WO2000071585A1 - Human antibodies to staphylococcus aureus - Google Patents

Abstract

Isolated human monoclonal antibodies and antigen-binding portions thereof which specifically bind to one or more S. aureus strains, including methicillin resistant S. aureus strains, are disclosed. The human antibodies can be produced in a non-human transgenic animal, e.g., a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies by undergoing V-D-J recombination and isotype switching. Also disclosed are pharmaceutical compositions comprising the human antibodies, non-human transgenic animals and hybridomas which produce the human antibodies, and therapeutic and diagnostic methods for using the human antibodies.

Description

HUMAN ANTIBODIES TO STAPHYLOCOCCUS AUREUS

Background of the Invention

Staphylococcus aureus is a human pathogen capable of causing symptoms ranging from skin boils to septicemia and death (1). Staphylococci are among the most common nosocomial pathogens in hospitals and long-term-care facilities. In particular, S. aureus is an important cause of infections associated with indwelling medical devices such as heart valves and joint prostheses. Examples of particularly susceptible class of patients include the elderly and immunocompromised hospital patients. In addition to being a major cause of nosocomial infections, S. aureus can also mediate diseases through community-acquired infections.

Antibiotic resistant strains of S. aureus have emerged since the widespread use of antibiotics. In addition, hospital strains are ofen resistant to multiple antibiotics. Methicillin resistance has been reported soon after this antibiotic was first introduced for treating penicillin-resistant S. aureus in 1959, and has gradually become a serious problem in many countries (2,3). Methicillin-resistant S. aureus (MRSA) strains are generally resistant to all β-lactam antibiotics by expression of a mutated penicillin binding protein (PBP 2a) that has a low binding affinity for these antibiotics (4). At present, MRSA is treated with the glycopeptide antibiotic vancomycin. Although "true" vancomycin resistance has not been seen in clinical cases of S. aureus, the genes for acquiring resistance to vancomycin are known and vancomycin resistant S. aureus strains have been artificially generated (5). Thus, the emergence of S. aureus resistant strains in clinical settings may be only a matter of time.

In spite of the fact that vancomycin treatment remains widely effective for MRSA infections, this treatment regimen induces significant renal toxicities (6). Few effective alternatives to glycopeptides have been developed, which include the use of the drugs Synercid and the oxazolidinones (7). These new types of antibiotics, although effective, may generate resistant organisms after their widespread use. Moreover, some patients are unable to take antibiotics because of allergic reactions. The polysaccharide capsule (CP) of S. aureus has been a major focus for groups developing immunotherapy for S. aureus infections (13,14). CP vaccines have been investigated in animal models (14) and given to human volunteers to raise hyperimmune serum (11). Hyperimmunized serum from human volunteers immunized with CP vaccines is one alternative for passive immunization of individuals at risk of S. aureus infections that is currently being investigated in clinical trials (11). However, this approach has several disadvantages, including the fact that 1) human immunizations are typically restricted to certain well characterized antigens, 2) human serum must be extensively screened and still may contain infectious agents, and 3) the relative activity of different batches of serum may be difficult to control. These drawbacks may result in a potentially infectious, expensive treatment that targets a single antigen and is difficult to standardize. Accordingly, the need exists for developing improved strategies for providing safer, less toxic alternatives to the current therapy for MRS A, and which are less likely to cause resistant organisms after their widespread use.

Summary of the Invention The present invention provides isolated human monoclonal antibodies which specifically bind to Staphylococcus aureus by binding to an antigen of S. aureus, as well as compositions containing one or a combination of such antibodies. Preferably, the human antibodies cross-react with an epitope present on multiple (i.e., two or more) S. aureus clinical isolates. In certain embodiments, the human antibodies are also characterized by binding to S. aureus or an S. aureus-antigcn with high affinity, and by inhibiting S. aureus growth and/or mediating phagocytosis and cell killing of S. aureus (in vitro and in vivo) in the presence of human effector cells. Accordingly, the human monoclonal antibodies of the invention can be used as diagnostic or therapeutic agents in vivo and in vitro. Isolated human antibodies of the invention encompass various antibody isotypes, such as IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD, and IgE. Typically, they include IgGl (e.g., IgGlκ) and IgM isotypes. The antibodies can be full-length (e.g., an IgGl or IgG4 antibody) or can include only an antigen-binding portion (e.g., a Fab, F(ab')₂, Fv or a single chain Fv fragment). In one embodiment, the human antibodies are recombinant human antibodies. In another embodiment, the human antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene, fused to an immortalized cell. In particular embodiments, the antibodies are produced by hybridomas referred to herein as 2GD12, 2H12, 8.1E5, 8.2C1, 7F1, 6D12 and 5H10. In another embodiment, human anti- S. aureus antibodies of the present invention can be characterized by one or more of the following properties: a) crossreactivity with at least one, preferably multiple, S aureus isolates, e.g., S. aureus clinical isolates; b) a binding affinity to S. aureus or an S. αwrews-antigen with an affinity constant of at least about 10⁷ M^"1, preferably about 10⁸ M^"1, and more preferably, about 10⁹ M^"1 to lO^ M^'1 or higher; c) an association constant (K_assoc) with S. aureus or an S. αwrews-antigen of at least about 10³, more preferably about 10⁴ and most preferably about 10⁵ M^S^"1; d) a dissociation constant (K_di_S) from S. aureus or an S. ourews-antigen of about 10^"3 s^"1, preferably about 10^"4 s^"1, more preferably, 10^"5 s^"1, and most preferably, 10^"6 s^"1; e) the ability to opsonize S. aureus; or f) the ability to inhibit growth and colonization of S. aureus and/or mediate phagocytosis and killing of S. aureus in the presence of human effector cells at a concentration of about 10 μg/ml or less (e.g., in vitro).

