WO2016073860A1

WO2016073860A1 - Binding molecules specific for staphylococcus protein a and uses thereof

Info

Publication number: WO2016073860A1
Application number: PCT/US2015/059480
Authority: WO
Inventors: Randall MACGILL; Peter Pavlik; Vaheh Oganesyan; Nancy Ulbrandt; Bret SELLMAN
Original assignee: Medimmune, Llc
Priority date: 2014-11-06
Filing date: 2015-11-06
Publication date: 2016-05-12

Abstract

This disclosure provides Staphylococcus aureus Protein A (SpA) binding molecules, e.g., anti-SpA antibodies and antigen-binding fragments thereof. In certain aspects, the anti-SpA antibodies and fragments thereof can be hybridoma-derived murine monoclonal antibodies, and humanized versions thereof. In certain aspects, the binding molecules provided herein, e.g., anti-SpA antibodies and antigen-binding fragments thereof, inhibit, suppress, or antagonize SpA activity. In addition, this disclosure provides SpA mutant proteins that can be used to elicit an effective vaccine response. Finally, this disclosure provides compositions and methods for diagnosing and treating diseases or disorders associated with a Staphylococcus infection.

Description

BINDING MOLECULES SPECIFIC FOR

STAPHYLOCOCCUS PROTEIN A AND USES THEREOF

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 5, 2015, is named SPA-100WOl_SL.txt and is 48 kilobytes in size.

BACKGROUND

[0002] This disclosure provides compositions that specifically bind to Staphylococcus Protein A (SpA) e.g., S. aureus Protein A, and methods for the use of such compositions, e.g., for the treatment or prevention of a condition or disease associated with a Staphylococcus infection.

[0003] Staphylococcus aureus is a gram-positive, facultatively aerobic, clump-forming cocci bacterium that commonly colonizes the nose and skin of healthy humans. Approximately 20-30% of the population is colonized with S. aureus at any given time. Staphylococcus aureus bacteria, sometimes also referred to as "staph", "Staph, aureus", or "S. aureus", are considered opportunistic pathogens that cause minor infections (e.g., pimples, boils) and systemic infections.

[0004] Mucosal and epidermal barriers (skin) normally protect against S. aureus infections.

Interruption of these natural barriers as a result of injuries (e.g., burns, trauma, surgical procedures and the like) dramatically increases the risk of infection. Diseases that compromise the immune system (e.g., diabetes, end-stage renal disease, cancer and the like) also increase the risk of infection. Opportunistic S. aureus infections can become serious, causing a variety of diseases or conditions, non-limiting examples of which include bacteremia, cellulitis, eyelid infections, food poisoning, joint infections, skin infections, scalded skin syndrome, toxic shock syndrome, pneumonia, osteomyelitis, endocarditis, meningitis and abscess formation. S. aureus also can cause infection and disease in animals. For example, S. aureus frequently is associated with bovine mastitis.

[0005] S. aureus expresses a number of virulence factors, including capsular polysaccharides and protein toxins. One major virulence factor often associated with S. aureus infection is Protein A (or SpA), which is a 56 kDa surface protein originally found in the cell wall of S. aureus. SpA is expressed by most strains (> 90%) of S. aureus (Fosgren, A., et al., 1970. Infect Immun 2: 672-73; Kronvall, G., et al., 1971. Infect Immun 3: 10-15; Peacock, S.J., et al., 2002. Infect Immun 70: 4987-96). SpA is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolality, and a two-component system called ArlS-ArlR. SpA is composed of five homologous IgG/VH3 binding domains (A-E); each domain contains a triple a helical bundle plus a C-terminal variable X region containing the cell wall anchor (Kim, H.K., et al., 2010. J Ex Med 207: 1863-1870). SpA, being a virulence factor, enables S. aureus to evade innate and adaptive immune responses (Cheng et al., 2009; Kim et al., 2011). SpA binds to the Fey portion of human and animal immunoglobulins, a defense mechanism that provides S. aureus with protection from opsonophagocytic killing (Forsgren A., 1970). Further, SpA associates with the Fab portion of VH3- type Fabs; the cross-linking of B cell receptors leads to the activation and clonal expansion of B cells and their subsequent apoptotic collapse, a mechanism that suppresses adaptive immune responses during staphylococcal infection (Goodyear and Silverman, 2003).

[0006] There was emergence of multidrug-resistant strains, methicillin-resistant S. aureus (MRSA), which made the therapy of both hospital- and community- acquired infections challenging (Klevens et al., 2008). S. aureus infection in humans does not lead to protective immunity, and up to 30% of skin and soft tissue infections reoccur even with antibiotic or surgical therapies (Lowy, 1998). Thus, there remains a need to find effective treatments for staphylococcal infection.

BRIEF SUMMARY

[0007] The disclosure provides Staphylococcus aureus protein A (SpA) binding molecules, e.g. , anti-SpA antibodies or antigen-binding fragments thereof, e.g. , monoclonal antibodies capable of inhibiting interaction of SpA and its ligands, and inhibiting SpA activities.

[0008] The disclosure provides an isolated binding molecule or antigen-binding fragment thereof that specifically binds to an epitope of Staphylococcus aureus protein A (SpA). In certain aspects, the binding molecule or fragment thereof can specifically bind to the same SpA epitope as an antibody or antigen-binding fragment thereof that contains a heavy chain variable region (VH) and light chain variable region (VL) with the amino acid sequences SEQ ID NO: 2 and 4, SEQ ID NO: 6 and 8, SEQ ID NO: 10 and 12, SEQ ID NO: 14 and 16, SEQ ID NO: 18 and 20, SEQ ID NO: 22 and 24, or SEQ ID NO: 26 and 28, respectively. In certain aspects the binding molecule or fragment thereof can competitively inhibit SpA binding by an antibody or antigen-binding fragment thereof that contains a heavy chain variable region (VH) and light chain variable region (VL) with the amino acid sequences SEQ ID NO: 2 and 4, SEQ ID NO: 6 and 8, SEQ ID NO: 10 and 12, SEQ ID NO: 14 and 16, SEQ ID NO: 18 and 20, SEQ ID NO: 22 and 24, or SEQ ID NO: 26 and 28, respectively.

[0009] In certain aspects the binding molecule or fragment thereof includes a VL complementarity determining region 1 (VL-CDR1), a VL-CDR2, and a VL-CDR3, where the VL-CDRs contain amino acid sequences identical to, or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more of the VL-CDRs to: SEQ ID NOs: 47, 48, and 49, SEQ ID NOs: 50, 51, and 52, SEQ ID NOs: 53, 56, and 55, SEQ ID NOs: 53, 54, and 55, SEQ ID NOs: 57, 58, and 59, SEQ ID NOs: 60, 61, and 62, or SEQ ID NOs: 63, 64, and 65, respectively. According to this aspect, the binding molecule or fragment thereof can further include a VH-CDRl, a VH-CDR2, and a VH-CDR3.

[0010] In certain aspects the binding molecule or fragment thereof includes a VH-CDRl, a VH- CDR2, and a VH-CDR3, where the VH-CDRs contain amino acid sequences identical to, or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more of the VH-CDRs to: SEQ ID NOs: 29, 30, and 31, SEQ ID NOs: 32, 33, and 34, SEQ ID NOs: 35, 36, and 37, SEQ ID NOs: 38, 39, and 40, SEQ ID NOs: 41, 42, and 43, or SEQ ID NOs: 44, 45, and 46, respectively. According to this aspect the binding molecule or fragment thereof can further include a VL-CDR1, a VL-CDR2, and a VL-CDR3.

[0011] In certain aspects the binding molecule or fragment thereof includes an antibody VL that contains an amino acid sequence at least 85%, 90%, 95%, or 100% identical to a reference amino acid sequence that can be SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, and SEQ ID NO: 28. According to this aspect the binding molecule can further include a VH.

[0012] In certain aspects the binding molecule or fragment thereof includes an antibody VH that contains an amino acid sequence at least 85%, 90%, 95%, or 100% identical to a reference amino acid sequence that can be SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO: 26. According to this aspect the binding molecule can further include a VH.

[0013] In certain aspects, a binding molecule or antigen-binding fragment thereof as described above can be an antibody or antigen-binding fragment thereof.

[0014] The disclosure further provides an isolated antibody or antigen-binding fragment thereof that specifically binds to SpA. In certain aspects the antibody or fragment thereof includes VL-CDR1, VL-CDR2, VL-CDR3, VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to: SEQ ID NOs: 47, 48, 49, 29, 30, and 31, SEQ ID NOs: 50, 51, 52, 32, 33, and 34, SEQ ID NOs: 53, 56, 55, 35, 36, and 37, SEQ ID NOs: 53, 54, 55, 35, 36, and 37, SEQ ID NOs: 57, 58, 59, 38, 39, and 40, SEQ ID NOs: 60, 61, 62, 41, 42, and 43, or SEQ ID NOs: 63, 64, 65, 44, 45, and 46, respectively. In certain aspects the antibody or fragment thereof includes VH and VL amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences containing SEQ ID NO: 2 and SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24, or SEQ ID NO: 26 and SEQ ID NO: 28, respectively. In one aspect, the antibody or fragment thereof includes a VH having the amino acid sequence SEQ ID NO: 10 and a VL including the amino acid sequence SEQ ID NO: 12.

5] An antibody or antigen-binding fragment thereof of as provided by the disclosure can further includes a heavy chain constant region or fragment thereof, e.g., an IgG constant region. In certain aspects the IgG constant region can have one or more amino acid substitutions relative to a wild- type IgG constant region. In certain aspects the modified IgG can have an increased half-life compared to the half-life of an IgG having a wild-type IgG constant region. For example, an IgG constant region can include one or more single amino acid substitutions at positions 251-257, 285- 290, 308-314, 385-389, or 428-436, where the amino acid position numbering is according to the EU index as set forth in Kabat. In one aspect, the one or more amino acid substitutions in the IgG constant region can be a substitution of the amino acid at Kabat position 252 with Tyrosine (Y), Phenylalanine (F), Tryptophan (W), or Threonine (T); a substitution of the amino acid at Kabat position 254 with Threonine (T); a substitution of the amino acid at Kabat position 256 with Serine (S), Arginine (R), Glutamine (Q), Glutamic acid (E), Aspartic acid (D), or Threonine (T); a substitution of the amino acid at Kabat position 257 with Leucine (L); a substitution of the amino acid at Kabat position 309 with Proline (P); a substitution of the amino acid at Kabat position 311 with Serine (S); a substitution of the amino acid at Kabat position 428 with Threonine (T), Leucine (L), Phenylalanine (F), or Serine (S); a substitution of the amino acid at Kabat position 433 with Arginine (R), Serine (S), Isoleucine (I), Proline (P), or Glutamine (Q); a substitution of the amino acid at Kabat position 434 with Tryptophan (W), Methionine (M), Serine (S), Histidine (H), Phenylalanine (F), or Tyrosine; or a combination of two or more of these substitutions. In one aspect the IgG constant region is a human IgG constant region that includes amino acid substitutions relative to a wild-type human IgG constant region at Kabat positions 252, 254, and 256, where the amino acid at Kabat position 252 is substituted with Tyrosine (Y); the amino acid at Kabat position 254 is substituted with Threonine (T); and the amino acid at Kabat position 256 is substituted with Glutamic acid (E), also referred to as a "YTE" IgG constant region. In certain aspects the IgG constant region is a human IgGl constant region. In certain aspects an antibody or fragment thereof as provided herein can further include a light chain constant region, e.g., a human kappa constant region or a human lambda constant region.

[0016] In further aspects, an antibody or fragment thereof as provided by this disclosure can be a murine antibody, a humanized antibody, a chimeric antibody, or an antigen-binding fragment thereof. Moreover, the antibody or fragment thereof can be monoclonal, polyclonal, recombinant, multispecific, or any combination thereof. An antibody fragment as provided herein can be, e.g., an Fv fragment, an Fab fragment, an F(ab')2 fragment, an Fab' fragment, a dsFv fragment, an scFv fragment, or an sc(Fv)2 fragment, or any combination thereof.

[0017] In certain aspects, an antibody or fragment thereof as provided by this disclosure can block SpA ligand binding, and/or can be an antagonist of SpA activity. In certain aspects the ligand can be human IgG Fc, VH3 Fabs, von Willebrand Factor, TNF receptor I, VH3 B-cell receptors, or a combination thereof. Further, an antibody or fragment thereof as provided by this disclosure can be attached, e.g., conjugated or fused, to a heterologous agent, e.g., an antimicrobial agent, a therapeutic agent, a prodrug, a protein, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, a polyethylene glycol (PEG) polymer, or any combination thereof.

[0018] This disclosure further provides a composition that includes an antibody or antigen-binding fragment as provided herein, and a carrier. The disclosure also provides a kit that includes an antibody or fragment thereof, or a composition as provided herein, as well as instructions for using the antibody or fragment thereof or using the composition or directions for obtaining instructions for using the antibody or antigen-binding fragment thereof or using the composition. Such a kit can further include reagents and supplies such as a buffer, a solid support, or both. The solid support can be, e.g., a bead, a filter, a membrane or a multiwall plate. In certain aspects the buffer provided by a kit as disclosed can be suitable for an enzyme-linked immunosorbent assay (ELISA). A kit as provided herein can, in addition, include instructions for isolating, purifying, detecting and quantifying a SpA polypeptide. The disclosure also provides a diagnostic reagent that includes an antibody or fragment of as provided herein. [0019] In certain aspects, the disclosure provides an isolated polynucleotide that includes a nucleic acid encoding a VL, where the VL contains a VL-CDR1, a VL-CDR2, and a VL-CDR3, where the VL-CDRs include amino acid sequences identical to, or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more of the VL-CDRs to: SEQ ID NOs: 47, 48, and 49, SEQ ID NOs: 50, 51, and 52, SEQ ID NOs: 53, 56, and 55, SEQ ID NOs: 53, 54, and 55, SEQ ID NOs: 57, 58, and 59, SEQ ID NOs: 60, 61, and 62, or SEQ ID NOs: 63, 64, and 65, respectively. In certain aspects, the disclosure provides an isolated polynucleotide that includes a nucleic acid encoding a VH, where the VH contains a VH-CDR1, a VH-CDR2, and a VH-CDR3, where the VH-CDRs include amino acid sequences identical to, or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more of the VH-CDRs to: SEQ ID NOs: 29, 30, and 31, SEQ ID NOs: 32, 33, and 34, SEQ ID NOs: 35, 36, and 37, SEQ ID NOs: 38, 39, and 40, SEQ ID NOs: 41, 42, and 43, or SEQ ID NOs: 44, 45, and 46, respectively.

[0020] In certain aspects, the disclosure provides an isolated polynucleotide that includes a nucleic acid encoding an antibody VL, where the VL has an amino acid sequence at least 85%, 90%, 95%, or 100% identical to reference amino acid sequence SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, or SEQ ID NO: 28. In certain aspects, the disclosure provides an isolated polynucleotide that includes a nucleic acid encoding an antibody VH, where the VH has an amino acid sequence at least 85%, 90%, 95%, or 100% identical to reference amino acid sequence SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 26.

[0021] In certain aspects, an antibody or fragment thereof, or subunit thereof that includes a VH, a VL, or a VH and VL encoded by a polynucleotide provided by this disclosure can specifically bind to SpA. For example, the antibody can specifically bind to the same epitope as an antibody or antigen-binding fragment thereof with a VH and VL having the amino acid sequences SEQ ID NO: 2 and 4, SEQ ID NO: 6 and 8, SEQ ID NO: 10 and 12, SEQ ID NO: 14 and 16, SEQ ID NO: 18 and 20, SEQ ID NO: 22 and 24, or SEQ ID NO: 26 and 28, respectively.

[0022] In certain aspects the disclosure provides a polynucleotide or a combination of polynucleotides that encodes a binding molecule or fragment thereof as provided by this disclosure or an antibody or fragment thereof as provided by this disclosure.

[0023] The disclosure also provides a vector containing an isolated polynucleotide provided by the disclosure. [0024] The disclosure further provides a polynucleotide composition that includes a polynucleotide containing a nucleic acid encoding a VH, and a polynucleotide containing a nucleic acid encoding a VL, where the VL and VH include VL-CDRl, VL-CDR2, VL-CDR3, VH-CDRl , VH-CDR2, and VH-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to: SEQ ID NOs: 47, 48, 49, 29, 30, and 31, SEQ ID NOs: 50, 51, 52, 32, 33, and 34, SEQ ID NOs: 53, 56, 55, 35, 36, and 37, SEQ ID NOs: 53, 54, 55, 35, 36, and 37, SEQ ID NOs: 57, 58, 59, 38, 39, and 40, SEQ ID NOs: 60, 61, 62, 41, 42, and 43, or SEQ ID NOs: 63, 64, 65, 44, 45, and 46, respectively. The disclosure further provides a polynucleotide composition that includes a polynucleotide containing a nucleic acid encoding a VH, and a polynucleotide containing a nucleic acid encoding a VL, where the VL and VH include, respectively, amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24, and SEQ ID NO: 26 or SEQ ID NO: 28, respectively. In certain aspects, the VH and VL are encoded by nucleic acid sequences at least 85%, 90%, 95%, or 100% identical to reference nucleic acid sequences SEQ ID NO: 1 and SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23, and SEQ ID NO: 25 or SEQ ID NO: 27, respectively. In certain aspects, the nucleic acid encoding a VH and the nucleic acid encoding a VL are in the same vector, and the disclosure provides such a vector. In certain aspects, the nucleic acid encoding a VH and the nucleic acid encoding a VL are in different vectors, and the disclosure provides such vectors. As provided herein, an antibody or fragment thereof encoded by the polynucleotides of the provided composition can specifically bind to SpA. For example, the antibody can specifically bind to the same epitope as an antibody or antigen- binding fragment thereof containing a VH and a VL that include the amino acid sequences SEQ ID NO: 2 and 4, SEQ ID NO: 6 and 8, SEQ ID NO: 10 and 12, SEQ ID NO: 14 and 16, SEQ ID NO: 18 and 20, SEQ ID NO: 22 and 24, or SEQ ID NO: 26 and 28, respectively.

