The Abs or antigen-binding fragments thereof can be of any type (e.g., human or humanized IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., human or humanized IgG1 (e.g., IgG1a or IgG1b), IgG2 (e.g., IgG2a or IgG2b), IgG3 (e.g., IgG3a or IgG3b), IgG4 (e.g., IgG4a or IgG4b), IgA1 and IgA2), or subclass, but have low Fc binding affinity (e.g., 1×10) for SpA under physiological conditions (e.g., phosphate buffered saline) (e.g., as determined using surface plasmon resonance using recombinant SpA). -7 M, 1 x 10 -6 M, 1 x 10 -5 M, 1 x 10 -4 M or 1×10 -3 K is bigger than M D or the K of SpA for the Fc region of human IgG1 D Larger K DPreferred is an Ab or antigen-binding fragment thereof. The antigen-binding fragment may be, for example, an antigen-binding fragment of human or humanized IgG1 (e.g., IgG1a or IgG1b), IgG2 (e.g., IgG2a or IgG2b), IgG4 (e.g., IgG4a or IgG4b), IgD, IgA (e.g., IgA1 or IgA2), IgE, or humanized IgM, and is preferably a fragment of human or humanized IgG3 (e.g., IgG3a or IgG3b). Amino acid mutations can be introduced into the constant regions of these IgG subclasses. Amino acid mutations that can be introduced include those that enhance binding to Fc receptors (e.g., as described in Proc. Natl. Acad. Sci. USA 103(11):4005-4010, 2006; MAb 1(6):572-579, 2009; U.S. Patent Application Publication Nos. 2010 / 0196362; 2013 / 0108623; 2014 / 0171623; 2014 / 0093496; and 2014 / 0093959), or those that enhance binding to Fc receptors (e.g., as described in J. Biol. Chem. 276(9):6591-6604, 2001; Int. Immunol. 18(12):1759-1769, 2006; and J. Biol. Chem. 281(33):23514-23524, 2006).

[0026] The two types of H chains can be heterologously associated to generate bispecific Abs. Knobs-into-holes technology (e.g., as described in J. Immunol. Methods 248(1-2):7-15, 2001; and J. Biol. Chem. 285(27):20850-20859, 2010), electrostatic repulsion technology (e.g., as described in WO 06 / 106905), and SEEDbody technology (e.g., as described in Protein Eng. Des. Sel. 23(4):195-202, 2010) can be used to heterologously associate the two types of H chains via the CH3 domain. Any of the antibodies described herein may have modified or deleted glycans. Examples of antibodies with modified carbohydrate chains include glycosylated recombinant antibodies (e.g., as described in WO 99 / 54342), antibodies with defucosylated carbohydrate chains (e.g., as described in WO 00 / 61739, WO 02 / 31140, WO 06 / 067847, and WO 06 / 067913), and bisecting GlcNAc (e.g., as described in WO 02 / 79255). Known examples of methods for generating glycosylation-deficient IgG antibodies include introducing a mutation to asparagine at EU numbering position 297 in the heavy chain (J. Clin. Pharmacol. 50(5):494-506, 2010) and producing IgG using Escherichia coli (E. coli) (J. Immunol. Methods 263(1-2):133-147, 2002; and J. Biol. Chem. 285(27):20850-20859, 2010). Furthermore, heterologous deletion of the C-terminal lysine in IgG and heterologous mispairing of disulfide bonds in the hinge region of IgG2 can be reduced by introducing amino acid deletions / substitutions (e.g., as described in WO 09 / 041613).Any of the Abs or antigen-binding fragments described herein include at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid that is not present in the corresponding human Ab (e.g., an added, inserted, or substituted amino acid that is not present in a CDR). Any of the Abs or antigen-binding fragments described herein can further have at least one deleted amino acid (compared to the corresponding human Ab), such as a deletion from the N-terminus or C-terminus of the light or heavy chain, or a deletion of one amino acid from the constant region (e.g., the Fc region).

[0027] SpA or a fragment thereof (e.g., at least 7, 8, 9, or 10 contiguous amino acids of SEQ ID NO:1 (e.g., starting at amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of SEQ ID NO:1, or the entirety of SEQ ID NO:1) can be used as an immunogen to generate Abs using standard techniques for polyclonal and monoclonal Ab preparation. Ab fragments can be generated from monoclonal Abs using methods well known in the art.

[0028] An immunogen is typically used to prepare Abs by immunizing a suitable subject (e.g., rabbit, goat, mouse, or other mammal). An appropriate immunogen preparation can contain, for example, a recombinantly expressed or chemically synthesized polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0029] As an alternative to preparing monoclonal Ab-secreting hybridomas, monoclonal Abs directed against a polypeptide can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an Ab phage display library) that contains the polypeptide of interest. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP* Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly suitable for use in generating and screening Ab display libraries can be found in, e.g., U.S. Pat. No. 5,223,409; WO 92 / 18619; WO 91 / 17271; WO 92 / 2079; WO 92 / 15679; WO 93 / 01288; WO 92 / 01047; WO 92 / 09690; WO 90 / 02809; Fuchs et al., Bio / Technology 9:1370-1372, 1991; Hay et al., Hum. Antibod. Hybridomas 3:81-85, 1992; Huse et al., Science 246:1275-1281, 1989; Griffiths et al., EMBO J. 12:725-734, 1993.