Examples of S. aureus clinical isolates which can be targeted by the human antibodies of the invention include, but are not limited to: IBERIAN, EMRSA,

ONTARIO and BRAZILIAN strains. In a particular embodiment, the antibodies bind to at least one methicillin-resistant S. aureus (MRSA) strain.

Isolated human antibodies of the invention can bind any S. aureus antigen including for example, antigens secreted by, or present on the surface of, S. aureus strains. Examples of such antigens include capsular polysaccharides (e.g., CP type 5 or CP type 8), fibronectin binding proteins, protein A, toxins (α-, β-, δ, and γ- toxin, enterotoxins, epidemolytic toxin), toxic shock syndrome toxin superantigens, coagulase, staphylokinase, penicillin binding protein 2a (PBP 2a) and adhesins, among others. In one embodiment, the human antibodies bind to S. aureus or an S. aureus antigen with an affinity constant of at least about 10⁷ M^"1, preferably about 10⁸ M^"1, and more preferably, about 10⁹ M^"1 to 10¹⁰ M^"1 or stronger, and are capable of mediating phagocytosis and killing of S. aureus by human effector cells, e.g., polymorphonuclear cells (PMNs), with an IC50 of about 1 x 10^"7 M or less, or at a concentration of about 10 μg/ml or less in vitro.

In another aspect, the invention provides nucleic acid molecules encoding the antibodies, or antigen-binding portions, of the invention. Accordingly, recombinant expression vectors which include the antibody-encoding nucleic acids of the invention, and host cells transfected with such vectors, are also encompassed by the invention, as are methods of making the antibodies of the invention by culturing these host cells.

In yet another aspect, the invention provides isolated B-cells from a transgenic non-human animal, e.g., a transgenic mouse, which are capable of expressing various isotypes (e.g., IgG, IgA and/or IgM) of human monoclonal antibodies that specifically bind to S. aureus or an S. aureus-antigen. Preferably, the isolated B cells are obtained from a transgenic non-human animal, e.g., a transgenic mouse, which has been immunized with whole S. aureus or a purified or enriched preparation of an S. aureus- antigen. Preferably, the transgenic non-human animal, e.g., a transgenic mouse, has a genome comprising a human heavy chain transgene and a human light chain transgene. The isolated B-cells are then immortalized to provide a source (e.g., a hybridoma) of human monoclonal antibodies to S. aureus or an S. aureus-antigen.

Accordingly, the present invention also provides a hybridoma capable of producing human monoclonal antibodies that specifically bind to S. aureus or an S. aureus-antigen. In one embodiment, the hybridoma includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene, fused to an immortalized cell. The transgenic non-human animal can be immunized with whole S. aureus or a purified or enriched preparation of an S. aureus-antigen to generate antibody-producing hybridomas. Particular hybridomas of the invention include 2GD12, 2H12, 8.1E5, 8.2C1, 7F1, 6D12, and 5H10.

In yet another aspect, the invention provides a transgenic non-human animal, such as a transgenic mouse (also referred to herein as "HuMab"), which express human monoclonal antibodies that specifically bind to S. ureus or an S. aureus-antigen. In a particular embodiment, the transgenic non-human animal is a transgenic mouse having a genome comprising a human heavy chain transgene and a human light chain transgene. The transgenic non-human animal can be immunized with whole S. aureus or a purified or enriched preparation of an S. aureus-antigen. Preferably, the transgenic non-human animal, e.g., the transgenic mouse, is capable of producing multiple isotypes of human monoclonal antibodies to S. aureus or an S. aureus antigen (e.g., IgG, IgA and/or IgM) by undergoing N-D-J recombination and isotype switching. Isotype switching may occur by, e.g., classical or non-classical isotype switching.