[0025] The disclosure further provides a host cell that can include a polynucleotide as provided herein, a composition as provided herein, or a vector or vectors as provided herein. The disclosure also provides a method of making an antibody or antigen-binding fragment as provided herein where the method includes culturing a provided host cell and isolating the antibody or fragment thereof. [0026] This disclosure further provides a method for preventing, treating or managing pneumonia in a subject, where the method includes administering to a subject in need thereof an effective amount of an antibody or fragment thereof or antibody composition as provided herein. In certain aspects the pneumonia can be associated with the presence of Staphylococcus bacteria in the subject's lungs, e.g., Staphylococcus is S. pneumoniae or S. aureus.

[0027] The disclosure further provides a method for preventing, treating or managing a skin condition, e.g., dermonecrosis, in a subject, where the method includes administering to a subject in need thereof an effective amount of an antibody or fragment thereof or antibody composition as provided herein. In certain aspects the skin condition is associated with the presence of Staphylococcus bacteria in the subject's skin layers.

[0028] The disclosure further provides a method for preventing, treating, or managing a condition associated with a Staphylococcus infection in a subject, where the method includes administering to a subject in need thereof an effective amount of an antibody or fragment thereof or antibody composition as provided herein. The Staphylococcus can be, e.g., S. aureus which can be antibiotic resistant, e.g., methicillin resistant. In certain aspects the amount of antibody or fragment thereof is effective to reduce SpA ligand binding, where the ligand can be human IgG Fc, VH3 Fabs, von Willebrand Factor, TNF receptor I, VH3 B-cell receptors, or a combination thereof.

[0029] Moreover, the disclosure provides a method for diagnosing a condition mediated by Staphylococcus bacteria in a subject, where the method includes administering to a subject in need thereof a diagnostically effective amount of an antibody or fragment thereof or antibody composition as provided herein; and detecting presence of a biological effect associated with the administration of the antibody or fragment thereof or the composition; where detection of a biological effect associated with the administration of the antibody or fragment thereof or the composition is indicative of presence of a condition mediated by Staphylococcus. The Staphylococcus can be, e.g., S. aureus which can be antibiotic resistant, e.g., methicillin resistant.

[0030] The disclosure further provides a method of preventing, reducing, or managing SpA-induced virulence in a subject infected with Staphylococcus bacteria, where the method includes administering to a subject in need thereof an effective amount of an antibody or fragment thereof or antibody composition as provided herein.

[0031] In another aspect, the disclosure provides a mutant SpA IgG/VH3 binding domain region polypeptide. In certain aspects the mutant SpA polypeptide includes the amino acid sequence 1 naaqhdeaqq naX^X^qvlnmp nlnadqrX₂sgf iqX₃₃lkddpsq sanvlgeaqk 50 51 lndsqapkad aqqnnfnkdq qsafyeilnm pnlneaqrng fiqslkddps 100

101 qstnvlgeak klnesqapka dnnfnkeqqn afyeilnmpn lneeqrngfi 150

151 qslkddpsqs anllseakkl nesqapkadn kfnkeqqnaf yeilhlpnln 200

201 eeqrngfiqs lkddpsqsan llaeakklnd aqapkadnkf nkeqqnafye 250

251 ilhlpnltee qrngfiqslk ddpsvskeil aeakklndaq apkeednnkp 300

301 gkednnkpgk ednnk (SEQ ID NO: 67),

where X₁₃ is F, R, H, or L; X_u is Y, R, H, K, A, or F; X₂₈ is N or A; and X₃₃ is S, I, L, A, G, V, R, H, or K, where the polypeptide includes at least one amino acid substitution relative to SEQ ID NO: 66, and where the polypeptide exhibits reduced binding to the Fc region of IgG, to a VH3 Fab region, or to both, compared to a wild-type IgG/VH3 binding domain region polypeptide including the amino acid sequence of SEQ ID NO: 66.

[0032] In certain aspects a mutant SpA polypeptide as provided herein contains a single amino acid substitution relative to SEQ ID NO: 66, for example the Tyrosine (Y) at position 14 can be substituted with Arginine (R) (SEQ ID NO: 68), or the Serine (S) at position 33 can be substituted with Isoleucine (I) (SEQ ID NO: 69). In certain aspects the SpA polypeptide contains two amino acid substitutions relative to SEQ ID NO: 66, for example the Phenylalanine (F) at position 13 is substituted with Arginine (R) and the Serine (S) at position 33 is substituted with Isoleucine (I) (SEQ ID NO: 70), or the Tyrosine (Y) at position 14 is substituted with Arginine (R) and the Serine (S) at position 33 is substituted with Isoleucine (I) (SEQ ID NO: 71).

[0033] In certain aspects an antibody or fragment thereof as provided herein can specifically bind to a mutant SpA polypeptide as provided herein, but can still block ligand binding to wild-type SpA. In other aspects the disclosure provides a composition for eliciting or modulating an immune response to Staphylococcus bacteria in a subject in need thereof, including administering an effective amount of a mutant SpA polypeptide provided by the disclosure as an antigen. Such a composition can further include one or more adjuvants, e.g., aluminum hydroxide and/or paraffin oil.

[0034] The disclosure further provides a method of modulating an immune response to Staphylococcus infection in a subject, including administering to a subject in need thereof an effective amount of an antibody or fragment thereof as provided herein, and/or an effective amount of a mutant SpA polypeptide as provided herein, where the antibody or SpA polypeptide can block B-cell superantigen activity.

[0035] In any of the therapeutic or diagnostic methods provided by this disclosure, the subject can be, e.g., a domestic animal or a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1A and IB illustrate IP challenge assays of wild-type SpA and doubly Fc & VH3 Fab ablated SpA mutants in kidney (Panel A) and spleen (Panel B). Data are the means + standard errors of the means (SEM). Results in left and right panels are representative of two independent analyses.

Statistical significance was determined by Kruskal-Wallis analysis. An asterisk (*) indicates statistical significance (p<0.05).

[0037] FIG. 2A and 2B illustrate IP challenge assays of Y14R-S33I component SpA mutants in kidney (Panel A) and spleen (Panel B). Data are the means + standard errors of the means (SEM).

Results in left and right panels are representative of two independent analyses. Statistical significance was determined by Kruskal-Wallis analysis. An asterisk (*) indicates statistical significance (p<0.05).

[0038] FIG. 3 illustrates IN challenge assays of doubly ablated SpA mutants in lung pneumonia model. Data are the means + standard errors of the means (SEM). Statistical significance was determined by Kruskal-Wallis analysis (p<0.05). An asterisk (*) indicates statistical significance (p<0.05).

[0039] FIG. 4 illustrates IN challenge assay of Y14R-S33I mutant SpA in lung pneumonia model.

Data are the means + standard errors of the means (SEM). Vaccination with Y14R-S33I lowered lung CFU versus CFA/IFA. Statistical significance was determined by the Mann Whitney U test. An asterisk (*) indicates statistical significance (p<0.05). LOD corresponds to limit of detection.

[0040] FIG. 5A illustrates HTRF Fab displacement of SpA 53.1 variants. FIG. 5B illustrates HTRF Fc displacement of SpA 53.1 variants. Data are the means + standard deviation (SD).

[0041] FIG. 6A illustrates retained binding of anti-SpA mAb to Protein A sensors in buffer (Panel A) and in 95% human serum (Panel B). FIG. 6B illustrates percent retained binding of various anti- SpA mAbs in 95% normal human serum (NHS). Data are the means + standard deviation (SD).

[0042] FIG. 7 illustrates opsonophagocytic killing (OPK) assay of various SpA monoclonal antibodies. Data are the means of three independent experiments, and error bars represent + standard errors of the mean (SEM).

[0043] FIGS. 8A and 8C show the binding of anti-SpA mAbs to in vitro-grown S. aureus SF8300.

FIGS. 8B and 8D show the binding of anti-SpA mAbs to bacteria recovered after 1 hr in vivo passage in mice. The positive control was an anti-LTA antibody. The negative control is the R347 antibody. [0044] FIG. 9A illustrates binding of SpA 53.10S at equilibrium to 5". aureus SF8300 on whole bacteria cells. Methicillin-resistant Staphylococcus aureus SF8300 (USA300) was provided by Binh Diep (University of California, San Francisco). FIG. 9B illustrates binding of 3F6 chimera at equilibrium to S. aureus SF8300 on whole cells. 3F6 is an anti-SpA positive control mAb against an SpA mutant (SpAKKAA; Kim, H.K., et al, 2012 Infect. Immun. 80:3460-3470). FIG. 9C illustrates binding of R347, an isotype-matched non-specific control antibody, at equilibrium to S. aureus SF8300 on whole cells. Data are the means + standard deviation (SD).

[0045] FIG. 10A illustrates binding of SpA 53.10S at equilibrium to 5". aureus SF8300 in 50% normal human serum (NHS). FIG. 10B illustrates binding of 3F6 chimera at equilibrium to S. aureus SF8300 in 50% NHS. FIG. IOC illustrates binding of R347 chimera at equilibrium to 5". aureus SF8300 in 50% NHS. Data are the means + standard deviation (SD).

[0046] FIG. 11A-B illustrates IP organ burden assay of SpA 53.10 variants in kidney (Panel A) and in spleen (Panel B). Data are the means + standard errors of the means (SEM). Statistical significance was determined by Kruskal-Wallis analysis. An asterisk (*) indicates statistical significance over the negative control antibody R347(p<0.05). Numbers on x axis correspond to mg/kg. LOD corresponds to limit of detection.

[0047] FIG. 12 illustrates IV kidney burden assay of SpA 53.10S. Values on the X-axis are the antibody dose in mg/kg. Data are the means + standard errors of the means (SEM). Statistical significance was determined by Kruskal-Wallis analysis. An asterisk (*) indicates statistical significance over the negative control antibody R347 (p<0.05). Numbers on x axis correspond to mg/kg. LOD corresponds to limit of detection.

[0048] FIG. 13 illustrates IN survival assay of SpA 53.10 variants in combination with anti-S. aureus alpha toxin antigen binding fragment LC10. An asterisk (*) indicates statistical significance over the negative control antibody R347 (p<0.05)

[0049] FIG. 14 illustrates IV survival assay of murine antibody SpA 53.1 in combination with LC10. An asterisk (*) indicates statistical significance over the negative control antibody R347 (p<0.05)

DETAILED DESCRIPTION

[0050] Provided herein are binding molecules and antigen-binding fragments thereof that specifically bind to SpA. In some embodiments, such binding molecules are antibodies and antigen- binding fragments thereof that specifically bind to SpA. Polynucleotides encoding anti-SpA binding molecules, e.g., antibodies or antigen-binding fragments thereof, compositions comprising anti-SpA binding molecules, e.g., antibodies or antigen-binding fragments thereof, and methods of making anti-SpA binding molecules, e.g., antibodies or antigen-binding fragments thereof are also provided. Methods of using anti-SpA binding molecules, e.g., antibodies or antigen-binding fragments thereof, such as methods of preventing, treating or managing a condition or disease associated with a Staphylococcus infection in a subject and diagnostic uses, are further provided.

I. Definitions

[0051] The term "SpA" as used herein refers to Staphylococcus aureus protein A, and/or active fragments thereof. The IgG/VH3 binding domain region of SpA, e.g., amino acids 35 to 349 of GenBank Accession Number gblEUZ31280.1 l, is shown below and is presented as SEQ ID NO: 66.

1 naaqhdeaqq nafyqvlnmp nlnadqrngf iqs lkddpsq sanvlgeaqk 50

51 lndsqapkad aqqnnfnkdq qsafyeilnm pnlneaqrng f iqslkddps 100

101 qstnvlgeak klnesqapka dnnfnkeqqn afyeilnmpn lneeqrngfi 150

151 qs lkddpsqs anllseakkl nesqapkadn kfnkeqqnaf yeilhlpnln 200

201 eeqrngfiqs lkddpsqsan llaeakklnd aqapkadnkf nkeqqnafye 250

251 ilhlpnltee qrngf iqslk ddpsvskeil aeakklndaq apkeednnkp 300

301 gkednnkpgk ednnk 315

[0052] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a binding molecule which specifically binds to SpA," is understood to represent one or more binding molecules which specifically bind to SpA. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0053] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "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).

[0054] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0055] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects or aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0056] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

[0057] A polypeptide as disclosed herein can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three- dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded. As used herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein via an oxygen-containing or a nitrogen- containing side chain of an amino acid, e.g. , a serine or an asparagine. [0058] By an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0059] Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms "fragment," "variant," "derivative" and "analog" when referring to a binding molecule such as an antibody which specifically binds to SpA as disclosed herein include any polypeptides which retain at least some of the antigen-binding properties of the corresponding native antibody or polypeptide. Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein. Variants of a binding molecule, e.g., an antibody which specifically binds to SpA as disclosed herein include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. In certain aspects, variants can be non-naturally occurring. Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of a binding molecule, e.g. , an antibody which specifically binds to SpA as disclosed herein are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins. Variant polypeptides can also be referred to herein as "polypeptide analogs." As used herein a "derivative" of a binding molecule, e.g. , an antibody which specifically binds to SpA refers to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those peptides which contain one or more derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

[0060] The term "polynucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g. , messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond (e.g. , an amide bond, such as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g. , DNA or RNA fragments, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a binding molecule, e.g. , an antibody which specifically binds to SpA contained in a vector is considered isolated as disclosed herein. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. In addition, polynucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

[0061] As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g. , on a single vector, or in separate polynucleotide constructs, e.g. , on separate (different) vectors. Furthermore, any vector can contain a single coding region, or can comprise two or more coding regions, e.g. , a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid can encode heterologous coding regions, either fused or unfused to a nucleic acid encoding an a binding molecule which specifically binds to SpA, e.g., an antibody, or antigen-binding fragment, variant, or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

[0062] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide normally can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, e.g. , a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter can be a cell- specific promoter that directs substantial transcription of the DNA in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell- specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

[0063] A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue- specific promoters and enhancers as well as lymphokine-inducible promoters (e.g. , promoters inducible by interferons or interleukins).

[0064] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

[0065] In other embodiments, a polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA).

[0066] Polynucleotide and nucleic acid coding regions can be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide as disclosed herein, e.g. , a polynucleotide encoding a binding molecule which specifically binds to SpA, e.g., an antibody, or antigen-binding fragment, variant, or derivative thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells can have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. For example, the wild- type leader sequence can be substituted with the leader sequence of human tissue plasminogen activator (TP A) or mouse β-glucuronidase.

[0067] Disclosed herein are certain binding molecules, or antigen-binding fragments, variants, or derivatives thereof. Unless specifically referring to full-sized antibodies, the term "binding molecule" encompasses full-sized antibodies as well as antigen-binding fragments, variants, analogs, or derivatives of such antibodies, e.g. , engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules, but which use a different scaffold.

[0068] As used herein, the term "binding molecule" refers in its broadest sense to a molecule that specifically binds an antigenic determinant. As described further herein, a binding molecule can comprise one of more of the antigen binding domains described herein. As used herein, an "antigen binding domain" includes a site that specifically binds to an antigenic determinant. A non-limiting example of an antigen binding molecule is an antibody or fragment thereof that retains antigen- specific binding. The term "antigen binding domain" is distinguished from the term "IgG/VH3 binding domain," which refers herein to one or more of the five homologous regions of staphylococcal protein A that can bind to immunoglobulin Fc-gamma and VH3 Fabs.

[0069] The terms "antibody" and "immunoglobulin" can be used interchangeably herein. An antibody (or a fragment, variant, or derivative thereof as disclosed herein comprises at least the variable domain of a heavy chain and at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g. , Harlow et al , Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0070] As will be discussed in more detail below, the term "immunoglobulin" comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) with some subclasses among them (e.g. , γ1-γ4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g. , IgGi, IgG₂, IgG₃, IgG₄, IgAi, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of this disclosure.

[0071] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C- terminus at the bottom of each chain.

[0072] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy- terminus of the heavy and light chain, respectively.

[0073] As indicated above, the variable region allows the binding molecule to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of a binding molecule, e.g. , an antibody combine to form the variable region that defines a three dimensional antigen binding site. This quaternary binding molecule structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VL chains.

[0074] The six "complementarity determining regions" or "CDRs" present in each antibody antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen binding domains, referred to as "framework" regions, show less inter-molecular variability. The framework regions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, /. Mol. Biol., 796:901-917 (1987), which are incorporated herein by reference in their entireties).