[0030] The Examples section below describes additional methods for isolating and sequencing human Abs (e.g., human IgG3) that specifically bind to SpA epitopes (e.g., epitopes located within or defined by the polypeptide of SEQ ID NO: 1). Additional general methods for generating Abs and antigen-binding fragments are described in U.S. Patent Application Publication No. 2011 / 0059085.

[0031] In some embodiments, the Abs or antigen-binding fragments provided herein are human or humanized Abs (e.g., human or humanized IgG3 Abs). In some embodiments, the humanized Abs are human Abs that have been engineered to contain at least one complementarity-determining region (CDR) present in a non-human Ab (e.g., a rat, mouse, rabbit, or goat Ab). In some embodiments, one humanized Ab or fragment thereof can contain all three CDRs of the light chain of a human or non-human Ab that specifically binds to an SpA epitope (e.g., an epitope located within the polypeptide of SEQ ID NO: 1 or defined). In some embodiments, the humanized Ab or fragment thereof can contain all three CDRs of the heavy chain of a human or non-human Ab that specifically binds to an SpA epitope (e.g., an epitope located within the polypeptide of SEQ ID NO: 1 or defined). In some embodiments, the humanized Ab or fragment thereof can contain all three CDRs of the heavy chain and all three CDRs of the light chain of a humanized or human monoclonal Ab that specifically binds to an SpA epitope (e.g., an epitope located or defined within the polypeptide of SEQ ID NO: 1).

[0032] The Abs of the present invention can also include multimeric forms of Abs. For example, the Abs of the present invention can take the form of Ab dimers, trimers, or higher multimers of monomeric immunoglobulin molecules. Dimers of complete immunoglobulin molecules or F(ab')2 fragments are tetravalent, while dimers of Fab fragments or scFv molecules are bivalent. The individual monomers within an Ab multimer can be the same or different, i.e., they can be heteromeric or homomeric Ab multimers. For example, the individual Abs within a multimer can have the same or different binding specificities.

[0033] Ab multimerization can be achieved through natural Ab aggregation or through chemical or recombinant conjugation techniques known in the art. For example, a certain percentage of purified Ab preparations (e.g., purified IgG1 molecules) naturally form protein aggregates containing Ab homodimers and other higher-order Ab multimers. Alternatively, Ab homodimers may be formed through chemical conjugation techniques known in the art. To form Ab multimers, heterobifunctional crosslinkers can be used, including, but not limited to, SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acetylthioacetate) (available, for example, from Pierce Biotechnology, Inc., Rockford, IL). A typical protocol for forming Ab homodimers is set forth in Ghetie et al. (Proc. Natl. Acad. Sci. USA 94:7509-7514, 1997). Ab homodimers can be converted to Fab'2 homodimers through digestion with pepsin. Another method for forming Ab homodimers is by using the autophilic T15 peptide described by Zhao et al. (J. Immunol. 25:396-404, 2002).

[0034] Alternatively, Abs can be engineered to multimerize through recombinant DNA technology. IgM and IgA naturally form Ab multimers through interaction with mature J chain polypeptides. Non-IgA or non-IgM molecules, such as IgG molecules, can be engineered to contain the IgA or IgM J chain-interacting domain, thereby conferring on the non-IgA or non-IgM molecule the ability to form higher-order multimers (see, e.g., Chintalacharuvu et al., Clin. Immunol. 101:21-31, 2001 and Frigerio et al., Plant Physiol. 123:1483-1494, 2000). IgA dimers are naturally secreted into the lumen of mucosally-lined organs. This secretion is mediated through the interaction of the J chain with the polymeric IgA receptor (plgR) on epithelial cells. If secretion of the IgA form of an Ab (or an Ab engineered to contain a J-chain interacting domain) is undesirable, it can be greatly reduced by expressing the Ab molecule in association with a mutant J-chain that interacts less well with pIgR (Johansen et al., J. Immunol., 167:5185-192, 2001). ScFv dimers can also be formed by recombinant techniques known in the art. An example of the construction of scFv dimers is set forth in Goel et al. (Cancer Res. 60:6964-71, 2000). Ab multimers can be purified using any suitable method known in the art, including, but not limited to, size exclusion chromatography.

[0035] Any of the Abs or antigen-binding fragments described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the Ab or its antigen-binding fragment in a cat or solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as feline serum albumin). Any of the Abs or antigen-binding fragments described herein can be conjugated to a label (e.g., a fluorophore, radioisotope, or luminescent molecule) or a therapeutic agent (e.g., a cytotoxic drug or radioisotope). Exemplary methods for attaching labels or therapeutic agents to Abs are described in U.S. Patent Application Publication No. 2013 / 0224228. Non-limiting examples of cytotoxic agents include agents known to induce cell death in microorganisms (e.g., gram-positive bacteria, such as Staphylococcus aureus).Non-limiting examples of cytotoxic drugs that can be conjugated to any of the Abs or antigen-binding fragments thereof provided herein include linezolid, erythromycin, mupirocin, ertapenem, doripenem, imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cephalothin, cephalexin, ceflacor, cefamandole, cefoxitin, cefamantole, cephalosporin ... Prozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, televancin, clindamycin, lincomycin, daptomycin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin G, temocillin, ticarcillin, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin These include fluoxacin, temafloxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sufamethizole, sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole, sulfonamide chrysoidine, demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline.