In another aspect, the present invention provides methods for producing human monoclonal antibodies which specifically react with S. aureus or an S. aureus-antigen. In one embodiment, the method includes immunizing a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene, with whole S. aureus or a purified or enriched preparation of an S. aureus-antλgen. B cells (e.g., splenic B cells) of the animal are then obtained and fused with myeloma cells to form immortal, hybridoma cells that secrete human monoclonal antibodies against S. aureus or an S. aureus-an gen.

Isolated anti-S. aureus human monoclonal antibodies of the invention, or antigen binding portions thereof, can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., an Fab' fragment). For example, an antibody or antigen-binding portion of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific or a multispecific antibody). Accordingly, in another aspect, the present invention features a bispecific or multispecific molecule comprising at least one first binding specificity for S. aureus or an S. aureus-antigen and a second binding specificity for an Fc receptor, e.g., human Fcγ RI or a human Fc receptor.

Multispecific molecules of the invention also include trispecific, tetraspecific and other multispecific molecules. In one embodiment the multispecific molecule includes an anti-enhancement factor (EF) portion, e.g., a molecule which binds to a surface protein involved in cytotoxic activity.

In a particular embodiment, bispecific and multispecific molecules of the invention comprise at least one antibody, or fragment thereof (e.g., an Fab, Fab', F(ab')₂, Fv, or a single chain Fv). In a particular embodiment, the antibody or fragment thereof is a completely human antibody or a portion thereof, or a "chimeric" or a "humanized" antibody or a portion thereof (e.g., has a variable region, or at least a complementarity determining region (CDR), derived from a non-human antibody (e.g., murine) with the remaining portion(s) being human in origin).

In one embodiment, the at least one antibody or fragment thereof of the bispecific or multispecific molecule binds to an Fc receptor, such as a human IgG receptor, e.g., an Fc-gamma receptor (FcγR), such as FcγRI (CD64), FcγRII(CD32), and FcγRJII (CD 16). A preferred Fcγ receptor is the high affinity Fcγ receptor, FcγRI. However, other Fc receptors, such as human IgA receptors (e.g. FcαRI) also can be targeted. The Fc receptor is preferably located on the surface of an effector cell, e.g., a monocyte, macrophage or an activated polymorphonuclear cell. In a preferred embodiment, the bispecific and multispecific molecules bind to an Fc receptor at a site which is distinct from the immunoglobulin (e.g., IgG or IgA) binding site of the receptor. Therefore, the binding of the bispecific and multispecific molecules is not blocked by physiological levels of immunoglobulins.

In another aspect, the present invention provides target-specific effector cells which comprise an effector cell expressing an Fc receptor, e.g., a macrophage or an activated PMN cell, and a bispecific or multispecific molecule of the invention.

In another aspect, the present invention provides compositions, e.g., pharmaceutical and diagnostic compositions, comprising a pharmaceutically acceptable carrier and at least one human monoclonal antibody of the invention, or an antigen- binding portion thereof, which specifically binds to S. aureus or an S. aureus-antigen. In one embodiment, the composition comprises a combination of the human antibodies or antigen-binding portions thereof, preferably each of which binds to a distinct epitope. For example, a pharmaceutical composition comprising a human monoclonal antibody that exhibits limited crossreactivity to S. aureus isolates, but that mediates highly effective phagocytosis of S. aureus, can be combined with another human monoclonal antibody that exhibits broad crossreactivity to S. aureus strains. Thus, the combination provides multiple therapies tailored to provide the maximum therapeutic benefit. Compositions, e.g., pharmaceutical compositions, comprising a combination of at least one human monoclonal antibody of the invention, or antigen-binding portions thereof, and at least one bispecific or multispecific molecule of the invention, are also within the scope of the invention. In yet another aspect, the invention provides a method for inhibiting infectivity of S. aureus by inhibiting growth and/or colonization, and/or by inducing phagocytosis and/or killing, of S. aureus bacteria by human effector cells, such as human polymorphonuclear cells (PMNs), using an antibody, or antigen-binding portion thereof (or a bispecific or multispecific antibody) of the invention. In one embodiment, the method comprises contacting S. aureus either in vitro or in vivo with one or a combination of human monoclonal antibodies of the invention, or an antigen-binding portion thereof, in the presence of a human effector cell. The method can be employed in culture, e.g. in vitro or ex vivo (e.g., cultures comprising S. aureus and effector cells). For example, a sample containing S. aureus and effector cells can be cultured in vitro, and combined with an antibody of the invention, or an antigen-binding portion thereof (or a bispecific or multispecific antibody of the invention). Alternatively, the method can be performed in a subject, e.g., as part of an in vivo (e.g., therapeutic or prophylactic) protocol. For in vivo methods, the antibody, or antigen-binding portion thereof (or a bispecific or multispecific antibody of the invention), can be administered to a human subject suffering from an S. αwrews-mediated disease such that growth inhibition, phagocytosis and/or killing of S. aureus is induced. In one embodiment, the subject can be additionally treated with an agent that modulates, e.g., enhances or inhibits, the expression or activity of Fc receptor, e.g., an Fcα receptor or an Fcγ receptor, by for example, treating the subject with a cytokine. Preferred cytokines for administration during treatment with the bispecific and multispecific molecules include granulocyte colony-stimulating factor (G-CSF), granulocyte- macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumor necrosis factor (TNF). Isolated human monoclonal antibody compositions of the invention also can be administered in combination with other known anti-bacterial therapies.