[0075] In the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al. , U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al. , J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference, where the definitions include overlapping or subsets of amino acids when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acids which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. The exact amino acid numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which amino acids comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0076] Table 1 CDR Definitions¹

Kabat Chothia VH CDR1 31-35 26-32

VH CDR2 50-65 52-58

VH CDR3 95-102 95-102

VL CDR1 24-34 26-32

VL CDR2 50-56 50-52

VL CDR3 89-97 91-96

'Numbering of all CDR definitions in Table 1 is according to the

numbering conventions set forth by Kabat et al. (see below).

[0077] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al, U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid positions in a binding molecule which specifically binds to SpA, e.g, an antibody, or antigen-binding fragment, variant, or derivative thereof as disclosed herein are according to the Kabat numbering system.

[0078] Binding molecules, e.g. , antibodies or antigen-binding fragments, variants, or derivatives thereof include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g. , Fab, Fab' and F(ab')₂, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, e.g. , in US patent 5,892,019. Immunoglobulin or antibody molecules encompassed by this disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

[0079] By "specifically binds," it is generally meant that a binding molecule, e.g. , an antibody or fragment, variant, or derivative thereof binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, a binding molecule is said to "specifically bind" to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity by which a certain binding molecule binds to a certain epitope. For example, binding molecule "A" can be deemed to have a higher specificity for a given epitope than binding molecule "B," or binding molecule "A" can be said to bind to epitope "C" with a higher specificity than it has for related epitope "D."

[0080] A binding molecule, e.g. , an antibody or fragment, variant, or derivative thereof disclosed herein can be said to bind a target antigen, e.g. , SpA disclosed herein or a fragment or variant thereof with an off rate (k(off)) of less than or equal to 5 X 10^~2 sec^"1, 10^~2 sec^"1, 5 X 10^~3 sec^"1 or 10^"3 sec^"1. A binding molecule as disclosed herein can be said to bind a target antigen, e.g. , SpA with an off rate (k(off)) less than or equal to 5 X 10^"4 sec^"1, 10^"4 sec^"1, 5 X 10^"5 sec^"1, or 10^"5 sec^"1 5 X 10^"6 sec^"1, 10^"6 sec^"1, 5 X 10^"7 sec^"1 or 10^"7 sec^"1.

[0081] A binding molecule, e.g. , an antibody or antigen-binding fragment, variant, or derivative disclosed herein can be said to bind a target antigen, e.g. , SpA with an on rate (k(on)) of greater than or equal to 10³ M^"1 sec^"1, 5 X 10³ M^"1 sec^"1, 10⁴ M^"1 sec^"1 or 5 X 10⁴ M^"1 sec^"1. A binding molecule as disclosed herein can be said to bind a target antigen, e.g. , SpA with an on rate (k(on)) greater than or equal to 10⁵ M^"1 sec^"1, 5 X 10⁵ M^"1 sec^"1, 10⁶ M^"1 sec^"1, or 5 X 10⁶ M^"1 sec^"1 or 10⁷ M^"1 sec^"1.

[0082] A binding molecule, e.g. , an antibody or fragment, variant, or derivative thereof is said to competitively inhibit binding of a reference antibody or antigen binding fragment to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody or antigen binding fragment to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays. A binding molecule can be said to competitively inhibit binding of the reference antibody or antigen binding fragment to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0083] As used herein, the term "affinity" refers to a measure of the strength of the binding of an individual epitope with the CDR of an immunoglobulin molecule. See, e.g. , Harlow et al , Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28. As used herein, the term "avidity" refers to the overall stability of the complex between a population of immunoglobulins and an antigen, that is, the functional combining strength of an immunoglobulin mixture with the antigen. See, e.g. , Harlow at pages 29-34. Avidity is related to both the affinity of individual immunoglobulin molecules in the population with specific epitopes, and also the valencies of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity. An interaction between a between a bivalent monoclonal antibody with a receptor present at a high density on a cell surface would also be of high avidity.

[0084] Binding molecules or antigen-binding fragments, variants or derivatives thereof as disclosed herein can also be described or specified in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances. Thus, a binding molecule is cross reactive if it binds to an epitope other than the one that induced its formation. The cross reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, can actually fit better than the original.

[0085] A binding molecule, e.g. , an antibody or fragment, variant, or derivative thereof can also be described or specified in terms of their binding affinity to an antigen. For example, a binding molecule can bind to an antigen with a dissociation constant or KD no greater than 5 x 10^~2 M, 10^~2 M, 5 x 10^"3 M, 10^"3 M, 5 x 10^"4 M, 10^"4 M, 5 x 10^"5 M, 10^"5 M, 5 x 10^"6 M, 10^"6 M, 5 x 10^"7 M, 10^"7 M, 5 x 10^"8 M, 10^"8 M, 5 x 10^"9 M, 10^"9 M, 5 x 10^"10 M, 10^"10 M, 5 x 10^"11 M, 10^"11 M, 5 x 10^"12 M, 10^"12 M, 5 x 10^"13 M, 10^"13 M, 5 x 10^"14 M, 10^"14 M, 5 x 10^"15 M, or 10^"15 M.

[0086] Antibody fragments including single-chain antibodies can comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains. Also included are antigen-binding fragments that comprise any combination of variable region(s) with a hinge region, CHI , CH2, and CH3 domains. Binding molecules, e.g. , antibodies, or antigen-binding fragments thereof disclosed herein can be from any animal origin including birds and mammals. The antibodies can be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be condricthoid in origin (e.g. , from sharks). As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.

[0087] As used herein, the term "heavy chain portion" includes amino acid sequences derived from an immunoglobulin heavy chain, a binding molecule, e.g. , an antibody comprising a heavy chain portion comprises at least one of: a CHI domain, a hinge (e.g. , upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, a binding molecule, e.g. , an antibody or fragment, variant, or derivative thereof can comprise a polypeptide chain comprising a CHI domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHI domain and a CH3 domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a binding molecule, e.g. , an antibody or fragment, variant, or derivative thereof comprises a polypeptide chain comprising a CH3 domain. Further, a binding molecule for use in the disclosure can lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As set forth above, it will be understood by one of ordinary skill in the art that these domains (e.g. , the heavy chain portions) can be modified such that they vary in amino acid sequence from the original immunoglobulin molecule.

[0088] The heavy chain portions of a binding molecule, e.g. , an antibody as disclosed herein can be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide can comprise a CHI domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion can comprise a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.

[0089] As used herein, the term "light chain portion" includes amino acid sequences derived from an immunoglobulin light chain. The light chain portion comprises at least one of a VL or CL domain.

[0090] Binding molecules, e.g. , antibodies or antigen-binding fragments, variants, or derivatives thereof disclosed herein can be described or specified in terms of the epitope(s) or portion(s) of an antigen, e.g. , a target SpA that they recognize or specifically bind. The portion of a target SpA which specifically interacts with the antigen binding domain of an antibody is an "epitope," or an "antigenic determinant." A target antigen, e.g., SpA can comprise a single epitope or at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen.

[0091] As previously indicated, the subunit structures and three dimensional configuration of the constant regions of the various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino terminal variable domain of an immunoglobulin heavy chain and the term "CHI domain" includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CHI domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.

[0092] As used herein the term "CH2 domain" includes the portion of a heavy chain molecule that extends, e.g. , from about amino acid 244 to amino acid 360 of an antibody using conventional numbering schemes (amino acids 244 to 360, Kabat numbering system; and amino acids 231-340, EU numbering system; see Kabat EA et al. op. cit. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 amino acids.

[0093] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 amino acids and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol. 767 :4083 (1998)).

[0094] As used herein the term "disulfide bond" includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. In certain IgG molecules, the CHI and CL regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).

[0095] As used herein, the term "chimeric antibody" will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which can be intact, partial or modified) is obtained from a second species. In some embodiments the target binding region or site will be from a non-human source (e.g. mouse or primate) and the constant region is human.

[0096] The term "bispecific antibody" as used herein refers to an antibody that has binding sites for two different antigens within a single antibody molecule. It will be appreciated that other molecules in addition to the canonical antibody structure can be constructed with two binding specificities. It will further be appreciated that antigen binding by bispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means. (Strohlein and Heiss, Future Oncol. 6: 1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9 (2010)). A bispecific antibody can also be a diabody.

[0097] As used herein, the term "engineered antibody" refers to an antibody in which the variable domain in either the heavy and light chain or both is altered by at least partial replacement of one or more CDRs from an antibody of known specificity and/or by partial framework region replacement or other sequence changes. Although the CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class, e.g. , from an antibody from a different species. An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity is grafted into a human heavy or light chain framework region is referred to herein as a "humanized antibody." It may not be necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those amino acids that are necessary to maintain the activity of the target binding site. Given the explanations set forth in, e.g., U. S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6, 180,370, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional engineered or humanized antibody.

[0098] As used herein the term "properly folded polypeptide" includes polypeptides (e.g., anti-SpA antibodies) in which the functional domains comprising the polypeptide are distinctly active. As used herein, the term "improperly folded polypeptide" includes polypeptides in which at least one of the functional domains of the polypeptide is not active. In one embodiment, a properly folded polypeptide comprises polypeptide chains linked by at least one disulfide bond and, conversely, an improperly folded polypeptide comprises polypeptide chains not linked by at least one disulfide bond.

[0100] As used herein the term "engineered" includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques).

[0101] As used herein, the terms "linked," "fused" or "fusion" are used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means. An "in-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments can be physically or spatially separated by, for example, in-frame linker sequence. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the "fused" CDRs are co-translated as part of a continuous polypeptide.

[0102] In the context of polypeptides, a "linear sequence" or a "sequence" is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which amino acids that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.

[0103] The term "expression" as used herein refers to a process by which a gene produces a biochemical, for example, a polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.

[0104] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In certain aspects, a subject is successfully "treated" for a pathologic disease, condition, or disorder mediated by Staphylococcus bacteria, e.g. , S. aureus, according to the methods of the present disclosure if the patient shows, e.g., an improved survival rate, lessened symptoms of the infection, etc.

[0105] Non-limiting examples of diseases, disorder, or conditions caused by S. aureus infection include burns, cellulitis, dermonecrosis, eyelid infections, food poisoning, joint infections, pneumonia, skin infections, surgical wound infection, scalded skin syndrome and toxic shock syndrome. In addition, it is a frequent pathogen in foreign body infections, such as intravascular lines, pacemakers, artificial heart valves and joint implants. Some conditions and diseases can involve the direct action of SpA as a component of infection or mediator of the condition or disease state, others can involve the indirect or secondary action of SpA (e.g., a primary virulence factor causes the main symptom or majority of symptoms associated with the condition, and SpA acts to further advance the disease through its disruption of opsonophagocytic killing). A binding molecule, e.g. , an antibody or antigen-binding fragment, variant, or derivative disclosed herein, can be used to treat, prevent, or alleviate the symptoms associated with the aforementioned conditions.

[0106] By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on.

[0107] As used herein, phrases such as "a subject that would benefit from administration of anti- SpA binding molecule or antibody" and "an animal in need of treatment" includes subjects, such as mammalian subjects, that would benefit from administration of an anti-SpA binding molecule, such as an antibody, comprising one or more antigen binding domains. Such binding molecules, e.g. , antibodies, can be used, e.g., for detection of SpA (e.g., for a diagnostic procedure) and/or for treatment, i.e. , palliation or prevention of a disease, with anti-SpA binding molecules. As described in more detail herein, the anti-SpA binding molecules can be used in unconjugated form or can be conjugated, e.g. , to a drug, prodrug, or a detectable label.

[0108] A "conservative amino acid substitution" is one in which one amino acid is replaced with another amino acid having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g. , aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. , threonine, valine, isoleucine) and aromatic side chains (e.g. , tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the present disclosure do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen(s), i.e. , the SpA to which the polypeptide or antibody binds. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen-binding are well-known in the art (see, e.g. , Brummell et al , Biochem. 32: 1180-1 187 (1993); Kobayashi et al , Protein Eng. 12(10):879-884 (1999); and Burks et al. , Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

II. Anti-SpA-binding molecules

[0109] SpA binding molecules are provided herein, e.g., anti-SpA antibodies and antigen-binding fragments thereof. SpA contains five homologous IgG/VH3 binding domains, A-E, and a C terminal variable domain that varies between Staphylococcus species, and between S. aureus serotypes. By "SpA binding molecule" or "SpA antibody" is meant a binding molecule, e.g., an antibody or antigen-binding fragment thereof that binds to SpA via a specific interaction between the binding molecule and SpA. Accordingly, as used herein, the terms "SpA binding molecule" and "SpA antibody" do not refer in general to antibodies that non-specifically bind to SpA via their Fc-gamma region or non-specifically through a VH3 Fab. In some embodiments, SpA binding molecules, e.g., anti-SpA antibodies or antigen-binding fragments thereof provided herein are murine antibodies, humanized antibodies or human antibodies. In some embodiments, SpA binding molecules, e.g., anti-SpA antibodies or antigen-binding fragments thereof, comprise a Fab, a Fab', a F(ab')2, a Fd, a single chain Fv or scFv, a disulfide linked Fv, a V-NAR domain, an IgNar, an intrabody, an IgGACH2, a minibody, a F(ab')3, a tetrabody, a triabody, a diabody, a single-domain antibody, DVD-Ig, Fcab, mAb2, a (scFv)2, or a scFv-Fc. In some embodiments, the antibody is of the IgGl subtype and comprises the triple mutant YTE, as disclosed supra in the Definitions section. In some instances, SpA binding molecules have other scaffolds, e.g., scaffolds of a lipocalin, a fibronectin domain, an ankyrin consensus repeat domain, or thioredoxin.

[0110] In certain aspects, this disclosure provides a SpA binding molecule or antigen-binding fragment thereof, e.g., an anti-SpA antibody or antigen-binding fragment thereof, which can specifically bind to the same SpA epitope as an antibody or antigen-binding fragment thereof comprising the heavy chain variable region (VH) and light chain variable region (VL) of SpA 33.6, SpA 03.5, SpA 53.10S, SpA 53.1, SpA 06.1, SpA 66.6, or SpA 07.27. Also provided is a SpA binding molecule or antigen-binding fragment thereof, e.g., an anti-SpA antibody or antigen-binding fragment thereof, which can competitively inhibit, or can bind to SpA with a greater affinity than an antibody or antigen-binding fragment thereof comprising the VH and VL of SpA 33.6, SpA 03.5, SpA 53.10S, SpA 53.1, SpA 06.1 , SpA 66.6, or SpA 07.27. As provided herein, the VH and VL of SpA 33.6 comprise SEQ ID NOs: 2 and 4, respectively, the VH and VL of SpA 03.5 comprise SEQ ID NOs: 6 and 8, respectively, the VH and VL of SpA 53.10S comprise SEQ ID NOs: 10 and 12, respectively, the VH and VL of SpA 53.1 comprise SEQ ID NOs: 14 and 16, respectively, the VH and VL of SpA 06.1 comprise SEQ ID NOs: 18 and 20, respectively, the VH and VL of SpA 66.6 comprise SEQ ID NOs: 22 and 24, respectively, and the VH and VL of SpA 07.27 comprise SEQ ID NOs: 26 and 28, respectively.

[0111] The disclosure further provides a SpA binding molecule or antigen-binding fragment, e.g., an anti-SpA antibody or antigen-binding fragment thereof, including a VL region. In certain aspects the VL region includes one, two, or three VL-CDRs such as a VLCDR1 identical to, or identical except for eight, seven, six, five, four, three, two, or one single amino acid substitutions, deletions, or insertions, to SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 60, or SEQ ID NO: 63, a VLCDR2 identical to or identical except for eight, seven, six, five, four, three, two, or one single amino acid substitutions, deletions, or insertions, to SEQ ID NO: 48, SEQ ID NO: 51 , SEQ ID NO: 54, or SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 61 , or SEQ ID NO: 64, or a VLCDR3 identical to or identical except for eight, seven, six, five, four, three, two, or one single amino acid substitutions, deletions, or insertions, to SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 59, SEQ ID NO: 62, or SEQ ID NO: 65. In certain aspects the VL region contains VL-CDR1, VL-CDR2, and VL-CDR3 amino acid sequences identical to, or identical except for eight, seven, six, five, four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more of the VL-CDRS to: SEQ ID NOs: 47, 48, and 49, SEQ ID NOs: 50, 51, and 52, SEQ ID NOs: 53, 56, and 55, SEQ ID NOs: 53, 54, and 55, SEQ ID NOs: 57, 58, and 59, SEQ ID NOs: 60, 61, and 62, or SEQ ID NOs: 63, 64, and 65, respectively.

[0112] In certain aspects, the VL region comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the reference amino acid sequence SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, or SEQ ID NO: 28. In certain aspects, the binding molecule further comprises a VH-CDR1, a VH-CDR2, and a VH- CDR3, or a heavy chain variable region (VH). [0113] Further provided is a binding molecule or antigen-binding fragment thereof that specifically binds to SpA, e.g., an anti-SpA antibody or antigen-binding fragment thereof, including a VH region. In certain aspects the VH region includes one, two, or three VH-CDRs such as a VHCDR1 identical to, or identical except for eight, seven, six, five, four, three, two, or one single amino acid substitutions, deletions, or insertions, to SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41 , or SEQ ID NO: 44, a VHCDR2 identical to or identical except for eight, seven, six, five, four, three, two, or one single amino acid substitutions, deletions, or insertions, to SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, or SEQ ID NO: 45, or a VHCDR3 identical to or identical except for eight, seven, six, five, four, three, two, or one single amino acid substitutions, deletions, or insertions, to SEQ ID NO: 31 , SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, or SEQ ID NO: 46. In certain aspects the VH region contains VH-CDR1 , VH-CDR2, and VH-CDR3 amino acid sequences identical to, or identical except for eight, seven, six, five, four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more of the VH-CDRS to: SEQ ID NOs: 29, 30, and 31, SEQ ID NOs: 32, 33, and 34, SEQ ID NOs: 35, 36, and 37, SEQ ID NOs: 38, 39, and 40, SEQ ID NOs: 41 , 42, and 43, or SEQ ID NOs: 44, 45, and 46, respectively.