[0036] For example, the Abs provided herein (e.g., human or humanized monoclonal IgG3) or antigen-binding fragments thereof (e.g., fragments of human or humanized monoclonal IgG3) that specifically bind to SpA include: (i) a heavy chain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 2, 3 and 4, respectively, and / or a light chain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 7, 8 and 9, respectively; (ii) a heavy chain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 12, 13 and 14, respectively, and / or a light chain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 17, 18 and 19, respectively; (iii) a heavy chain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 22, 23 and 24, respectively, and / or a light chain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 27, 28 and 29, respectively; (iv) a heavy chain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 32, 33 and 34, respectively, and / or a light chain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 37, 38 and 39, respectively; (v) a heavy chain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 42, 43 and 44, respectively, and / or a light chain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 47, 48 and 49, respectively; (vi) a heavy chain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 52, 53 and 54, respectively, and / or a light chain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 57, 58 and 59, respectively; (vii) a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 62, 63, and 64, respectively, and / or a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NOs: 67, 68, and 69, respectively; or (viii) may comprise a heavy chain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 72, 73 and 74, respectively, and / or a light chain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 77, 78 and 79, respectively.

[0037] In some examples, any of the Abs provided herein has an Ab heavy chain comprising SEQ ID NO:6 and / or a light chain comprising SEQ ID NO:11; an Ab heavy chain comprising SEQ ID NO:16 and / or a light chain comprising SEQ ID NO:21; an Ab heavy chain comprising SEQ ID NO:26 and / or a light chain comprising SEQ ID NO:31; an Ab heavy chain comprising SEQ ID NO:36 and / or a light chain comprising SEQ ID NO:41; an Ab heavy chain comprising SEQ ID NO:46 and / or a light chain comprising SEQ ID NO:51; an Ab heavy chain comprising SEQ ID NO:56 and / or a light chain comprising SEQ ID NO:61; an Ab heavy chain comprising SEQ ID NO:66 and / or a light chain comprising SEQ ID NO:71; or an Ab heavy chain comprising SEQ ID NO:76 and / or a light chain comprising SEQ ID NO:81.

[0038] In additional examples provided herein, an Ab (e.g., a human or humanized IgG3) or antigen-binding fragment (e.g., an antigen-binding fragment of a human or humanized IgG3) may be administered at a concentration of 1×10 -10 Less than M (e.g., 1 × 10 -11 Less than M or 1 x 10 -12 K (less than M) D can bind to SpA and / or can replace a human Ab (e.g., one or more of IgG1, IgG, and IgG4) that binds to SpA, wherein the antigen or antigen-binding fragment comprises a set of six CDRs: (i) SEQ ID NOs: 2, 3, 4, 7, 8 and 9; (ii) SEQ ID NOs: 12, 13, 14, 17, 18 and 19; (iii) SEQ ID NOs: 22, 23, 24, 27, 28 and 29; (iv) SEQ ID NOs: 32, 33, 34, 37, 38 and 39; (v) SEQ ID NOs: 42, 43, 44, 47, 48 and 49; (vi) SEQ ID NOs: 52, 53, 54, 57, 58 and 59; (vii) SEQ ID NOs: 62, 63, 64, 67, 68, and 69; or (viii) SEQ ID NOs: 72, 73, 74, 77, 78, and 79 The set of six CDRs has no more than 1, 2, 3, 4, 5, or 6 total amino acid substitutions (e.g., conservative amino acid substitutions) within the set of six CDRs (total set) selected from the group consisting of:

[0039] For example, an Ab (e.g., a human or humanized IgG3) or antigen-binding fragment (e.g., an antigen-binding fragment of a human or humanized IgG3) provided herein can comprise a set of six CDRs with one, two, three, or no more than four total amino acid substitutions within the set of six CDRs (the entire set) of SEQ ID NOs: 2, 3, 4, 7, 8, and 9. For example, an Ab (e.g., a human or humanized IgG3) or antigen-binding fragment (e.g., an antigen-binding fragment of a human or humanized IgG3) provided herein can comprise: (i) a set of six CDRs of SEQ ID NOs: 2, 3, 4, 7, 8 and 9; (ii) a set of six CDRs of SEQ ID NOs: 12, 13, 14, 17, 18 and 19; (iii) a set of six CDRs of SEQ ID NOs: 22, 23, 24, 27, 28 and 29; (iv) a set of six CDRs of SEQ ID NOs: 32, 33, 34, 37, 38 and 39; (v) a set of six CDRs of SEQ ID NOs: 42, 43, 44, 47, 48 and 49; (vi) a set of six CDRs of SEQ ID NOs: 52, 53, 54, 57, 58 and 59; (vii) a set of six CDRs of SEQ ID NOs: 62, 63, 64, 67, 68, and 69; or (viii) a set of six CDRs of SEQ ID NOs: 72, 73, 74, 77, 78, and 79 It may comprise or consist of:

[0040] In additional examples, the Abs (e.g., human or humanized monoclonal IgG3) or antigen-binding fragments (e.g., antigen-binding fragments of human or humanized IgG3) provided herein that specifically bind to SpA include: (i) a variable domain comprising or consisting of SEQ ID NO:5; (ii) a variable domain comprising or consisting of SEQ ID NO:10; (iii) a variable domain comprising or consisting of SEQ ID NO:15; (iv) a variable domain comprising or consisting of SEQ ID NO:20; (v) a variable domain comprising or consisting of SEQ ID NO:25; (vi) a variable domain comprising or consisting of SEQ ID NO:30; (vii) a variable domain comprising or consisting of SEQ ID NO:35; (xiii) a variable domain comprising or consisting of SEQ ID NO:65; (xiv) a variable domain comprising or consisting of SEQ ID NO:70; (xv) a variable domain comprising or consisting of SEQ ID NO:75; or (xvi) a variable domain comprising or consisting of SEQ ID NO:80.For example, an Ab (e.g., a human or humanized monoclonal IgG3) or antigen-binding fragment (e.g., an antigen-binding fragment of a human or humanized IgG3) can have: (i) a variable domain comprising or consisting of SEQ ID NO:5 and / or a variable domain comprising or consisting of SEQ ID NO:10; (ii) a variable domain comprising or consisting of SEQ ID NO:15 and / or a variable domain comprising or consisting of SEQ ID NO:20; (iii) a variable domain comprising or consisting of SEQ ID NO:25 and / or a variable domain comprising or consisting of SEQ ID NO:30; (iv) a variable domain comprising or consisting of SEQ ID NO:35 and / or a variable domain comprising or consisting of SEQ ID NO:35. (v) a variable domain comprising or consisting of SEQ ID NO:45 and / or a variable domain comprising or consisting of SEQ ID NO:50; (vi) a variable domain comprising or consisting of SEQ ID NO:55 and / or a variable domain comprising or consisting of SEQ ID NO:60; (vii) a variable domain comprising or consisting of SEQ ID NO:65 and / or a variable domain comprising or consisting of SEQ ID NO:70; or a variable domain comprising or consisting of SEQ ID NO:75 and / or a variable domain comprising or consisting of SEQ ID NO:80.

[0041] Some embodiments of any of the Abs (e.g., human or humanized monoclonal IgG3) or antigen-binding fragments (e.g., antigen-binding fragments of human or humanized IgG3) described herein specifically bind to SpA in strains of MRSA; specifically bind to the epitope defined by SEQ ID NO: 1; or bind to SpA at a concentration of 1×10 -10 Less than M (e.g., 1 × 10 -11 Less than M or 1 x 10 -12 (less than) K Dand the ability to displace human Abs bound to SpA in the cell wall of Staphylococcus aureus bacteria (e.g., MRSA bacteria).

[0042] Pharmaceutical Composition Provided herein are pharmaceutical compositions containing at least one pharmaceutically acceptable carrier (e.g., a non-naturally occurring pharmaceutically acceptable carrier) and at least one (e.g., two, three, or four) of any of the Abs or antigen-binding fragments thereof provided herein. Non-limiting examples of pharmaceutically acceptable carriers include sterile water, physiological saline, stabilizers, excipients, antioxidants (e.g., ascorbic acid), buffers (e.g., phosphate, citrate, histidine, and other organic acids), preservatives, surfactants (e.g., PEG and Tween), chelating agents (e.g., EDTA or EGTA), and binders. Additional examples of pharmaceutically acceptable carriers also include low molecular weight polypeptides, proteins (e.g., serum albumin and gelatin), amino acids (e.g., glycine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, arginine, and lysine), sugars and carbohydrates (e.g., polysaccharides and monosaccharides), and sugar alcohols (e.g., mannitol and sorbitol).When preparing an injectable aqueous solution, physiological saline and isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used in combination with suitable solubilizers, such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol and PEG), and nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer 188, and HCO-50). Larger fluid volumes can be administered subcutaneously by incorporating hyaluronidase into the preparation (see, e.g., Expert. Opin. Drug. Deliv. 4(4):427-440, 2007).

[0043] The Abs and antigen-binding fragments provided herein can be, for example, encapsulated within microcapsules (made, for example, from hydroxymethylcellulose, gelatin, and poly(methyl methacrylate)) or incorporated as components of colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, for example, "Remington's Pharmaceutical Science 16th edition," Oslo Ed. (1980)). Methods for preparing pharmaceutical compositions as controlled-release pharmaceutical formulations are also well known, and such methods can be applied to the Abs and antigen-binding fragments of the present invention (see, e.g., Langer et al., J. Biomed. Mater. Res. 15:267-277, 1981; Langer, Chemtech. 12:98-105, 1982; U.S. Pat. No. 3,773,919; EP 58,481; Sidman et al., Biopolymers 22:547-556, 1983; and EP 133,988).

[0044] The pharmaceutical compositions provided herein can be formulated for intravenous, intraarterial, intradermal, subcutaneous, intramuscular, intraperitoneal or oral administration.

[0045] The dosage of the pharmaceutical composition of the present invention can be appropriately determined by considering the dosage form, administration method, patient age and weight, patient symptoms, and the severity of SA infection or risk level of SA infection. Generally, the daily dose for an adult may be, for example, 0.1 mg to 10,000 mg, administered in a single dose or in divided doses. The dosage may be, for example, 0.2 to 10,000 mg / day (e.g., 1 to 10 g / day, 2 to 8 g / day, 1 to 5 g / day, 0.5 to 2.5 g / day, 0.5 to 500 mg / day, 1 to 300 mg / day, 3 to 100 mg / day, or 5 to 50 mg / day). These dosages may vary depending on the patient's weight and age and the administration method. However, selecting an appropriate dosage is clearly within the knowledge of those skilled in the art. Similarly, the administration period can be appropriately determined depending on the progress of treatment.