[0114] In certain aspects, the VH region comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the reference amino acid sequence SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 19, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 26. In certain aspects, the binding molecule further comprises a VL-CDR1, a VL-CDR2, and a VL-CDR3, or a light chain variable region (VL).

[0115] In particular aspects, a SpA binding molecule provided herein comprises an anti-SpA antibody or antigen-binding fragment thereof. For example, the disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to SpA comprising a VL region and a VH region, where the VL and VH collectively contain VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1 , VH-CDR2, and VH-CDR3 amino acid sequences identical or identical except for eight, seven, six, five, four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to: SEQ ID NOs: 47, 48, 49, 29, 30, and 31, SEQ ID NOs: 50, 51, 52, 32, 33, and 34, SEQ ID NOs: 53, 56, 55, 35, 36, and 37, SEQ ID NOs: 53, 54, 55, 35, 36, and 37, SEQ ID NOs: 57, 58, 59, 38, 39, and 40, SEQ ID NOs: 60, 61, 62, 41, 42, and 43, or SEQ ID NOs: 63, 64, 65, 44, 45, and 46, respectively. [0116] In some instances, the disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to SpA comprising a VH and a VL, where the VH and VL contain, respectively, amino acid sequences at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24, or SEQ ID NO: 26 and SEQ ID NO: 28, respectively.

[0117] In one aspect, the disclosure provides an anti-SpA antibody or antigen-binding fragment thereof comprising the VH and VL of SpA 53.1 OS, i.e., the VH amino acid sequence SEQ ID NO: 10 and the VL amino acid sequence SEQ ID NO: 12.

[0118] An anti-SpA antibody or antigen-binding fragment thereof provided herein, e.g., as described above can be, e.g., a murine antibody, a humanized antibody, a chimeric antibody, or an antigen-binding fragment thereof. Alternatively, an anti-SpA antibody or antigen-binding fragment thereof provided herein can be, e.g., a monoclonal, polyclonal, recombinant, multispecific, or any combination thereof. An anti-SpA antibody antigen-binding fragment can be, e.g., an Fv fragment, an Fab fragment, an F(ab')2 fragment, an Fab' fragment, a dsFv fragment, an scFv fragment, or an sc(Fv)2 fragment.

[0119] An anti-SpA antibody or antigen-binding fragment thereof provided herein, e.g., as described above, can include, in addition to a VH and a VL, a heavy chain constant region or fragment thereof. In certain aspects the heavy chain constant region is a human heavy chain constant region, e.g., a human IgG constant region, e.g., a human IgGl constant region. As described elsewhere herein, in certain aspects a heavy chain constant region or fragment thereof, e.g., a human IgG constant region or fragment thereof, can include one or more amino acid substitutions relative to a wild-type IgG constant region, where the modified IgG has an increased half-life compared to the half-life of an IgG having the wild-type IgG constant region. For example, the IgG constant region can contain one or more amino acid substitutions of amino acids at positions 251-257, 285- 290, 308-314, 385-389, and 428-436, wherein the amino acid position numbering is according to the EU index as set forth in Kabat. In certain aspects the IgG constant region can contain one or more of a substitution of the amino acid at Kabat position 252 with Tyrosine (Y), Phenylalanine (F), Tryptophan (W), or Threonine (T), a substitution of the amino acid at Kabat position 254 with Threonine (T), a substitution of the amino acid at Kabat position 256 with Serine (S), Arginine (R), Glutamine (Q), Glutamic acid (E), Aspartic acid (D), or Threonine (T), a substitution of the amino acid at Kabat position 257 with Leucine (L), a substitution of the amino acid at Kabat position 309 with Proline (P), a substitution of the amino acid at Kabat position 311 with Serine (S), a substitution of the amino acid at Kabat position 428 with Threonine (T), Leucine (L), Phenylalanine (F), or Serine (S), a substitution of the amino acid at Kabat position 433 with Arginine (R), Serine (S), Isoleucine (I), Proline (P), or Glutamine (Q), or a substitution of the amino acid at Kabat position 434 with Tryptophan (W), Methionine (M), Serine (S), Histidine (H), Phenylalanine (F), or Tyrosine. For example, the IgG constant region can contain amino acid substitutions relative to a wild-type human IgG constant region including a substitution of the amino acid at Kabat position 252 with Tyrosine (Y), a substitution of the amino acid at Kabat position 254 with Threonine (T), and a substitution of the amino acid at Kabat position 256 with Glutamic acid (E).

[0120] An anti-SpA antibody or antigen-binding fragment thereof provided herein, e.g., as described above, can include, in addition to a VH and a VL, and optionally a heavy chain constant region or fragment thereof, a light chain constant region or fragment thereof. In certain aspects the light chain constant region is a kappa lambda light chain constant region, e.g., a human kappa constant region or a human lambda constant region. In a specific aspect, the light chain constant region is a human kappa constant region.

[0121] This disclosure provides an anti-SpA antibody or antigen-binding fragment thereof where the light chain contains a human kappa constant region. In certain aspects the disclosure provides an anti-SpA antibody or antigen-binding fragment thereof comprising a human IgGl YTE heavy chain and a human kappa light chain.

[0122] Anti-SpA antibodies or fragments thereof provided herein can have beneficial properties.

For example, the antibody or fragment thereof can inhibit, suppress, or block IgG Fc domains or VH3 Fabs from binding to SpA on the surface of S. aureus, thereby facilitating, rather than preventing opsonization. Specifically, SpA binds to the Fey portion of human and animal immunoglobulins as well as VH3 Fabs, a defense mechanism that provides S. aureus with protection from opsonophagocytic killing (Forsgren A, Infect. Immun. 2:672- 673 (1970)). The antibody or fragment thereof as disclosed herein specifically binds to SpA, and can, in certain aspects, inhibit, suppress, or block IgG Fc domains or VH3 Fabs from binding to SpA. As a result, the defense mechanism of S. aureus is broken and the bacteria are no longer protected from opsonophagocytic killing. That is, the antibody or fragment thereof as disclosed herein can facilitate opsonization or phagocytosis. [0123] As noted above, a VH and/or VL amino acid sequence can be, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% similar to a sequence set forth herein, and/or comprise, e.g., 1, 2, 3, 4, 5 or more single amino acid substitutions, insertions or deletions, e.g., conservative substitutions, relative to a sequence set forth herein. An SpA antibody having VH and VL regions having a certain percent similarity to a VH region or VL region, or having one or more substitutions, insertions, or deletions, e.g., conservative substitutions, can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding VH and/or VL regions described herein, followed by testing of the encoded altered antibody for binding to SpA and optionally testing for retained function using the functional assays described herein.

[0124] The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method well known in the art, e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., Octet or KINEXA® or BIACORE™ analysis). Direct binding assays as well as competitive binding assay formats can be readily employed. See, for example, Berzofsky et al., "Antibody- Antigen Interactions," In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Immunology, W. H. Freeman and Company: New York, N.Y. (1992); and methods described herein. The measured affinity of a particular antibody- antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH, temperature). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD or ¾, k_on, k_cff) are made with standardized solutions of antibody and antigen, and a standardized buffer, as known in the art and such as the buffer described herein.

[0125] The disclosure further provides an anti-SpA antibody or fragment thereof as described above, where the antibody is conjugated to a heterologous agent. In certain aspects the agent can be an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, a polyethylene glycol (PEG), or a combination of two or more of any the agents.

[0126] In certain embodiments, the SpA-binding molecule is a polypeptide that is not an antibody.

A variety of methods for identifying and producing non- antibody polypeptides that bind with high affinity to a protein target are known in the art. See, e.g., WO 2009/058379, WO 2011/130324, WO 2011/130328, Skerra, Curr. Opin. Biotechnol., 18:295-304 (2007), Hosse et al., Protein Science, 15: 14-27 (2006), Gill et al, Curr. Opin. Biotechnol., 17:653-658 (2006), Nygren, FEBS J., 275:2668-76 (2008), and Skerra, FEBS J., 275:2677-83 (2008), each of which is incorporated by reference herein in its entirety. In certain embodiments, phage display technology can been used to identify/produce a SpA-binding polypeptide. In certain embodiments, the polypeptide comprises a protein scaffold such as, e.g., a protein A scaffold, a lipocalin scaffold, a fibronectin domain scaffold, an ankyrin consensus repeat domain scaffold, or a thioredoxin scaffold.

III. Binding Molecules that Bind to the Same Epitope as, or Competitively Inhibit, Anti-SpA Antibodies and Antigen-binding Fragments Thereof

7] In certain embodiments, this disclosure provides a SpA-binding molecule, e.g., an anti-SpA antibody or antigen-binding fragment thereof, which binds to the same epitope as do the various anti-SpA antibodies described herein. The term "epitope" as used herein refers to a target protein determinant capable of binding to an antibody of the present disclosure. Epitopes can consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter can be lost in the presence of denaturing solvents. The antibodies that bind to overlapping epitopes can be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of, in a statistically significant manner) with antibodies such as SpA 33.6, SpA 03.5, SpA 53.10S, SpA 53.1, SpA 06.1, SpA 66.6, or SpA 07.27, in standard SpA binding or activity assays (although certain antibodies can "competitively inhibit" other antibodies without binding to the same epitope, e.g., through allosteric inhibition). Accordingly, in one embodiment, provided herein are anti-SpA antibodies and antigen-binding fragments thereof, e.g., monoclonal antibodies, that compete for binding to SpA with another anti-SpA antibody or antigen-binding fragment thereof, such as murine monoclonal antibodies SpA 33.6, SpA 03.5, SpA 53.1, SpA 06.1, SpA 66.6, or SpA 07.27, or humanized variants as disclosed herein (e.g. SpA 53.10S). The ability of a test antibody to inhibit the binding of, e.g., SpA 33.6, SpA 03.5, SpA 53.10S, SpA 53.1, SpA 06.1, SpA 66.6, or SpA 07.27 demonstrates that the test antibody can compete with that antibody for binding to SpA; such an antibody can, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on SpA as the anti-SpA antibody or antigen-binding fragment thereof with which it competes. In one embodiment, the anti-SpA antibody or antigen-binding fragment thereof that binds to the same epitope on SpA as, e.g., murine monoclonal antibodies SpA 33.6, SpA 03.5, SpA 53.10S, SpA 53.1, SpA 06.1, SpA 66.6, or SpA 07.27. IV. Preparation of Anti-SpA Antibodies and Antigen-binding Fragments

[0128] Monoclonal anti-SpA antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495. Using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized as described above to elicit the production by lymphocytes of antibodies that will specifically bind to an immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen as determined by immunoprecipitation, immunoblotting, or by an in vitro binding assay {e.g. radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)) can then be propagated either in in vitro culture using standard methods (Coding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in an animal. The monoclonal antibodies can then be purified from the culture medium or ascites fluid as described for polyclonal antibodies above.

[0129] Alternatively anti-SpA monoclonal antibodies can also be made using recombinant DNA methods as described in U.S. Patent No. 4,816,567. The polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cell, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, monoclonal antibodies are generated by the host cells. Also, recombinant anti-SpA monoclonal antibodies or antigen-binding fragments thereof of the desired species can be isolated from phage display libraries expressing CDRs of the desired species as described (McCafferty et al., 1990, Nature, 348:552-554; Clackson et al, 1991, Nature, 352:624-628; and Marks et al, 1991, J. Mol. Biol., 222:581-597).

[0130] The polynucleotide(s) encoding an anti-SpA antibody or an antigen-binding fragment thereof can further be modified in a number of different manners using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant regions of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted (1) for those regions of, for example, a human antibody to generate a chimeric antibody or (2) for a non- immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.

[0131] In certain embodiments, the anti-SpA antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. Human antibodies can be directly prepared using various techniques known in the art. Immortalized human B lymphocytes immunized in vitro or isolated from an immunized individual that produce an antibody directed against a target antigen can be generated (See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol., 147 (l):86-95; and U.S. Patent 5,750,373).

[0132] Also, the anti-SpA human antibody or antigen-binding fragment thereof can be selected from a phage library, where that phage library expresses human antibodies, as described, for example, in Vaughan et al., 1996, Nat. Biotech., 14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162, Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381, and Marks et al., 1991, J. Mol. Biol., 222:581). Techniques for the generation and use of antibody phage libraries are also described in U.S. Patent Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731 ; 6,555,313; 6,582,915; 6,593,081 ; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al., 2007, J. Mol. Bio., doi: 10.1016/j.jmb.2007.12.018 (each of which is incorporated by reference in its entirety).

[0133] Affinity maturation strategies and chain shuffling strategies (Marks et al., 1992, Bio/Technology 10:779-783, incorporated by reference in its entirety) are known in the art and can be employed to generate high affinity human antibodies or antigen-binding fragments thereof.

[0134] In some embodiments, an anti-SpA monoclonal antibody can be a humanized antibody.

Methods for engineering, humanizing or resurfacing non-human or human antibodies can also be used and are well known in the art. A humanized, resurfaced or similarly engineered antibody can have one or more amino acids from a source that is non-human, e.g., but not limited to, mouse, rat, rabbit, non-human primate or other mammal. These non-human amino acids are replaced by amino acids that are often referred to as "import" amino acids, which can be taken from an "import" variable, constant or other region of a known human sequence. Such imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art. In general, the CDR amino acids are directly and most substantially involved in influencing SpA binding. Accordingly, part or all of the non-human or human CDR sequences are maintained while the non- human sequences of the variable and constant regions can be replaced with human or other amino acids.

[0135] Antibodies can also optionally be humanized, resurfaced, or human antibodies engineered with retention of high affinity for the antigen SpA and other favorable biological properties. To achieve this goal, humanized (or human) or engineered anti-SpA antibodies and resurfaced antibodies can be optionally prepared by a process of analysis of the parental sequences and various conceptual humanized and engineered products using three-dimensional models of the parental, engineered, and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the amino acids in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of amino acids that influence the ability of the candidate immunoglobulin to bind its antigen, such as SpA. In this way, framework (FW) amino acids can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.

[0136] Humanization, resurfacing or engineering of anti-SpA antibodies or antigen-binding fragments thereof of the present disclosure can be performed using any known method, such as but not limited to those described in, Jones et al., Nature 321 :522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239: 1534 (1988)), Sims et al., J. Immunol. 151 : 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151 :2623 (1993), U.S. Pat. Nos. 5,639,641, 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763, 192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101 ; 5,585,089; 5,225,539; 4,816,567, 7,557,189; 7,538,195; and 7,342,110; International Application Nos. PCT/US98/16280; PCT/US96/18978; PCT/US91/09630; PCT/US91/05939; PCT/US94/01234; PCT/GB89/01334; PCT/GB91/01134; PCT/GB92/01755; International Patent Application Publication Nos. WO90/14443; WO90/14424; WO90/14430; and European Patent Publication No. EP 229246; each of which is entirely incorporated herein by reference, including the references cited therein. [0137] Anti-SpA humanized antibodies and antigen-binding fragments thereof can also be made in transgenic mice containing human immunoglobulin loci that are capable upon immunization of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

[0138] In certain embodiments an anti-SpA antibody fragment is provided. Various techniques are known for the production of antibody fragments. Traditionally, these fragments are derived via proteolytic digestion of intact antibodies (for example Morimoto et al., 1993, Journal of Biochemical and Biophysical Methods 24: 107-117; Brennan et al, 1985, Science, 229:81). In certain embodiments, anti-SpA antibody fragments are produced recombinantly. Fab, Fv, and scFv antibody fragments can be expressed in and secreted from E. coli or other host cells, thus allowing the production of large amounts of these fragments. Such anti-SpA antibody fragments can also be isolated from the antibody phage libraries discussed above. The anti-SpA antibody fragments can also be linear antibodies as described in U.S. Patent No. 5,641,870. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

[0139] According to the present disclosure, techniques can be adapted for the production of single- chain antibodies specific to SpA (see, e.g., U.S. Pat. No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see, e.g., Huse et al, Science 246: 1275- 1281 (1989)) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for SpA, or derivatives, fragments, analogs or homologs thereof. Antibody fragments can be produced by techniques in the art including, but not limited to: (a) a F(ab')2 fragment produced by pepsin digestion of an antibody molecule; (b) a Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment, (c) a Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent, and (d) Fv fragments.

[0140] In certain aspects, an anti-SpA antibody or antigen-binding fragment thereof can be modified in order to increase its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody or antibody fragment by mutation of the appropriate region in the antibody or antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody or antibody fragment at either end or in the middle {e.g., by DNA or peptide synthesis), or by YTE mutation. Other methods to increase the serum half- life of an antibody or antigen-binding fragment thereof, e.g., conjugation to a heterologous molecule such as PEG are known in the art. [0141] Modified anti-SpA antibodies or antigen-binding fragments thereof as provided herein can comprise any type of variable region that provides for the association of the antibody or polypeptide with SpA. In this regard, the variable region can comprise or be derived from any type of mammal that can be induced to mount a humoral response and generate immunoglobulins against a desired antigen. As such, the variable region of an anti-SpA antibody or antigen-binding fragment thereof can be, for example, of human, murine, or lupine origin. In some embodiments both the variable and constant regions of the modified anti-SpA antibodies or antigen-binding fragments thereof are human. In other embodiments the variable regions of compatible antibodies (e.g., derived from a non-human source) can be engineered or specifically tailored to improve the binding properties or reduce the immunogenicity of the molecule. In this respect, variable regions useful in the present disclosure can be humanized or otherwise altered through the inclusion of imported amino acid sequences.

[0142] In certain embodiments, the variable domains in both the heavy and light chains of an anti- SpA antibody or antigen-binding fragment thereof are altered by at least partial replacement of one or more CDRs and/or by partial framework region replacement and sequence changing. Although the CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and in certain embodiments from an antibody from a different species. In some instances, it is not necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen-binding capacity of one variable domain to another. Given the explanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional antibody with reduced immunogenicity.

[0143] Alterations to the variable region notwithstanding, those skilled in the art will appreciate that the modified anti-SpA antibodies or antigen-binding fragments thereof of this disclosure will comprise antibodies (e.g., full-length antibodies or antigen-binding fragments thereof) in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as reduced serum half-life when compared with an antibody of approximately the same immunogenicity comprising a native or unaltered constant region. In some embodiments, the constant region of the modified antibodies will comprise a human constant region. Modifications to the constant region compatible with this disclosure comprise additions, deletions or substitutions of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein can comprise alterations or modifications to one or more of the three heavy chain constant domains (CHI , CH2 or CH3) and/or to the light chain constant domain (CL). In some embodiments, modified constant regions wherein one or more domains are partially or entirely deleted are contemplated. In some embodiments, the modified antibodies will comprise domain deleted polypeptides or variants wherein the entire CH2 domain has been removed (ACH2 polypeptides). In some embodiments, the omitted constant region domain can be replaced by a short amino acid spacer (e.g., 10 amino acids) that provides some of the molecular flexibility previously imparted by the absent constant domain.

[0144] Anti-SpA antibodies or fragments thereof provided herein can inhibit, suppress, or block IgG Fc domains and/or VH3 Fabs from binding to SpA on the surface of S. aureus, thereby facilitating opsonization. Specifically, SpA binds to IgG Fc regions, which provides S. aureus with protection from opsonophagocytic killing. Such detrimental effects can be inhibited, suppressed, or blocked by the binding of the antibody or fragment thereof as disclosed herein to SpA.

[0145] In certain embodiments, an anti-SpA antibody or an antigen-binding fragment thereof provides for altered effector functions that, in turn, affect the biological profile of the administered antibody or antigen-binding fragment thereof. For example, the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating modified antibody. In other cases it can be that constant region modifications, consistent with this disclosure, moderate complement binding and thus reduce the serum half-life and nonspecific association of a conjugated cytotoxin. Yet other modifications of the constant region can be used to eliminate disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. Similarly, modifications to the constant region in accordance with this disclosure can easily be made using well-known biochemical or molecular engineering techniques well within the purview of the skilled artisan.

[0146] In certain embodiments, an anti-SpA antibody or antigen-binding fragment thereof can be engineered to fuse the CH3 domain directly to the hinge region of the respective modified antibodies or fragments thereof. In other polypeptides a peptide spacer can be inserted between the hinge region and the modified CH2 and/or CH3 domains. For example, compatible polypeptides can be expressed in which the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5-20 amino acid spacer. Such a spacer can be added, for instance, to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. Amino acid spacers can, in some cases, prove to be immunogenic and elicit an unwanted immune response against the polypeptide. Accordingly, in certain embodiments, any spacer added to the polypeptide can be relatively non-immunogenic, or even omitted altogether, so as to maintain the desired biochemical qualities of the modified antibodies.

[0147] Besides the deletion of whole constant region domains, anti-SpA antibodies or antigen- binding fragments thereof provided herein can be modified by the partial deletion or substitution of a few or even a single amino acid in a constant region. For example, the mutation of a single amino acid in selected areas of the CH2 domain can be enough to substantially reduce Fc binding. Similarly one or more constant region domains that control the effector function (e.g., complement C1Q binding) can be fully or partially deleted. Such partial deletions of one or more constant region domains can improve selected characteristics of the antibody or antigen-binding fragment thereof (e.g., serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Moreover, the constant regions of the disclosed anti-SpA antibodies and antigen-binding fragments thereof can be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting polypeptide. In this respect it is possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody or antigen-binding fragment thereof. Certain embodiments can comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as decreasing or increasing effector function or provide for more cytotoxin or carbohydrate attachment. In such embodiments it can be desirable to insert or replicate specific sequences derived from selected constant region domains.

[0148] The present disclosure further embraces variants and equivalents that are substantially homologous to the murine, chimeric, humanized or human anti-SpA antibodies, or antigen-binding fragments thereof, set forth herein. These can contain, for example, conservative substitution mutations, i.e., the substitution of one or more amino acids by similar amino acids. For example, conservative substitution refers to the substitution of an amino acid with another within the same general class such as, for example, one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid or one neutral amino acid by another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art.

[0149] An anti-SpA antibody or antigen-binding fragment thereof can be further modified to contain additional chemical moieties not normally part of the protein. Those derivatized moieties can improve the solubility, the biological half-life or absorption of the protein. The moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for those moieties can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000).

V. Polynucleotides Encoding SpA-Binding Molecules and Expression Thereof

[0150] This disclosure provides polynucleotides comprising nucleic acid sequences that encode a polypeptide that specifically binds SpA or an antigen-binding fragment thereof. For example, the disclosure provides a polynucleotide, or two or more polynucleotides, comprising a nucleic acid sequence that encodes an anti-SpA antibody or a subunit of an anti-SpA antibody, or encodes an antigen-binding fragment of such an antibody. The polynucleotides of the disclosure can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, e.g., modified genomic DNA, and synthetic DNA; and can be double- stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.

[0151] In certain embodiments, a polynucleotide can be isolated. In certain embodiments, a polynucleotide can be substantially pure. In certain embodiments a polynucleotide can be cDNA or are derived from cDNA. In certain embodiments a polynucleotide can be recombinantly produced. In certain embodiments a polynucleotide can comprise the coding sequence for the mature polypeptide fused in the same reading frame to a polynucleotide which aids, for example, in expression and secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell). The polypeptide having a leader sequence is a preprotein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides can also encode for a SpA-binding proprotein which is the mature protein plus additional 5' amino acids.

[0152] In certain aspects this disclosure provides an isolated polynucleotide comprising a nucleic acid encoding an antibody VL, wherein the VL comprises VL-CDR1 , VL-CDR2, and VL-CDR3 amino acid sequences identical to, or identical except for eight, seven, six, five, four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more of the VL-CDRS to: SEQ ID NOs: 47, 48, and 49, SEQ ID NOs: 50, 51 , and 52, SEQ ID NOs: 53, 56, and 55, SEQ ID NOs: 53, 54, and 55, SEQ ID NOs: 57, 58, and 59, SEQ ID NOs: 60, 61 , and 62, or SEQ ID NOs: 63, 64, and 65, respectively.

[0153] The disclosure further provides an isolated polynucleotide comprising a nucleic acid encoding an antibody VH, wherein the VH comprises VH-CDR1 , VH-CDR2, and VH-CDR3 amino acid sequences identical to, or identical except for eight, seven, six, five, four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more of the VH-CDRS to: SEQ ID NOs: 29, 30, and 31, SEQ ID NOs: 32, 33, and 34, SEQ ID NOs: 35, 36, and 37, SEQ ID NOs: 38, 39, and 40, SEQ ID NOs: 41, 42, and 43, or SEQ ID NOs: 44, 45, and 46, respectively.

[0154] The disclosure further provides an isolated polynucleotide comprising a nucleic acid encoding an antibody VL, wherein the VL comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the reference amino acid sequence SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, or SEQ ID NO: 28. In certain aspects, the polynucleotide comprises a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the reference nucleic acid sequence SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 27.

[0155] Moreover, the disclosure provides an isolated polynucleotide comprising a nucleic acid encoding an antibody VH, wherein the VH comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the reference amino acid sequence SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 26. In certain aspects, the polynucleotide comprises a nucleotide sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the reference nucleic acid sequence SEQ ID NO: 1 , SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, or SEQ ID NO: 25.

[0156] In certain aspects, an antibody or antigen-binding fragment thereof comprising a VH or VL encoded by a polynucleotide as described above, can specifically bind to SpA. In certain cases, such an antibody or antigen-binding fragment thereof can specifically bind to the same epitope as an antibody or antigen-binding fragment thereof comprising the VH and VL of SpA 33.6, SpA 03.5, SpA 53.10S, SpA 53.1, SpA 06.1, SpA 66.6, or SpA 07.27. In certain aspects the disclosure provides a polynucleotide or combination of polynucleotides encoding a binding molecule, e.g., an antibody or antigen-binding fragment thereof that specifically binds to SpA.

[0157] Further provided herein is a vector comprising a polynucleotide as described above. Suitable vectors are described elsewhere herein, and are known to those of ordinary skill in the art.

[0158] In certain aspects, the disclosure provides a composition, e.g., a pharmaceutical composition, comprising a polynucleotide or vector as described above, optionally further comprising one or more carriers, diluents, excipients, or other additives.

[0159] In certain aspects, the disclosure provides a polynucleotide composition comprising: a polynucleotide that comprises a nucleic acid encoding a VH, and polynucleotide that comprises a nucleic acid encoding a VL. According to this aspect, the VL and VH together can comprise VL- CDR1 , VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences identical or identical except for eight, seven, six, five, four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to: SEQ ID NOs: 47, 48, 49, 29, 30, and 31, SEQ ID NOs: 50, 51 , 52, 32, 33, and 34, SEQ ID NOs: 53, 56, 55, 35, 36, and 37, SEQ ID NOs: 53, 54, 55, 35, 36, and 37, SEQ ID NOs: 57, 58, 59, 38, 39, and 40, SEQ ID NOs: 60, 61, 62, 41 , 42, and 43, or SEQ ID NOs: 63, 64, 65, 44, 45, and 46, respectively.

[0160] In further aspects the disclosure provides a polynucleotide composition comprising: a polynucleotide that comprises a nucleic acid encoding a VH and polynucleotide that comprises a nucleic acid encoding a VL, wherein the VL and VH comprise, respectively, amino acid sequences at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to reference amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24, or SEQ ID NO: 26 and SEQ ID NO: 28, respectively.

[0161] In certain aspects, the VH and VL are encoded by nucleic acid sequences at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to reference nucleic acid sequences SEQ ID NO: 1 and SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23, or SEQ ID NO: 25 and SEQ ID NO: 27, respectively.

[0162] In a polynucleotide composition as described above, the polynucleotide comprising a nucleic acid encoding a VH and the polynucleotide comprising a nucleic acid encoding a VL can reside in a single vector, or can be on separate vectors. Accordingly the disclosure provides one or more vectors comprising the polynucleotide composition described above.

[0163] In some cases, a polynucleotide composition encoding a VH and VL as described above can encode an antibody or antigen-binding fragment thereof that can specifically bind to SpA. In some aspects, the polynucleotide composition encodes an antibody or antigen-binding fragment thereof that can specifically bind to the same epitope as an antibody or antigen-binding fragment thereof comprising the VH and VL of SpA 33.6, SpA 03.5, SpA 53.1 OS, SpA 53.1, SpA 06.1, SpA 66.6, or SpA 07.27.

[0164] In some instances, the polynucleotide can encode a binding molecule that has other scaffold(s), e.g., a scaffold of a lipocalin, a fibronectin domain, an ankyrin consensus repeat domain, or thioredoxin. [0165] This disclosure further provides a host cell comprising a polynucleotide, polynucleotide composition, or vector as provided above, where the host cell can, in some instances, express an antibody or antigen-binding fragment thereof that specifically binds to SpA. Such a host cell can be utilized in a method of making an antibody or antigen-binding fragment thereof as provided herein, which method includes (a) culturing the host cell and (b) isolating the antibody or antigen-binding fragment thereof expressed from the host cell.

[0166] In certain embodiments, the polynucleotides comprise the coding sequence for the mature SpA-binding polypeptide, e.g., an anti-SpA antibody or an antigen-binding fragment thereof, fused in the same reading frame to a marker sequence that allows, for example, for purification of the encoded polypeptide. For example, the marker sequence can be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used.

[0167] Polynucleotide variants are also provided. Polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli). Vectors and cells comprising the polynucleotides described herein are also provided.

[0168] In some embodiments, a DNA sequence encoding a SpA-binding molecule, e.g., an anti-SpA antibody or an antigen-binding fragment thereof, can be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides can contain 5' or 3' overhangs for complementary assembly. [0169] Once assembled (by synthesis, site-directed mutagenesis or another method), the polynucleotide sequences encoding a particular isolated polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed, e.g., by nucleotide sequencing, restriction mapping, and/or expression of a biologically active polypeptide in a suitable host. In order to obtain high expression levels of a transfected gene in a host, the gene can be operatively linked to or associated with transcriptional and translational expression control sequences that are functional in the chosen expression host.

[0170] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding anti-SpA antibodies or antigen-binding fragments thereof. Recombinant expression vectors are replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of an anti-SpA antibody or and antigen-binding fragment thereof, operatively linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. In one example, a transcriptional unit can comprise an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences, as described in detail below. Such regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, conferred, e.g., by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are operatively linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where a recombinant protein is expressed without a leader or transport sequence, the protein can include an N-terminal methionine. This methionine can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

[0171] The choice of expression control sequence and expression vector will depend upon the choice of host. A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR 1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as Ml 3 and filamentous single-stranded DNA phages.

[0172] Suitable host cells for expression of a SpA-binding molecule, e.g., an anti-SpA antibody or antigen-binding fragment thereof include prokaryotes, yeast, insect or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems could also be employed. Additional information regarding methods of protein production, including antibody production, can be found, e.g., in U.S. Patent Publication No. 2008/0187954, U.S. Patent Nos. 6,413,746 and 6,660,501, and International Patent Publication No. WO 04009823, each of which is hereby incorporated by reference herein in its entirety.

[0173] Various mammalian or insect cell culture systems can also be employed to express recombinant SpA-binding molecules, e.g., anti-SpA antibodies or antigen-binding fragments thereof. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly folded, appropriately modified and completely functional. Examples of suitable mammalian host cell lines include HEK-293 and HEK-293T, the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23: 175, 1981), and other cell lines including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian expression vectors can comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, BioTechnology 6:47 (1988).

[0174] SpA-binding molecules, e.g., anti-SpA antibodies or antigen-binding fragments thereof produced by a transformed host, can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence and glutathione- S -transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance and x-ray crystallography.

[0175] For example, supernatants from systems that secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a SpA-binding molecule. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.

[0176] A recombinant SpA-binding protein, e.g., an anti-SpA antibody or antigen-binding fragment thereof produced in bacterial culture, can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

[0177] Methods known in the art for purifying antibodies and other proteins also include, for example, those described in U.S. Patent Publication Nos. 2008/0312425, 2008/0177048, and 2009/0187005, each of which is hereby incorporated by reference herein in its entirety.

VI. Treatment Methods Using Therapeutic Anti-SpA Antibodies

[0178] Methods are provided for the use of SpA binding molecules, e.g., anti-SpA antibodies, including antigen-binding fragments, variants, and derivatives thereof, to treat patients having a disease or condition associated with a Staphylococcus infection, or to prevent, reduce, or manage SpA-induced virulence in a subject infected with Staphylococcus bacteria. [0179] The following discussion refers to diagnostic methods and methods of treatment of various diseases and disorders with a SpA-binding molecule, e.g., an anti-SpA antibody or antigen-binding fragment, variant, or derivative that retains the desired properties of anti-SpA antibodies provided herein, e.g., capable of specifically binding SpA and antagonizing SpA activity. In some embodiments, SpA-binding molecules are murine, human, or humanized antibodies. In some embodiments, the SpA-antibody is identical to one of the monoclonal antibodies SpA 33.6, SpA 03.5, SpA 53.10S, SpA 53.1, SpA 06.1, SpA 66.6, or SpA 07.27. In some embodiments, the SpA- antibody is derived from one of the monoclonal antibodies SpA 33.6, SpA 03.5, SpA 53.10S, SpA 53.1, SpA 06.1 , SpA 66.6, or SpA 07.27. In certain embodiments the derived antibody is a humanized antibody such as SpA 53.10S. In other embodiments, the SpA-binding molecule comprises a YTE-mutated human IgGl constant region. In specific embodiments, the SpA-binding molecule is SpA 53.1 OS.

[0180] In one embodiment, treatment includes the application or administration of a SpA binding molecule, e.g., an anti-SpA antibody or antigen-binding fragment, variant, or derivative thereof as provided herein, to a subject or patient, where the subject or patient has a disease, a symptom of a disease, or a predisposition toward a disease. In another embodiment, treatment can also include the application or administration of a pharmaceutical composition comprising a SpA binding molecule, e.g., an anti-SpA antibody or antigen-binding fragment, variant, or derivative thereof as provided herein, to a subject or patient, or application or administration of a pharmaceutical composition comprising a SpA binding molecule to an isolated tissue or cell line from a subject or patient, who has a disease, a symptom of a disease, or a predisposition toward a disease.