[0046] Any of the pharmaceutical compositions provided herein can include one or more additional antibacterial agents. Non-limiting examples of such antibacterial agents include linezolid, erythromycin, mupirocin, ertapenem, doripenem, imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalothin, cephalexin, cefracor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefamantha Foperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telvancin, clindamycin, lincomycin, daptomycin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, Nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin G, temocillin, ticarcillin, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, te These include mafloxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole, sulfonamide chrysoidine, demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline.

[0047] Methods of treating a subject with a Staphylococcus aureus (S. aureus) infection or reducing the risk of developing a Staphylococcus aureus (S. aureus) infection in a subject Further provided are methods of treating a subject having an SA infection (MRSA infection, SA bacteremia, SA skin infection, SA mastitis, SA cellulitis or folliculitis, or SA-associated wound infection, abscess, osteomyelitis, endocarditis, pneumonia, septic shock, food poisoning, toxic shock syndrome), comprising administering to a subject in need thereof (e.g., a human or other mammal, such as a cow, sheep, dog, cat, horse, goat, rabbit, pig, or bird) a therapeutically effective amount of at least one of any of the pharmaceutical compositions provided herein or at least one of any of the Abs or antigen-binding fragments thereof provided herein. In some examples, the subject has been diagnosed or identified as having an SA infection (e.g., an MRSA infection). Some embodiments can further comprise (prior to the administering step) diagnosing, identifying, or selecting a subject having an SA infection (e.g., an MRSA or VRSA infection), or diagnosing, identifying, or selecting a subject as having an SA infection (e.g., an MRSA or VRSA infection). In some examples, the SA infection is a hospital-acquired infection. In some cases, the subject has previously been treated with an antibacterial therapy, and the previous treatment was unsuccessful.

[0048] Further provided are methods for reducing a subject's risk of developing an SA infection (e.g., an MRSA infection), comprising administering to the subject an effective amount of at least one of any of the pharmaceutical compositions provided herein or at least one of any of the Abs or antigen-binding fragments thereof provided herein. In some embodiments, the SA infection is a hospital-acquired infection. Some embodiments further comprise the step of selecting or identifying the subject as having an increased risk of developing an SA infection (e.g., an MRSA infection) prior to the administering step. For example, the subject may be a healthcare worker (e.g., a doctor, nurse, laboratory technician, or physician's assistant) (e.g., a healthcare worker who comes into physical contact with a subject with an SA infection (e.g., an MRSA infection)). The subject in these methods may be a subject admitted to hospital or inpatient treatment (e.g., a nursing home) that further includes (accommodates) at least one other subject with an SA infection (e.g., an MRSA infection). Subjects may be hospitalized patients, such as patients in intensive care units, immunocompromised patients and patients undergoing or about to undergo a surgical procedure.

[0049] In any of the methods provided herein, the subject can be male or female. For example, the subject can be an infant, a toddler, an adolescent, a teenager or an adult (e.g., at least 18 years old, at least 20 years old, at least 25 years old, at least 30 years old, at least 35 years old, at least 40 years old, at least 45 years old, at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, at least 75 years old, at least 80 years old, at least 85 years old, at least 90 years old, at least 95 years old or at least 100 years old). In some cases, the subject has a suppressed or reduced immune system (e.g., humoral or cellular immune system).

[0050] In some examples, at least one pharmaceutical composition provided herein or at least one Ab or antigen-binding fragment provided herein is administered intravenously, intraarterially, intradermally, subcutaneously, intramuscularly, intraperitoneally, or orally. For example, in a method for reducing the risk of developing an SA infection, a subject is administered at least one pharmaceutical composition provided herein or at least one Ab or antigen-binding fragment thereof provided herein before or shortly after physical contact with a subject who has been identified, diagnosed, has, or is suspected of having an SA infection (e.g., an MRSA infection).

[0051] In any of the methods described herein, the subject is administered at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) dose of at least one of the pharmaceutical compositions provided herein or at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) dose of any of the Abs or antigen-binding fragments provided herein. The subject can be administered at least two doses of two or more doses of any of the pharmaceutical compositions or any of the Abs or antigen-binding fragments provided herein at a frequency of at least once per month (e.g., at least twice per month, at least three times per month, at least four times per month, at least once per week, at least twice per week, at least three times per week, at least four times per week, at least five times per week, at least once per day, at least twice per day, or at least three times per day).

[0052] Some embodiments further comprise co-administering to the subject an Ab described herein and one or more additional antibacterial agents. Non-limiting examples of such antibacterial agents include linezolid, erythromycin, mupirocin, ertapenem, doripenem, imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalothin, cephalexin, cefracor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefamantha Foperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telvancin, clindamycin, lincomycin, daptomycin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, Nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin G, temocillin, ticarcillin, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, te These include mafloxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole, sulfonamide chrysoidine, demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline. Additional examples of therapeutic agents that can be included in any of the pharmaceutical compositions provided herein are one or more of the Abs described in U.S. Patent Application Publication No. 2011 / 0059085.