[0181] SpA binding molecules, e.g., anti-SpA antibodies or antigen-binding fragments, variants, or derivatives thereof provided herein, are useful for the treatment of various diseases or disorders. In one embodiment, SpA binding molecules, e.g., anti-SpA antibodies or antigen-binding fragments, variants, or derivatives thereof, are provided for use in the treatment, prevention, or management of a condition or disease associated with a Staphylococcus infection. Examples of the conditions or diseases include, but not limited to, pneumonia, or a skin condition such as dermonecrosis. Staphylococcus infections can be caused by, without limitation, S. aureus, which can be resistant to antibiotics such as methicillin. In certain aspects, the amount of the SpA binding molecules, e.g., anti-SpA antibodies or antigen-binding fragments, variants, or derivatives thereof provided herein, is effective to reduce B-cell receptor cross linking induced by SpA. [0182] In accordance with the methods of the present disclosure, at least one SpA binding molecule, e.g., an anti-SpA antibody or antigen-binding fragment, variant, or derivative thereof as defined elsewhere herein, is used to promote a positive therapeutic response. By "positive therapeutic response" is intended any improvement in the disease conditions associated with the activity of these binding molecules, e.g., anti-SpA antibodies or antigen-binding fragments, variants, or derivatives thereof, and/or an improvement in the symptoms associated with the disease. Thus, for example, an improvement in the disease can be characterized as a complete response. By "complete response" is intended an absence of clinically detectable disease with normalization of any previously test results. Such a response can in some cases persist, e.g., for at least one month following treatment according to the methods of the disclosure. Alternatively, an improvement in the disease can be categorized as being a partial response.

VII. Pharmaceutical Compositions and Administration Methods

[0183] Methods of preparing and administering SpA binding molecules, e.g., anti-SpA antibodies, or antigen-binding fragments, variants, or derivatives thereof provided herein, to a subject in need thereof are well known to or are readily determined by those skilled in the art. The route of administration of the SpA binding molecule, e.g., the anti-SpA antibody, or antigen-binding fragment, variant, or derivative thereof, can be, for example, oral, parenteral, by inhalation or topical. The term parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While these forms of administration are contemplated as suitable forms, another example of a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. A suitable pharmaceutical composition can comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. In other methods compatible with the teachings herein, SpA binding molecules, e.g., anti-SpA antibodies, or antigen-binding fragments, variants, or derivatives thereof as provided herein, can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent. In one embodiment, the administration is directly to the airway, e.g., by inhalation or intranasal administration.

[0184] As discussed herein, SpA binding molecules, e.g., anti-SpA antibodies, or antigen-binding fragments, variants, or derivatives thereof provided herein, can be administered in a pharmaceutically effective amount for the in vivo treatment of diseases or disorders associated with Staphylococcus infection. In this regard, it will be appreciated that the disclosed binding molecules can be formulated so as to facilitate administration and promote stability of the active agent. Pharmaceutical compositions accordingly can comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. A pharmaceutically effective amount of a SpA binding molecule, e.g., an anti-SpA antibody, or antigen-binding fragment, variant, or derivative thereof, conjugated or unconjugated, means an amount sufficient to achieve effective binding to a target and to achieve a benefit, e.g., to ameliorate symptoms of a disease or condition or to detect a substance or a cell. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).

[0185] Certain pharmaceutical compositions provided herein can be orally administered in an acceptable dosage form including, e.g., capsules, tablets, aqueous suspensions or solutions. Certain pharmaceutical compositions also can be administered by nasal aerosol or inhalation. Such compositions can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.

[0186] The amount of a SpA binding molecule, e.g., an anti-SpA antibody, or fragment, variant, or derivative thereof that can be combined with carrier materials to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration. The composition can be administered as a single dose, multiple doses or over an established period of time in an infusion. Dosage regimens also can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).

[0187] In keeping with the scope of the present disclosure, anti-SpA antibodies, or antigen-binding fragments, variants, or derivatives thereof can be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic effect. The anti-SpA antibodies or antigen-binding fragments, variants, or derivatives thereof provided herein can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody or antigen-binding fragment, variant, or derivative thereof of the disclosure with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. The form and character of the pharmaceutically acceptable carrier or diluent can be dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. A cocktail comprising one or more species of SpA binding molecules, e.g., anti-SpA antibodies, or antigen-binding fragments, variants, or derivatives thereof, can also be used.

[0188] By "therapeutically effective dose or amount" or "effective amount" is intended an amount of a SpA binding molecule, e.g., antibody or antigen-binding fragment, variant, or derivative thereof, that when administered brings about a positive therapeutic response with respect to treatment of a patient with a disease or condition to be treated.

[0189] Therapeutically effective doses of the compositions disclosed herein, for treatment of diseases or disorders associated with Staphylococcus infection, vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. In certain aspects, the subject or patient is a human, but non-human mammals including transgenic mammals can also be treated. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[0190] The amount of at least one SpA binding molecule, e.g., antibody or binding fragment, variant, or derivative thereof, to be administered is readily determined by one of ordinary skill in the art without undue experimentation given this disclosure. Factors influencing the mode of administration and the respective amount of at least one SpA binding molecule, e.g., antibody, antigen-binding fragment, variant or derivative thereof, include, but are not limited to, the severity of the disease, the history of the disease, and the age, height, weight, health, and physical condition of the individual undergoing therapy. Similarly, the amount of a SpA binding molecule, e.g., antibody, or fragment, variant, or derivative thereof, to be administered will be dependent upon the mode of administration and whether the subject will undergo a single dose or multiple doses of this agent.

[0191] This disclosure also provides for the use of a SpA binding molecule, e.g., an anti-SpA antibody or antigen-binding fragment, variant, or derivative thereof, in the manufacture of a medicament for treating, preventing, or managing a disease or disorder associated with Staphylococcus infection, e.g., pneumonia, or a skin condition.

VIII. Diagnostics

[0192] This disclosure further provides a diagnostic method useful during diagnosis of a condition mediated by Staphylococcus bacteria, which involves administering to a subject in need thereof a diagnostically effective amount of a SpA binding molecule or composition disclosed herein, e.g., an anti-SpA antibody or antigen-binding fragment, variant, or derivative thereof, and detecting presence of a biological effect associated with the administration of the SpA binding molecule or the composition, whereby detection of a biological effect associated with the administration of the SpA binding molecule or the composition is indicative of presence of a condition mediated by Staphylococcus.

IX. Kits comprising SpA-binding Molecules

[0193] This disclosure further provides kits that comprise a SpA binding molecule, e.g., an anti-SpA antibody or antigen-binding fragment thereof, described herein and that can be used to perform the methods described herein. In certain embodiments, a kit comprises at least one purified anti-SpA antibody or an antigen-binding fragment thereof in one or more containers. In some embodiments, the kits contain all of the components necessary and/or sufficient to perform a detection assay, including controls, directions for performing assays, and any desired software for analysis and presentation of results. One skilled in the art will readily recognize that the disclosed SpA-binding molecules, e.g., anti-SpA antibodies or antigen-binding fragments thereof, can be readily incorporated into one of the established kit formats which are well known in the art.

X. Immunoassays

[0194] SpA binding molecules provided herein, e.g., anti-SpA antibodies or antigen-binding fragments, variants, or derivatives thereof, can be assayed for immunospecific binding by any method known in the art. The immunoassays that can be used include but are not limited to competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol. 1, which is incorporated by reference herein in its entirety).

[0195] SpA-binding molecules, e.g., anti-SpA antibodies or antigen-binding fragments, variants, or derivatives thereof provided herein, can be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of SpA or conserved variants or peptide fragments thereof. In situ detection can be accomplished by removing a histological specimen from a patient, and applying thereto a labeled SpA-binding molecule, e.g., an anti-SpA antibody or antigen-binding fragment thereof, variant, or derivative thereof, e.g., applied by overlaying the labeled SpA-binding molecule (e.g., and antibody or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of SpA, or conserved variants or peptide fragments, but also its distribution in the examined tissue. With the present disclosure, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

[0196] The binding activity of a given lot of a SpA-binding molecule, e.g., an anti-SpA antibody or antigen-binding fragment, variant, or derivative thereof, can be determined according to well-known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

[0197] Methods and reagents suitable for determination of binding characteristics of an isolated SpA-binding molecule, e.g., an anti-SpA antibody or antigen-binding fragment, variant, or an altered/mutant derivative thereof, are known in the art and/or are commercially available. Equipment and software designed for such kinetic analyses are commercially available (e.g., BIAcore®, BIAevaluation® software, GE Healthcare; KINEXA® Software, Sapidyne Instruments, ForteBio Octet).

XL Mutant SpA and Antigens

In one aspect, the disclosure provides a mutant SpA IgG/VH3 binding domain region polypeptide, wherein the polypeptide exhibits reduced binding to the Fc region of IgG, to a VH3 Fab region, or to both compared to a wild-type IgG/VH3 binding domain region polypeptide comprising the amino acid sequence of SEQ ID NO: 66. For example, the mutant SpA polypeptide can comprise the amino acid sequence:

1 naaqhdeaqq naX₁₃X₁₄qvlnmp nlnadqrX₂sgf iqX₃₃ lkddpsq sanvlgeaqk 50

51 lndsqapkad aqqnnfnkdq qsafyeilnm pnlneaqrng fiqslkddps 100

101 qstnvlgeak klnesqapka dnnfnkeqqn afyeilnmpn lneeqrngfi 150

151 qslkddpsqs anllseakkl nesqapkadn kfnkeqqnaf yeilhlpnln 200

201 eeqrngfiqs lkddpsqsan llaeakklnd aqapkadnkf nkeqqnafye 250

251 ilhlpnltee qrngfiqslk ddpsvskeil aeakklndaq apkeednnkp 300

301 gkednnkpgk ednnk (SEQ ID NO: 67), where X₁₃ is F, R, H, or L; X₁₄ is Y, R, H, K, A, or F; X₂s is N or A; and X₃₃ is S, I, L, A, G, V, R, H, or K. The polypeptide can include at least one amino acid substitution relative to SEQ ID NO: 66, and the polypeptide can exhibit reduced binding to the Fc region of IgG, to a VH3 Fab region, or to both, compared to a wild-type IgG/VH3 binding domain region polypeptide including the amino acid sequence of SEQ ID NO: 66.

[0198] In some instances, the polypeptide comprises (a) substitution of Serine (S) at position 33 with Isoleucine (I), Leucine (L), Alanine (A), Glycine (G), Valine (V) Arginine (R), Histidine (H), or Lysine (K); (b) substitution of Asparagine (N) at position 28 with Alanine (A); (c) substitution of Phenylalanine (F) at position 13 with Arginine (R), Histidine (H), or Lysine (K); (d) substitution of Tyrosine (Y) at position 14 with Arginine (R) , Histidine (H), or Lysine (K), Alanine (A), or Phenylalanine (F); (e) substitutions of Phenylalanine (F) at position 13 with Arginine (R) and Tyrosine (Y) at position 14 with Phenylalanine (F); (f) substitution of Phenylalanine (F) at position 13 with Arginine (R) and Serine (S) at position 33 with Isoleucine (g) substitutions of Tyrosine (Y) at position 14 with Arginine (R) and Serine (S) at position 33 with Isoleucine (I); or any combination of (a)-(g).

[0199] In certain embodiments a mutant SpA polypeptide comprises a single amino acid substitution relative to SEQ ID NO: 66, for example the Tyrosine (Y) at position 14 can be substituted with Arginine (R) (SEQ ID NO: 68), or the Serine (S) at position 33 can be substituted with Isoleucine (I) (SEQ ID NO: 69).

[0200] In certain embodiments a mutant SpA polypeptide comprises two amino acid substitutions relative to SEQ ID NO: 66. For example, a mutant SpA polypeptide can comprise substitutions at the Phenylalanine (F) at position 13 with Arginine (R) and the Serine (S) at position 33 with Isoleucine (I) (SEQ ID NO: 70) (F13R-S33I), or substitutions of the Tyrosine (Y) at position 14 with Arginine (R) and Serine (S) at position 33 with Isoleucine (I) (SEQ ID NO: 71) (Y14R-S33I).

[0201] The disclosure further provides a composition for eliciting or modulating an immune response to Staphylococcus bacteria in a subject in need thereof. Such composition comprises a mutant SpA polypeptide described above as an antigen. In some instances, the composition further comprises one or more adjuvants. Examples of adjuvants include, but are not limited to aluminum hydroxide and paraffin oil.

[0202] In some instances, a method of modulating an immune response to Staphylococcus infection in a subject disclosed herein includes administering to a subject in need thereof an effective amount of an antibody described above, wherein the antibody blocks B-cell superantigen activity. In certain embodiments, the subject is a domestic animal or a human. In certain embodiments, the domestic animals include food animals and companion animals.

[0203] The practice employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0204] General principles of antibody engineering are set forth in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). General principles of protein engineering are set forth in Rickwood et al., eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). General principles of antibodies and antibody-hapten binding are set forth in: Nisonoff (1984) Molecular Immunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward (1984) Antibodies, Their Structure and Function (Chapman and Hall, New York, N.Y.). Additionally, standard methods in immunology known in the art and not specifically described can be followed as in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al., eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange, Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods in Cellular Immunology (W.H. Freeman and Co., NY). [0205] Standard reference works setting forth general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination (John Wiley & Sons, NY); Kennett et al., eds. (1980) Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses (Plenum Press, NY); Campbell (1984) "Monoclonal Antibody Technology" in Laboratory Techniques in Biochemistry and Molecular Biology, ed. Burden et al., (Elsevier, Amsterdam); Goldsby et al., eds. (2000) Kuby Immunology (4th ed.; H. Freemand & Co.); Roitt et al. (2001) Immunology (6th ed.; London: Mosby); Abbas et al. (2005) Cellular and Molecular Immunology (5th ed. ; Elsevier Health Sciences Division); Kontermann and Dubel (2001) Antibody Engineering (Springer Verlag); Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press); Lewin (2003) Genes VIII (Prentice Hall, 2003); Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

[0206] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

[0207] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

[0208] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain antibodies of the present disclosure and methods for using antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.

Example 1: Construction of SpA Mutants

[0209] Since it is nearly impossible to pan efficiently on a protein that can bind non-specifically to IgG through B-cell superantigen activity and through binding of VH3 B-cell receptors, ligand- ablated mutant SpA was used for generating anti-SpA monoclonal antibodies. Anti-SpA antibodies can have the capability of blocking SpA ligand binding, allowing and/or facilitating opsonophagocytic killing, or improving immune responses to other Staph antigens during infection by blocking B-cell superantigen activity, and/or having broad strain coverage. This study explored constructing SpA mutants with Fc and/or VH3 Fab ablation(s). [0210] DNA sequences containing the desired mutations were designed and constructed using standard methods. Plasmid DNA encoding mutant SpA proteins were cloned into E. coli using standard methods. Mutant SpA proteins were tested for lack of Fc and/or VH3 Fab binding using ELISA and Octet binding methods.

[0211] Various SpA mutants were constructed comprising ablated Fc-binding, ablated VH3 Fab- binding, or both, compared to a wild-type SpA. For ablation of VH3 Fab binding, S33 I, L, & R mutations were constructed. For ablation of IgG Fc binding, F13R and Y14R mutants were constructed. For Fc and Fab double ablation, mutations at both amino acid positions 13 and 33 or at both amino acid positions 14 and 33 were constructed (see Table 2).

Table 2: Potential Fc and Fab Ablations

Example 2: Properties of SpA Mutants [0212] CD-I mice were actively immunized by subcutaneous (SC) injection on days 1 & 11 with immunogen CFA/IFA (complete Freund's adjuvant/incomplete Freund's adjuvant; Difco), Wild- type SpA (WT SpA), double ablated mutant KKAA (Kim, H.K., et al , 2012 Infect. Immun. 80:3460-3470), double ablated mutant F13R-S33I, or double ablated mutant Y14R-S33I on days 1 & 11. Four days post-immunization the animals (n=20) were challenged with 1 X 10⁸ CFU of S. aureus USA300. At 24 hours post-challenge, the animals were euthanized and the staphylococcal load was enumerated in kidneys (FIG. 1A) and spleens (FIG. IB). The results indicate that mutant SpA can lower bacterial burden in the kidney (e.g., all of the mutants tested) and in the spleen (e.g., Y14R-S33I) as compared to CFA/IFA control mice.

[0213] CD-I mice were actively immunized by subcutaneous (SC) injection on days 1 & 11 with immunogen CFA/IFA, single Fc ablated mutant Y14R, single ablated VH3 Fab mutant S33I, or double ablated mutant Y14R-S33I. Four days post-immunization, the animals (n=10) were challenged with 1 X 10⁸ CFU of S. aureus USA300. 1 day post-challenge, the animals were euthanized and the staphylococcal load was enumerated in kidneys (FIG. 2A) and spleens (FIG. 2B). The results indicate that mutant SpA can lower bacterial burden in the kidney (e.g., all of the mutants tested) and in the spleen (e.g., Y14R-S33I) as compared to CFA/IFA control mice.

[0214] C57B1/6 mice were actively immunized by subcutaneous (SC) injection on days 1 & 11 with immunogen CFA/IFA, the KKAA mutant (Kim, H.K., et al, 2012 Infect. Immun. 80:3460- 3470), mutant F13R-S33I, or mutant Y14R-S33I. Four days post-immunization, the animals (n=10) were challenged with 5.2 X 10⁷ CFU of 5". aureus USA300. At 1 day post-challenge, the animals were euthanized, and the bacterial loads in the lungs of infected animals were determined. The results are shown in FIG. 3. The results indicate that mutant SpA (e.g., Y14R-S33I) can lower bacterial burden in the lungs of IN-immunized animals as compared to CFA/IFA control mice.

[0215] C57B1/6 mice were actively immunized by subcutaneous (SC) injection on days 1 & 11 with immunogen CFA/IFA or mutant Y14R-S33I. Four days post-immunization the animals (n=9) were challenged with 8 X 10⁷ CFU of 5". aureus USA300. At 48 hours post-challenge, the animals were euthanized, and the bacterial loads in the lungs of infected animals were determined. The results are shown in FIG. 4. The results indicate that Y14R-S33I can lower lung bacterial burden in a pneumonia model as compared to CFA/IFA control mice.

Example 3: Production of Anti-SpA Murine Monoclonal Antibodies

Immunization of Mice and Anti-SpA Hybridoma Generation [0216] Eight mice of each of the three strains Balb/C, SJL, and CD-I were immunized with Y14R- S33I mutant SpA Thirteen (13) days of Ultra short RIMMS (repetitive immunization at multiple sites) followed by RIMMS immunization protocol with Y14R-S33I mutant SpA in CFA/IFA and TiterMax adjuvant was used. Sera from the immunized mice were tested periodically for binding to Y14R-S33I mutant SpA and ability to compete with binding of human Fc and Fab to WT SpA. Splenocytes and lymph node cells were isolated, enriched for either antigen-specific or B-cell specific population and fused with myeloma fusion partner on days 16, 41 & 61.

Isolation, Cloning, Sequencing, Expression, and Purification of Anti-SpA Murine mAbs

[0217] Roughly 7,500 hybridomas were constructed, which were then screened for HTRF binding to YRSI SpA, and Fab & Fc antagonism from WT SpA. About 100 hybridomas were selected for another round of screening, which included HTRF Fab & Fc displacement assays, octet binding assays to SpA domains, and ELISA binding assay in normal human serum. 23 hybridomas were selected for limited dilution cloning and resulting monoclonal antibodies were selected for further evaluation.

[0218] The 23 monoclonal antibodies were scaled up, purified and tested for HTRF Fab & Fc displacement assays, affinity to WT SpA, Percent Retained Binding in Normal Human Serum, Opsonophagocytic Killing, Ex vivo binding, and relevant in vivo intraperitoneal and intranasal models on infection.

[0219] Variable domain genes of 6 antibodies, SpA 33.6, SpA 03.5, SpA 53.1, SpA 06.1, SpA 66.6, or SpA 07.27, were cloned using standard protocols. Briefly, mRNA was isolated using oligo-dT magnetic beads (Invitrogen), converted into cDNA using Superscript III reverse transcriptase (Invitrogen), and V-genes amplified by PCR using mouse Ig primer set (Novagen). PCR products were cloned into pCR2.1 vector using TOPO TA cloning kit (Invitrogen) and sequenced using M13R primer.

[0220] Variable mouse Ig genes of each of the antibodies were cloned into the pOE mammalian expression vector which contains human constant IgGl genes. The vectors were transfected into 293F cells using standard protocol (Invitrogen). Chimeric antibodies were expressed into culture supernatant and harvested on day 9. Chimeric antibodies were purified using Protein G column chromatography (GE).

Humanization of Anti-SpA Murine mAbs [0221] Murine SpA 53.1 monoclonal antibody was humanized by grafting its CDRs onto closest human frameworks. 15 humanized SpA53.1 variants were constructed using molecular cloning and gene synthesis techniques. Addressing potential aspartic acid isomerization risk, Asp56 in VLCDR2 sequence was mutated using standard site-specific mutagenesis protocol to glutamic acid, SpA53.10E, or to serine, SpA53.10S.

Example 4: Properties of Anti-SpA Monoclonal Antibodies

HTRF Fab & Fc Displacement

[0222] Selected anti-SpA antibodies as listed in Figure 5 were tested for their ability to displace corresponding Fab and Fc fragments. The assay measures the ability the anti-SpA mAbs to displace already bound ligands at 15 to 500-fold lower concentrations than the bound ligands. A good anti- SpA response results in a loss of signal in this assay where the ligand is in excess of the anti-SpA monoclonal antibody. Recombinant biotinylated WT SpA, Eu³⁺ Cryptate conjugated Streptavidin (Cisbio) and fluorescently labeled pooled human Fab and Fc of selected 53.1 variants were incubated for 2 hours at room temperature to form complexes. Indicated amounts of the anti-SpA 53.1 IgG variants were added to measure their ability to displace the ligands from WT SpA. HTRF signal was measured and calculated according to manufacturer's protocol (Cisbio). The results for Fab are shown in FIG. 5A and for Fc are shown in FIG. 5B.

Octet Affinity to Wild-type SpA

[0223] Biotinylated WT SpA was immobilized onto Octet Streptavidin sensors at 0.2 μg/mL.

Association and disassociation was measured in seven 2-fold dilutions of selected murine anti-SpA mAbs from 100 to 1.56 nM, plus zero mAb. Association / disassociation data was exported to Graphpad Prism for global curve fitting of data to determine k_cff, k_on, and ¾. The results show that most of the anti-SpA monoclonal antibodies tested have affinity to wild-type SpA in the range of 30 -200 pM, whereas SpA 03.5 exhibits notably low affinity of ~7 nM and SpA 06.1 exhibits remarkably high affinity of ~ 15 pM (see Table 3 Table 3: Octet Affinity Data

Octet Binding Assay to SpA Domains

[0224] Five individual SpA wild-type domains were cloned, expressed, and purified by standard methods. The domains were all biotinylated during expression using the Avitag system.

Comparable to the affinity assays above, biotinylated SpA domains were immobilized onto streptavidin Octet sensors (ForteBio) at 1 μg/mL. Association and disassociation of anti-SpA mAbs was measured at a single concentration, 10 μg/mL (67 nM).

[0225] The results are shown in Table 4. Twelve (12) mAbs bound to all five domains well. The k_on and k₀ff were scored by subjective observation of the binding curves to each domain to summarize domain binding data (see domain binding score). Table 4: Domain Binding Score of Various Anti-SpA mAbs

Retained Binding to SpA in Human Serum

[0226] Octet assay was conducted comparing binding of various anti-SpA mAbs to Protein A sensors at 100 μg/mL in buffer or in 95% normal human serum (NHS) (10-20 mg/mL hlgG, 100 to 200-fold excess). Bound murine anti-SpA mAbs were then detected using goat anti-mouse IgG. Anti-SpA mAbs in buffer or 95% NHS were allowed to bind Octet Protein A sensors (ForteBio) prior to quantifying the amount of bound murine anti-SpA mAbs with goat anti-mouse detection (Jackson Immuno Research). The results are shown in FIG. 6A (buffer) and FIG. 6B (95% NHS). The Octet signal in buffer and NHS of the goat anti-mouse IgG detection steps were then compared to determine the Percent Retained Binding in NHS. Data are Means + standard deviations.

[0227] The results show that eight (8) of twenty-three (23) anti-SpA mAbs exhibited minimal or no apparent retained binding, two (2) of twenty-three (23) anti-SpA mAbs did not meet the 50% threshold, thirteen (13) out of twenty-three (23) anti-SpA mAbs exhibited greater than 50% retained binding in normal human serum (NHS) (FIG. 6C). Opsonophagocytic Killing

[0228] Assays were run to test if the anti-SpA mAbs could promote opsonophagocytosis, The opsonophagocytic killing (OPK) assay involved combining ΙΟμΙ of S. aureus (10⁶ cells/ml), ΙΟμΙ of monoclonal antibody, and 60μ1 of DMEM plus 0.1 % gelatin. The solution was incubated for 30 minutes at 4°C. After 30 minutes, 10 μΐ of human promyelocytic leukemia (HL-60) cells at 10⁷ cells/ml were added, along with 10 μΐ of human sera pre-absorbed against S. aureus. At time TO, 10 μΐ of solution was plated and then incubated at 37°C with 1500 rpm of shaking for 60 minutes. At time T60, the HL-60 cells were lysed with 1% saponin, replated, and CFU concentration determined. The percentage OPK was calculated as calculated as follows: 100 x (1- (T60/T0)), where T60 refers to the CFU concentration at the end of the assay (i.e., at 60 minutes) and TO refers to the CFU concentration at the beginning of the assay. The results are shown in FIG. 7. The six monoclonal anti-SpA antibodies were opsonic against S. aureus strain Newman as compared to control antibody R347.

Ex vivo Binding

[0229] To determine whether the SpA protein targeted by the antibodies was expressed by S. aureus in vivo, antibody binding was assessed following in vivo passage in mice. Mice were challenged intraperitoneally with approximately 5xl0⁸ CFU of S. aureus strain SF8300. After 1 hour, the mice were exsanguinated and blood was pooled into ice cold citrate. Eukaryotic cells were lysed with 1 % NP-40. Lysed cells were washed three times with phosphate buffered saline (PBS) and sonicated, followed by resuspension of S. aureus bacteria in buffer (approximately 0.5-10 x 10⁶ CFU were recovered after lysis and resuspension). Anti-SpA antibodies were administered to S. aureus- containing cell lysates, and to in vitro-grown S. aureus, and antibody binding was evaluated by staining and FACS sorting. R347 mAb was run as a negative control, and anti-LTA mAb as a positive control.

[0230] FIGS. 8A and 8C show the binding of anti-SpA mAbs to in vitro grown S. aureus SF8300.

Chimeric anti-SpA antibodies 3.5 and 66.6 (Panel A) as well as the murine anti-SpA antibodies (Panel C) could bind to culture-grown bacteria, as could the LTA positive control and antibody. FIGS. 8B and 8D show the binding of anti-SpA mAbs to bacteria recovered after a 1-hr in vivo passage in mice. Chimeric anti-SpA antibody 66.6 (Panel B) as well as the murine anti-SpA antibodies (Panel D) could bind to in v vo-passaged bacteria, as could the LTA positive control antibody. Whole Cell Affinity

[0231] The whole cell affinity was measured at equilibrium as opposed to kinetic measurements.

Such assay can also be used to measure binding in competition with normal human serum

[0232] Various concentrations of antibodies 53.10S, 3F6, and negative control R347 were Europium-labeled according to manufacturer's instructions. (Eu³⁺, Delfia, Perkin Elmer). Antibody 3F6 is an anti-SpA positive control mAb against an SpA mutant (SpAKKAA; Kim, H.K., et al., 2012 Infect. Immun. 80:3460-3470). The labeled antibodies were incubated with fixed amounts of S. aureus strain SF8300 cells (Methicillin-resistant Staphylococcus aureus SF8300 (a USA300 strain)), provided by Binh Diep (University of California, San Francisco), ~ 4xl0⁵ cells CFU), and allowed to bind on ice for 1 nr. Unbound antibody was removed by washing, and the amount of bound antibody determined using time resolved fluorescence of the Eu³⁺ tag.

[0233] Non-specific signal (NSS, signal in the absence of cells) was subtracted to calculate specific binding, which was then fit using Graphpad Prism using One site specific binding with Hill slope (a measure of binding heterogeneity). The results, including the calculated binding affinities, are shown in FIGS. 9A-C.

[0234] These data show that while both anti-SpA mAbs lose affinity on whole cells compared to the biochemical Octet assays shown in Table 3, SpA 53.10S had a ~10-fold loss, compared to the -60- fold loss for the benchmark 3F6 anti-SpA mAb, which in the context of a whole bacterium no had an affinity no better than the irrelevant negative control IgG, R347.

Whole Cell Retained Binding Assay

[0235] A similar assay to the Whole Cell Affinity assay above was run to compare binding in ligand-receptor buffer (LRB, Perkin Elmer) and 50% normal human serum (NHS) in LRB. As above, non-specific signal controls were subtracted from total binding in LRB and total binding in

50% NHS (labeled 'Total' in FIGS. 10 A-C).

[0236] Bound ng mAb at 150 nM in LRB and 50% NHS were compared to determine whole cell percent retained binding. The results are shown in FIGS. lOA-C.

[0237] These data indicate SpA 53.1 OS is able to retain a large portion of its binding while in the context of whole cells and the relevant milieu of human serum. The negative control R347 and the benchmark 3F6 mAbs had no detectable binding at clinically achievable serum [mAb] of 150 nM while in the excess human IgG of serum. [0238] The data in figures 9 and 10 illustrate that S A 53.1 OS can retain binding to whole bacteria while in the relevant context of normal human serum, containing an excess of irrelevant IgG. At the highest mAb concentrations, human IgG from serum can be in ~200-fold excess

Example 5: In vivo Studies of Anti-SpA mAbs

IP Organ Burden

[0239] Cd-1 mice were passively vaccinated by intraperitoneal (IP) injection with R347, SpA 53.10, SpA 53.10E, or SpA 53.10S. 24 hours post- vaccination, the animals were challenged with 8 X 10⁷ CFU of S. aureus USA300. Twenty-four (24) hours post challenge, the animals were euthanized and the staphylococcal load in the kidneys and spleens of the infected animals was enumerated. The results are shown in FIGS. 11A (kidney) and 11B (spleen).

[0240] The data show the D56E and D56S versions of SpA 53.10 (53.10E and 53.10S) could significantly lower kidney bacterial burdens, and 53.10S could significantly lower organ CFU in both kidneys and spleens (Statistical significance was determined by Kruskal-Wallis analysis (p<0.05)).

IV Kidney Burden

[0241] Balb/C mice were passively vaccinated by intraperitoneal (IP) injection with R347 or SpA 53.10S. 24 hours post- vaccination, the animals were challenged with about 5 X 10⁶ CFU of 5". aureus SF8300 intravenously (IV). 96 hours post challenge, the animals were then euthanized and the staphylococcal load was enumerated in kidneys of the infected animals.

[0242] The data show the D56S version of SpA 53.10 (53.10S) can lower kidney burdens following IV challenge. Statistical significance was determined by Kruskal-Wallis analysis (p<0.05).

Example 6: In vivo Studies of Anti-SpA mAbs in Combination with LC10

[0243] IN Survival Assay C57B1/6 mice were passively vaccinated with control antibody R347, SpA 53.10, or SpA 53.10S in combination with anti-S. aureus alpha toxin antigen binding fragment LC10 at 3 mg/kg each antibody. Twenty-four (24) hours post-vaccination, the animals were challenged with 1.8 X 10⁸ CFU of S. aureus SF8300 intra-nasally (IN), and then monitored for survival over 6 days. The results are shown in FIG. 14. LC10 in combination with any antibody can provide a survival advantage over R347 controls [0244] LC10 in combination with any antibody could improve survival over R347 controls (Log Rank Test, p<0.05).

IV Survival Assay

[0245] Balb/C mice were passively vaccinated with control antibody R347, SpA 53.1, or anti-ClfA mAb 11H10, alone or in combination with LC10 at 1 mg/kg each antibody. Twenty-four (24) hours post-vaccination, the animals were challenged with 3 X 10⁶ CFU of 5". aureus SF8300 intravenously, and then monitored for survival over 14 days.

[0246] SpA 53.1 in combination with LC10 (as well as 11H10 in combination) could improve survival over LC10 alone (Log Rank Test, p<0.05)

***

[0247] The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

Claims

1. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to an epitope of Staphylococcus aureus protein A (SpA), wherein the binding molecule specifically binds to the same SpA epitope as an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and light chain variable region (VL) comprising, respectively, the amino acid sequences SEQ ID NO: 2 and 4, SEQ ID NO: 6 and 8, SEQ ID NO: 10 and 12, SEQ ID NO: 14 and 16, SEQ ID NO: 18 and 20, SEQ ID NO: 22 and 24, or SEQ ID NO: 26 and 28.

2. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to SpA, and competitively inhibits SpA binding by an antibody or antigen-binding fragment thereof comprising a VH and VL comprising, respectively, the amino acid sequences SEQ ID NO: 2 and 4, SEQ ID NO: 6 and 8, SEQ ID NO: 10 and 12, SEQ ID NO: 14 and 16, SEQ ID NO: 18 and 20, SEQ ID NO: 22 and 24, or SEQ ID NO: 26 and 28.

3. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to SpA comprising a VL complementarity determining region 1 (VL-CDR1), a VL-CDR2, and a VL-CDR3, wherein the VL-CDRs comprise, respectively, amino acid sequences identical to, or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more of the VL-CDRs to: SEQ ID NOs: 47, 48, and 49, SEQ ID NOs: 50, 51, and 52, SEQ ID NOs: 53, 56, and 55, SEQ ID NOs: 53, 54, and 55, SEQ ID NOs: 57, 58, and 59, SEQ ID NOs: 60, 61, and 62, or SEQ ID NOs: 63, 64, and 65.

4. The binding molecule of claim 3, further comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3.

5. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to SpA comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3, wherein the VH-CDRs comprise, respectively, amino acid sequences identical to, or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more of the VH-CDRs to: SEQ ID NOs: 29, 30, and 31, SEQ ID NOs: 32, 33, and 34, SEQ ID NOs: 35, 36, and 37, SEQ ID NOs: 38, 39, and 40, SEQ ID NOs: 41, 42, and 43, or SEQ ID NOs: 44, 45, and 46.