[0053] kit Further provided herein are kits containing at least one (e.g., two, three, four, or five) of any of the Abs or antigen-binding fragments provided herein. In some examples, the kits can contain recombinant SpA or peptides comprising or consisting of SEQ ID NO: 1 or antigenic fragments of SEQ ID NO: 1 (e.g., at least seven contiguous amino acids of SEQ ID NO: 1 (e.g., starting at amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of SEQ ID NO: 1)). In some examples, at least one Ab or antigen-binding fragment is attached to a solid substrate (e.g., a well, chip, film, bead, or chromatography resin). Such kits can include commercial packaging and / or printed information about the Abs and how to use them. [Example]

[0054] Example 1. Generation of human Abs that specifically bind SpA and displace human IgG immunoglobulins bound to SpA via their Fc region Human IgG3 Abs that bind to SpA epitopes were generated as described below. Five synthetic peptides spanning the IgG-binding sequence and Xr repeat sequence within SpA were used to screen anti-peptide Abs in the blood of 311 healthy adult volunteers. The five SpA-derived synthetic peptides used for screening had sequences designated SEQ ID NOs: 82, 83, 84, 85, and 1 (peptides 1, 2, 3, 4, and 5, respectively). Approximately 4% of healthy subjects had levels of anti-peptide Abs (anti-SpA) greater than 10-fold higher than background levels determined using enzyme-linked immunosorbent assay (ELISA) (hereafter referred to as "positive donors"). Plasma from these positive donors was obtained and used to isolate fully human Abs that specifically bind to peptides spanning the IgG-binding sequence and Xr repeat sequence of SpA using the method described in U.S. Patent Application Publication No. 2013 / 0018173. In summary, Abs of interest were isolated using antigen affinity chromatography and further de novo sequenced using mass spectrometry. Concurrently, Abs were isotyped using a human isotyping kit.

[0055] One of the isolated Abs was identified as belonging to the VH3 subfamily and possessing an IgG2 heavy chain and a VK1 light chain. B cells were isolated from donor blood using a kit obtained from STEMCELL Technologies, Inc. Their RNA was extracted using the Trizol extraction protocol, and cDNA was generated using SuperScript III. Leader-specific primers were used to amplify the corresponding heavy and light chains of the Abs, generating a "directed" ScFv library. The library was panned against wild-type SpA antigen for seven rounds. Clones were screened using direct and sandwich ELISAs with wild-type SpA. Selected clones were sequenced, and the heavy and light chains were cloned into a vector containing the IgG3 constant (Fc) region (a region lacking the SpA recognition site within the Fab region). The vector was transfected into a CHO cell line, and high-producing clones were selected. The purified Ab was tested for high SpA activity. The clones were expanded for large-scale production, and the produced Abs were purified and used for subsequent analysis. Examples of eight such Abs are listed below. PA8-G3 Ab Heavy chain variable domain of SEQ ID NO:5. Heavy chain CDR1, 2 and 3 of SEQ ID NOs: 2, 3 and 4, respectively. Heavy chain of SEQ ID NO:6. Light chain variable domain of SEQ ID NO: 10. Light chain CDR1, 2 and 3 of SEQ ID NOs: 7, 8 and 9, respectively. Light chain of SEQ ID NO: 11. PA4-G3 Ab Heavy chain variable domain of SEQ ID NO: 15. Heavy chain CDR1, 2 and 3 of SEQ ID NOs: 12, 13 and 14, respectively. Heavy chain of SEQ ID NO: 16. Light chain variable domain of SEQ ID NO: 20. Light chain CDR1, 2 and 3 of SEQ ID NOs: 17, 18 and 19, respectively. Light chain of SEQ ID NO:21. PA7.2-G3 Ab Heavy chain variable domain of SEQ ID NO: 25. Heavy chain CDR1, 2 and 3 of SEQ ID NOs: 22, 23 and 24, respectively. Heavy chain of SEQ ID NO:26. Light chain variable domain of SEQ ID NO: 30. Light chain CDR1, 2 and 3 of SEQ ID NOs: 27, 28 and 29, respectively. Light chain of SEQ ID NO: 31. PA15-G3 Ab The heavy chain variable domain of SEQ ID NO: 35. Heavy chain CDR1, 2 and 3 of SEQ ID NOs: 32, 33 and 34, respectively. Heavy chain of SEQ ID NO: 36. Light chain variable domain of SEQ ID NO: 40. Light chain CDR1, 2 and 3 of SEQ ID NOs: 37, 38 and 39, respectively. Light chain of SEQ ID NO: 41. PA21-G3 Ab Heavy chain variable domain of SEQ ID NO: 45. Heavy chain CDR1, 2 and 3 of SEQ ID NOs: 42, 43 and 44, respectively. Heavy chain of SEQ ID NO:46. Light chain variable domain of SEQ ID NO: 50. Light chain CDR1, 2 and 3 of SEQ ID NOs: 47, 48 and 49, respectively. Light chain of SEQ ID NO: 51. PA27-G3 Ab Heavy chain variable domain of SEQ ID NO: 55. Heavy chain CDR1, 2 and 3 of SEQ ID NOs: 52, 53 and 54, respectively. Heavy chain of SEQ ID NO:56. Light chain variable domain of SEQ ID NO: 60. Light chain CDR1, 2 and 3 of SEQ ID NOs: 57, 58 and 59, respectively. Light chain of SEQ ID NO: 61. PA32-G3 Ab Heavy chain variable domain of SEQ ID NO: 65. Heavy chain CDR1, 2 and 3 of SEQ ID NOs: 62, 63 and 64, respectively. Heavy chain of SEQ ID NO: 66. Light chain variable domain of SEQ ID NO: 70. Light chain CDR1, 2 and 3 of SEQ ID NOs: 67, 68 and 69, respectively. Light chain of SEQ ID NO: 71. PA37-G3 Ab The heavy chain variable domain of SEQ ID NO: 75. Heavy chain CDR1, 2 and 3 of SEQ ID NOs: 72, 73 and 74, respectively. Heavy chain of SEQ ID NO: 76. Light chain variable domain of SEQ ID NO: 80. Light chain CDR1, 2 and 3 of SEQ ID NOs: 77, 78 and 79, respectively. Light chain of SEQ ID NO: 81.

[0056] A set of experiments was performed to determine whether the PA8-G3 Ab could bind to SpA on the cell wall of SA. In these experiments, SA stain ATCC #25923 or clinical isolate 00X was incubated with either (i) biotinylated PA8-G3 followed by streptavidin-APC (Figure 2) to fluorometrically quantify the amount of biotin-PA-G3 bound to the SA surface, or (ii) purified unlabeled PA8-G3 Ab followed by biotinylated recombinant Fcγ receptor 1 and then streptavidin-APC (Figure 3) to fluorometrically quantify the amount of PA8-G3 bound to the SA surface, which would result in phagocytosis. Anti-interleukin-1a Ab (MABp1) was used as a negative control in these experiments. The data in Figure 2 show that PA8-G3 binds to SpA within the cell wall of SA, and the data in Figure 3 show that the bound PA8-G3 Ab has its Fc region available to interact with FcR, suggesting that the Ab can mediate opsinophagocytosis of SA in human subjects (as opposed to Abs that are bound to SpA and have Fc regions that cannot engage FcR and mediate bacterial opsinophagocytosis).

[0057] Another set of experiments was performed to test whether binding of PA8-G3 Ab to the surface of SA was recognized by Fcγ receptors on phagocytes. In these experiments, two different strains of pH-loaded green-labeled Staphylococcus aureus (S. aureus) (clinical isolate 00X or ATCC #25923) were incubated with either unlabeled PA8-G3 Ab or a control Ab (MABp1) and then incubated with differentiated HL-60 cells. The resulting fluorescence of HL-60 cells was determined using fluorescence-assisted cell sorting (FACS). The data indicate that PA8-G3 binds to the cell wall of both SA strains and mediates phagocytosis through Fcγ receptors on the surface of HL-60 cells (Figure 4). Successful phagocytosis of PA8-G3-bound S. aureus by differentiated HL-60 cells was also evident from fluorescence microscopy experiments.

[0058] Surface plasmon resonance was used to determine the binding kinetics of PA8-G3 to SpA. In these experiments, PA8-G3 Ab was immobilized using an anti-human capture sensor and commercial wild-type SpA. These data show that PA8-G3 has a K of 5.38 pM. D This affinity is approximately 1,000-fold higher than the nanomolar affinity of human serum IgG1, IgG2, and IgG4 for SpA.

[0059] An additional set of experiments was performed to determine whether the PA8-G3 Ab could successfully compete with human IgG bound to SpA through their Fc receptors for binding to SpA. In these experiments, two different S. aureus strains were incubated with human serum (containing high concentrations of Ig that bind to SpA through their Fc regions) for 15 min, followed by incubation with biotinylated PA8-G3 Ab or biotinylated MABp1-IgG3 Ab (an isotype-adapted negative control), then treated with streptavidin-APC, and fluorescence was determined by flow cytometry. The data show that the PA8-G3 Ab was able to bind to SpA with human IgG Abs bound to SpA through their Fc regions (Figure 5).

[0060] In another set of experiments, the PA8-G3 antibody was shown to compete with the binding of MABp1-IgG1 (which binds to SpA via its Fc region) to SpA-coated beads. Preincubation of SpA beads with PA8-G3 reduced the binding of subsequently added MABp1-IgG1 by 80.3%. Conversely, when SpA beads were preincubated with MABp1-IgG1, subsequently added PA8-G3 bound to more than 30% of the SpA bead surface within 15 minutes, whereas subsequently added MABp1-IgG3 (an isotype-adapted negative control with MABp1 Fab and human IgG3 Fc) did not significantly bind to SpA beads preincubated with MABp1-IgG1.

[0061] An additional set of experiments was performed to test the ability of additional anti-SpA Abs to promote the phagocytosis of SA by differentiated HL-60 cells. In these experiments, differentiated HL-60 cells were co-incubated with pH-loaded green-labeled S. aureus and one of the following Abs: PA7.2-G3, PA4-G3, PA8-G3, PA15-G3, PA21-G3, PA27-G3, PA32-G3, PA37-G3, or MABp1. MABp1 was used as a negative control in these experiments. The data demonstrate that all of the tested anti-SpA Abs were able to promote opsinization and phagocytosis of S. aureus by differentiated HL-60 cells (Figure 6).

[0062] K of seven additional anti-SpA Abs D Additional biolayer interferometry (ForteBio Octet Red 96 instrument) experiments were performed to determine the .01% ... -12 K less than M D and PA4-G3 has 5.38 x 10 -12 K of M D and PA15-G3 has 1×10 -12 K less than M D and PA21-G3 has 1 × 10 -12 K less than M D and PA27-G3 has 1×10 -12 K less than M D and PA32-G3 has 1×10 -12 K less than M D and PA37-G3 has 1 x 10 -12 K less than M D It was shown that

[0063] Taken together, the data demonstrate that the Abs provided herein can bind with very high affinity to SpA within the cell wall of SA and promote phagocytosis by immune cells, and can do so in the presence of human IgG bound to SpA via their Fc region.