6. The binding molecule of claim 5, further comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3.

7. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to SpA comprising an antibody VL, wherein the VL comprises an amino acid sequence at least 85%, 90%, 95%, or 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, and SEQ ID NO: 28.

8. The binding molecule of claim 7, further comprising a VH.

9. An isolated binding molecule or antigen-binding fragment thereof that specifically binds to SpA comprising an antibody VH, wherein the VH comprises an amino acid sequence at least 85%, 90%, 95%, or 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO: 26.

10. The binding molecule of claim 9, further comprising a VL.

11. The isolated binding molecule or antigen-binding fragment thereof of any one of claims 1 to 10, which comprises an antibody or antigen-binding fragment thereof.

12. An isolated antibody or antigen-binding fragment thereof that specifically binds to SpA, comprising VL-CDR1, VL-CDR2, VL-CDR3, VH-CDR1, VH-CDR2, and VH-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to: SEQ ID NOs: 47, 48, 49, 29, 30, and 31, SEQ ID NOs: 50, 51, 52, 32, 33, and 34, SEQ ID NOs: 53, 56, 55, 35, 36, and 37, SEQ ID NOs: 53, 54, 55, 35, 36, and 37, SEQ ID NOs: 57, 58, 59, 38, 39, and 40, SEQ ID NOs: 60, 61, 62, 41, 42, and 43, or SEQ ID NOs: 63, 64, 65, 44, 45, and 46, respectively.

13. An isolated antibody or antigen-binding fragment thereof that specifically binds to SpA, comprising VH and VL amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24, and SEQ ID NO: 26 and SEQ ID NO: 28, respectively.

14. The antibody or antigen-binding fragment thereof of claim 13, wherein the VH comprises the amino acid sequence SEQ ID NO: 10 and the VL comprises the amino acid sequence SEQ ID NO: 12.

15. The antibody or antigen-binding fragment thereof of any one of claims 12 to 14, which comprises a heavy chain constant region or fragment thereof.

16. The antibody or antigen-binding fragment thereof of claim 15, wherein the heavy chain constant region or fragment thereof is an IgG constant region.

17. The antibody or antigen-binding fragment of claim 16, wherein the IgG constant region comprises one or more amino acid substitutions relative to a wild-type IgG constant region, wherein the modified IgG has an increased half-life compared to the half-life of an IgG having the wild-type IgG constant region.

18. The isolated antibody or antigen-binding fragment of claim 17, wherein the IgG constant region comprises one or more amino acid substitutions of amino acids at positions 251- 257, 285-290, 308-314, 385-389, or 428-436, wherein the amino acid position numbering is according to the EU index as set forth in Kabat.

19. The isolated antibody or antigen-binding fragment of claim 18, wherein at least one IgG constant region amino acid substitution is:

(a) a substitution of the amino acid at Kabat position 252 with Tyrosine (Y),

Phenylalanine (F), Tryptophan (W), or Threonine (T);

(b) a substitution of the amino acid at Kabat position 254 with Threonine (T);

(c) a substitution of the amino acid at Kabat position 256 with Serine (S), Arginine (R), Glutamine (Q), Glutamic acid (E), Aspartic acid (D), or Threonine (T);

(d) a substitution of the amino acid at Kabat position 257 with Leucine (L);

(e) a substitution of the amino acid at Kabat position 309 with Proline (P);

(f) a substitution of the amino acid at Kabat position 311 with Serine (S); (g) a substitution of the amino acid at Kabat position 428 with Threonine (T), Leucine (L), Phenylalanine (F), or Serine (S);

(h) a substitution of the amino acid at Kabat position 433 with Arginine (R), Serine (S), Isoleucine (I), Proline (P), or Glutamine (Q);

(i) a substitution of the amino acid at Kabat position 434 with Tryptophan (W), Methionine (M), Serine (S), Histidine (H), Phenylalanine (F), or Tyrosine; or j) a combination of two or more of the substitutions.

20. The isolated antibody or antigen-binding fragment of claim 19, wherein the IgG constant region is a human IgG constant region comprising amino acid substitutions relative to a wild-type human IgG constant region at Kabat positions 252, 254, and 256, wherein

(a) the amino acid at Kabat position 252 is substituted with Tyrosine (Y);

(b) the amino acid at Kabat position 254 is substituted with Threonine (T); and

(c) the amino acid at Kabat position 256 is substituted with Glutamic acid (E).

21. The antibody or antigen-binding fragment thereof of any one of claims 16 to 20, wherein the IgG constant region is a human IgGl constant region.

22. The antibody or antigen-binding fragment thereof of any one of claims 12 to 21, further comprising a light chain constant region selected from the group consisting of a human kappa constant region and a human lambda constant region.

23. The antibody or antigen-binding fragment thereof of claim 22, wherein the light chain constant region is a human kappa constant region.

24. The antibody or antigen-binding fragment thereof of any one of claims 12 to 23, wherein the antibody or antigen-binding fragment thereof is a murine antibody, a humanized antibody, a chimeric antibody, or an antigen-binding fragment thereof.

25. The antibody or antigen-binding fragment thereof of any one of claims 12 to 24, which is monoclonal, polyclonal, recombinant, multispecific, or any combination thereof.

26. The antibody or antigen-binding fragment thereof of any one of claims 12 to 25, wherein the antigen-binding fragment thereof is an Fv fragment, an Fab fragment, an F(ab')2 fragment, an Fab' fragment, a dsFv fragment, an scFv fragment, or an sc(Fv)2 fragment, or any combination thereof.

27. The antibody or antigen-binding fragment thereof of any one of claims 12 to 26, which blocks SpA ligand binding.

28. The antibody or antigen-binding fragment thereof of any one of claims 12 to 27, which is an antagonist of SpA activity.

29. The antibody or antigen-binding fragment thereof of any one of claims 12 to 28, which is attached to an agent selected from the group consisting of an antimicrobial agent, a therapeutic agent, a prodrug, a protein, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, a polyethylene glycol (PEG) polymer, and any combination thereof.

30. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 12 to 29, and a carrier.

31. A kit, comprising

(a) the antibody or antigen binding fragment thereof of any one of claims 12 to 29 or the composition of claim 30; and

(b) instructions for using the antibody or antigen-binding fragment thereof or using the composition or directions for obtaining instructions for using the antibody or antigen-binding fragment thereof or using the composition.

32. The kit of claim 31, further comprising a buffer, a solid support, or both.

33. The kit of claim 32, wherein the solid support is a bead, a filter, a membrane or a multiwall plate.

34. The kit of claim 32 or claim 33, wherein the buffer is suitable for an enzyme- linked immunosorbent assay (ELISA).

35. The kit of any one of claims 31 to 34, wherein the instructions include one or more instructions for isolating, purifying, detecting and quantifying a SpA polypeptide.

36. An isolated polynucleotide comprising a nucleic acid encoding a VL, wherein the VL comprises a VL-CDR1, a VL-CDR2, and a VL-CDR3, wherein the VL-CDRs comprise, respectively, amino acid sequences identical to, or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more of the VL-CDRs to: SEQ ID NOs: 47, 48, and 49, SEQ ID NOs: 50, 51, and 52, SEQ ID NOs: 53, 56, and 55, SEQ ID NOs: 53, 54, and 55, SEQ ID NOs: 57, 58, and 59, SEQ ID NOs: 60, 61, and 62, or SEQ ID NOs: 63, 64, and 65.

37. An isolated polynucleotide comprising a nucleic acid encoding a VH, wherein the VH comprises a VH-CDR1, a VH-CDR2, and a VH-CDR3, wherein the VH-CDRs comprise, respectively, amino acid sequences identical to, or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more of the VH-CDRs to: SEQ ID NOs: 29, 30, and 31, SEQ ID NOs: 32, 33, and 34, SEQ ID NOs: 35, 36, and 37, SEQ ID NOs: 38, 39, and 40, SEQ ID NOs: 41, 42, and 43, or SEQ ID NOs: 44, 45, and 46.

38. An isolated polynucleotide comprising a nucleic acid encoding an antibody VL, wherein the VL comprises an amino acid sequence at least 85%, 90%, 95%, or 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, and SEQ ID NO: 28.

39. An isolated polynucleotide comprising a nucleic acid encoding an antibody VH, wherein the VH comprises an amino acid sequence at least 85%, 90%, 95%, or 100% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO: 26.

40. The isolated polynucleotide of any one of claims 36 to 39, wherein an antibody or antigen-binding fragment thereof comprising the VH, the VL, or a combination of the VH and the VL can specifically bind to SpA.

41. The isolated polynucleotide of claim 40, wherein the antibody specifically binds to the same epitope as an antibody or antigen-binding fragment thereof comprising a VH and VL that comprise the amino acid sequences SEQ ID NO: 2 and 4, SEQ ID NO: 6 and 8, SEQ ID NO: 10 and 12, SEQ ID NO: 14 and 16, SEQ ID NO: 18 and 20, SEQ ID NO: 22 and 24, or SEQ ID NO: 26 and 28, respectively.

42. A vector comprising the isolated polynucleotide of any one of claims 36 to 41.

43. A polynucleotide or a combination of polynucleotides encoding the binding molecule or antigen-binding fragment thereof of any one of claims 1 to 11.

44. A polynucleotide or combination of polynucleotides encoding the antibody or antigen-binding fragment thereof of any one of claims 12 to 29.

45. A composition comprising: a polynucleotide that comprises a nucleic acid encoding a VH, and a polynucleotide that comprises a nucleic acid encoding a VL, wherein the VL and VH comprise VL-CDRl, VL-CDR2, VL-CDR3, VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences identical or identical except for four, three, two, or one single amino acid substitutions, deletions, or insertions in one or more CDRs to: SEQ ID NOs: 47, 48, 49, 29, 30, and 31, SEQ ID NOs: 50, 51, 52, 32, 33, and 34, SEQ ID NOs: 53, 56, 55, 35, 36, and 37, SEQ ID NOs: 53, 54, 55, 35, 36, and 37, SEQ ID NOs: 57, 58, 59, 38, 39, and 40, SEQ ID NOs: 60, 61, 62, 41, 42, and 43, or SEQ ID NOs: 63, 64, 65, 44, 45, and 46, respectively.

46. A composition comprising: a polynucleotide that comprises a nucleic acid encoding a VH, and a polynucleotide that comprises a nucleic acid encoding a VL, wherein the VH and VL comprise, respectively, amino acid sequences at least 85%, 90%, 95%, or 100% identical to reference amino acid sequences selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16, SEQ ID NO: 18 and SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24, and SEQ ID NO: 26 and SEQ ID NO: 28, respectively.

47. The composition of claim 46, wherein the VH and VL are encoded by nucleic acid sequences at least 85%, 90%, 95%, or 100% identical to reference nucleic acid sequences selected from the group consisting SEQ ID NO: 1 and SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23, and SEQ ID NO: 25 and SEQ ID NO: 27, respectively.

48. The composition of any one of claims 45 to 47, wherein the polynucleotide comprising a nucleic acid encoding a VH and the polynucleotide comprising a nucleic acid encoding a VL are in the same vector.

49. The vector comprising the composition of claim 48.

50. The composition of any one of claims 45 to 48, wherein the polynucleotide comprising a nucleic acid encoding a VH and the polynucleotide comprising a nucleic acid encoding a VL are in different vectors.

51. The vectors comprising the composition of claim 50.

52. The composition of any one of claims 45 to 48, wherein an antibody or antigen- binding fragment thereof comprising the VH and the VL can specifically bind to SpA.

53. The composition of claim 52, wherein the antibody or antigen-binding fragment thereof can specifically bind to the same epitope as an antibody or antigen-binding fragment thereof comprising the VH and VL comprising, respectively, the amino acid sequences SEQ ID NO: 2 and 4, SEQ ID NO: 6 and 8, SEQ ID NO: 10 and 12, SEQ ID NO: 14 and 16, SEQ ID NO: 18 and 20, SEQ ID NO: 22 and 24, or SEQ ID NO: 26 and 28.

54. A host cell comprising the polynucleotide of any one of claims 36 to 41, 43, or 44, the composition of any one of claims 45 to 48, or the vector or vectors of any one of claim 42, 59, or 51.

55. A method of making the antibody or antigen-binding fragment of any one of claims 12 to 29, comprising

(a) culturing the cell of claim 54; and

(b) isolating the antibody or antigen-binding fragment thereof.

56. A diagnostic reagent comprising the antibody or antigen-binding fragment of any one of claims 12 to 29.

57. A method for preventing, treating or managing pneumonia in a subject, comprising administering to a subject in need thereof an effective amount of the antibody or antigen binding fragment thereof of any one of claims 12 to 29 or the composition of claim 30.

58. The method of claim 57, wherein the pneumonia is associated with the presence of Staphylococcus bacteria in the subject's lungs.

59. The method of claim 58, wherein the Staphylococcus is S. pneumoniae or S.

aureus.

60. A method for preventing, treating or managing a skin condition in a subject, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 12 to 29 or the composition of claim 30.

61. The method of claim 60, wherein the skin condition is dermonecrosis.

62. The method of claim 60 or claim 61, wherein the skin condition is associated with the presence of Staphylococcus bacteria in the subject's skin layers.

63. A method for preventing, treating, or managing a condition associated with a Staphylococcus infection in a subject, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 12 to 29 or the composition of claim 30.

64. The method of any one of claims 63, wherein the Staphylococcus is S. aureus.

65. The method of claim 64, wherein the S. aureus is antibiotic resistant.

66. The method of claim 65, wherein the S. aureus is resistant to methicillin.

67. The method of any one of claims 57 to 66, wherein the amount is effective to reduce SpA ligand binding, wherein the ligand can be human IgG Fc, VH3 Fabs, von Willebrand Factor, TNF receptor I, VH3 B-cell receptors, or a combination thereof.

68. A method for diagnosing a condition mediated by Staphylococcus bacteria in a subject, comprising: (a) administering to a subject in need thereof a diagnostically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 12 to 29 or the composition of claim 30; and

(b) detecting presence of a biological effect associated with the administration of the antibody or antigen-binding fragment thereof or the composition; wherein detection of a biological effect associated with the administration of the antibody or antigen-binding fragment thereof or the composition is indicative of presence of a condition mediated by Staphylococcus.

69. The method of claim 68, wherein the Staphylococcus is S. pneumoniae or S. aureus.

70. The method of claim 69, wherein the Staphylococcus is S. aureus.

71. The method of claim 70, wherein the S. aureus is antibiotic resistant.

72. The method of claim 71, wherein the S. aureus is resistant to methicillin.

73. A method of preventing, reducing, or managing SpA-induced virulence in a subject infected with Staphylococcus bacteria, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 12 to 29 or the composition of claim 30.

74. The method of any one of claims 56 to 73, wherein the subject is a domestic animal or a human.

75. A mutant SpA IgG/VH3 binding domain region polypeptide comprising the amino acid sequence of SEQ ID NO: 67, wherein X13 is F, R, H, or L; X14 is Y, R, H, K, A, or F; X₂8 is N or A; and X33 is S, I, L, A, G, V, R, H, or K, wherein the polypeptide comprises at least one amino acid substitution relative to SEQ ID NO: 66, and wherein the polypeptide exhibits reduced binding to the Fc region of IgG, to a VH3 Fab region, or to both, compared to a wild-type IgG/VH3 binding domain region polypeptide comprising the amino acid sequence of SEQ ID NO: 66.

76. The mutant SpA polypeptide of claim 75, wherein the polypeptide contains a single amino acid substitution relative to SEQ ID NO: 66.

77. The mutant SpA polypeptide of claim 76, wherein the Tyrosine (Y) at position 14 is substituted with Arginine (R) (SEQ ID NO: 68).

78. The mutant SpA polypeptide of claim 76, wherein the Serine (S) at position 33 is substituted with Isoleucine (I) (SEQ ID NO: 69).

79. The mutant SpA polypeptide of claim 75, wherein the polypeptide contains two amino acid substitutions relative to SEQ ID NO: 66.

80. The mutant SpA polypeptide of claim 79, wherein the Phenylalanine (F) at position 13 is substituted with Arginine (R) and the Serine (S) at position 33 is substituted with Isoleucine (I) (SEQ ID NO: 70).

81. The mutant SpA polypeptide of claim 79, wherein the Tyrosine (Y) at position 14 is substituted with Arginine (R) and the Serine (S) at position 33 is substituted with Isoleucine (I) (SEQ ID NO: 71).

82. An antibody or antigen-binding fragment thereof that specifically binds to the mutant SpA polypeptide of any one of claims 75 to 81 , wherein the antibody can block SpA ligand binding.

83. A composition for eliciting or modulating an immune response to Staphylococcus bacteria in a subject, comprising administering to a subject in need thereof an effective amount of the mutant SpA polypeptide of any one of claims 75 to 81 as an antigen.

84. The composition of claim 83, further comprising one or more adjuvants.

85. The composition of claim 84, wherein the one or more adjuvants comprise aluminum hydroxide and/or paraffin oil.

86. A method of modulating an immune response to Staphylococcus infection in a subject, comprising administering to a subject in need thereof an effective amount of the antibody of any one of claims 12 to 29 or 82, wherein the antibody can block B-cell superantigen activity.

87. A method of eliciting or modulating an immune response to Staphylococcus infection in a subject, comprising administering to a subject in need thereof an effective amount of the mutant SpA polypeptide of any one of claims 75 to 81 , wherein the SpA polypeptide blocks B-cell superantigen activity.

88. The method of claim 86 or 87, wherein the subject is a domestic animal or a human.