[0064] In Vivo Survival Study of Monoclonal Antibody PA8 in a Mouse Bacteremia / Sepsis Model: Survival of mice from S. aureus bacteremia was studied using a prophylactic dose of PA8 (a monoclonal antibody designated PA8-G3, described in Example 1).

[0065] Female Balb / C mice (6-8 weeks old) were purchased from Charles River Laboratory, NIH, Maryland. Upon arrival, mice were lab-housed and group-housed (10 mice / cage) in cages with absorbent bedding. All mice were maintained under the required husbandry standards found in the NIH Guide for the Care and Use of Laboratory Animals.

[0066] The protective effect of PA8 is 2 x 10 7 The SA sepsis model was investigated, induced by intravenous injection (iv) of 100 CFU of MRSA strain NR-46223. Mice were treated intravenously with PA8 at a specific dose (5 mg or 10 mg) or two 5 mg doses 3 hours before MRSA infection on days 0 and 3, respectively. Control mice were treated with formulation buffer alone. Mice were followed for 10 days (twice daily), at which point all remaining mice were sacrificed.

[0067] Three hours after intravenous administration of PA8 / preparation buffer (0.1 mL), mice were infected with Staphylococcus aureus strain NR-46223 (2 × 10 in 0.1 mL). 7 Mice were challenged with a single intravenous (IV) injection of 2 × 10 CFU of methicillin-resistant Staphylococcus aureus (MRSA) strain NR-46223. One set of mice received two doses of 5 mg on days 0 and 3, respectively. Significant differences in relative survival were detected between treatment groups. Referring to Figures 7A-D, a single dose of 5 mg (A) or 10 mg (B) of mAb PA8, or passive administration (intravenous) of two doses of 5 mg on days 0 and 3, significantly reduced survival times of 2 × 10 CFU of MRSA strain NR-46223.7 The survival rate of BALB / c mice with Staphylococcus aureus sepsis (induced by intravenous injection of 10 colony-forming units) was significantly higher than that of formulation buffer treatment in a dose-dependent manner (10 mice per group). Section (D) shows the survival rates using all different treatments on one graph. 10% (1 / 10) of mice receiving formulation buffer (1 / 10) survived bacterial challenge with S. aureus NR-46223, compared to 50% (5 / 10) of mice receiving 5 mg of mAb PA8 (p=0.016), 60% (p=0.09) of mice receiving two doses of 5 mg each, and 70% (7 / 10) of mice receiving 10 mg of mAb PA8 (p=0.003). Statistical analysis of the animal study data was performed using Kaplan-Meier survival analysis with the Mantel-Cox (log-rank) test. These results clearly demonstrate that PA8 provides a significant level of protection against lethal infection with S. aureus MRSA strains.

[0068] Example 3. Female Balb / C mice (10 per group) from Charles River Laboratories were administered 0.5 mg of vancomycin via the intraperitoneal route along with different suboptimal doses of PA8-G3 (0 mg, 2.5 mg, and 5 mg via the intravenous route) against MRSA (3 x 10 7CFU (iv) of PA8-G3 were injected 2 hours before infection with NR 46223. Mice were observed for 14 days. See Figure 8A-C. On day 14, only 10% of PBS-treated mice survived, while 30% of vancomycin-treated mice survived. However, when 2.5 mg of PA8-G3 was injected together with vancomycin treatment, 60% of animals survived (p=0.027). When 5 mg of PA8-G3 was injected together with vancomycin, 60% of animals survived, and those that died survived longer than the low-dose group (p=0.016). These data indicate that a low-effective dose of PA8-G3 can rescue animals from SA-mediated bacteremia when co-treated with a suboptimal dose of vancomycin. Statistical analysis of the animal experiment data was performed using Kaplan-Meier survival analysis with the Mantel-Cox (log-rank) test.

[0069] Other embodiments While the present invention has been described in conjunction with its detailed description, the above description is illustrative and is not intended to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A pharmaceutical composition for treating a Staphylococcus aureus infection or reducing the risk of developing a Staphylococcus aureus infection in a subject, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and a purified human or humanized IgG3 monoclonal antibody, the purified human or humanized IgG3 monoclonal antibody being administered via a Fab region paratope at a concentration of 1×10 －１０ K less than M Ｄ a purified human or humanized IgG3 monoclonal antibody that specifically binds to Staphylococcus aureus protein A (SpA) and has an Fc region that is capable of interacting with an Fc receptor when the monoclonal antibody binds to SpA via its Fab region paratope; A pharmaceutical composition, characterized in that said monoclonal antibody is capable of displacing human IgG immunoglobulins bound via their Fc region to SpA on Staphylococcus aureus bacteria.

2. 2. The pharmaceutical composition of claim 1, wherein the monoclonal antibody is capable of mediating opsonization of SpA-expressing Staphylococcus aureus bacteria in the presence of at least 1 mg / mL of IgG immunoglobulins that bind to SpA via their Fc region.

3. 2. The pharmaceutical composition of claim 1, wherein the Staphylococcus aureus infection is bacteremia.

4. 2. The pharmaceutical composition of claim 1, wherein the Staphylococcus aureus infection is caused by methicillin-resistant Staphylococcus aureus.

5. 2. The pharmaceutical composition of claim 1, wherein the monoclonal antibody is administered intravenously to a subject.

6. 10. The pharmaceutical composition of claim 1, wherein vancomycin is administered to a subject in conjunction with the monoclonal antibody.

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