Exemplary diseases that can be treated (e.g., ameliorated) or prevented using the methods and compositions of the invention include, but are not limited to, invasive or toxigenic infectious diseases. Such invasive diseases include Bacteremia, osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis. Such toxigenic diseases include Staphylococcol food poisoning, scalded skin syndrome and toxic shock syndrome. Additional examples of diseases that can be treated (e.g., prevented or ameliorated) using the methods of the invention include infections of the upper and/or lower respiratory, heart, gastrointestinal tract, CNS, eye, kidney and urinary tract, skin, and bone and joint.

In yet another aspect, the present invention provides a method for detecting in vitro or in vivo the presence of S. aureus in a sample, e.g., for diagnosing an S. aureus- mediated disease. In one embodiment, this is achieved by contacting a sample to be tested, along with a control sample, with a human monoclonal antibody of the invention, or an antigen-binding portion thereof ( or a bispecific or multispecific molecule), under conditions that allow for formation of a complex between the antibody and S. aureus. Complex formation is then detected (e.g., using an ELISA) in both samples, and any statistically significant difference in the formation of complexes between the samples is indicative the presence of S. aureus in the test sample.

Other features and advantages of the instant invention be apparent from the following detailed description and claims.

Brief Description of the Drawings

Figure 7 is a bar graph depicting the levels of anti-S. aureus human antibodies present in the plasma of HuMAb mice immunized with heat killed S. aureus, as compared to non-immunized controls. Shown are results of optical density with respect to the indicated dilution of pooled plasma from S. aureus immunized mice (hatched bar) and non-immunized mice (solid bar) analyzed by ELISA.

Figure 2 is a bar graph depicting the binding of the supernatants from six mixed hybridoma cultures, 2GD12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12, to S. aureus strain 209, compared to human IgG controls. The anti-FcγRI antibody H22 was used as a control.

Figure 3 is a bar graph depicting the binding of the supernatants from mixed hybridoma cultures (2GD12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12) to Methicillin resistant S. aureus strain BK2058 compared to media alone (bkd), as measured by ELISA. Figure 4 is a bar graph depicting the binding of the supernatants from the mixed hybridoma cultures (2GD12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12) to mixed bacteria (Escherichia coli, Pseudomonas aeroginosa, or Micrococcus luteus) as measured by ELISA. Figure 5 is a bar graph comparing the binding of the supernatants from the mixed hybridoma cultures (2GD12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12) to S. aureus (speckled bar) or E. coli (solid bar), as measured by flow cytometry.

Figure 6 is a bar graph depicting the binding of the supernatants from the indicated subcloned hybridoma cultures to S. aureus FDA 209, as measured by ELISA. Figure 7 is a bar graph showing binding of supernatants from subcloned hybridoma cultures to S. aureus ATCC 27661, as measured by ELISA.

Figures 8A-8C are histograms showing neutrophil-mediated phagocytosis of S. aureus upon incubation of polymorphonuclear cells (PMNs) with mixed hybridoma supernatants. FACScan analyses of control PMNs alone (Figure 8A), PMNs and S. aureus, with or without supernatant from mixed hybridoma cultures (Figure 8B), and PMNs and control E. coli, with or without supernatant from mixed hybridoma cultures (Figure 8C) are shown.

Figures 9A-9B show binding of purified human monoclonal antibody 6D12 to S. aureus FDA 209 compared to IgG controls. Figure 9A is a bar graph depicting the binding of two concentrations (1 and 10 μg/ml) of purified human monoclonal antibody 6D12 (open bars) to S. aureus FDA 209, compared to IgG controls (speckled bars) as measured using ELISA. Figure 9B is a histogram showing direct binding of FITC- labeled 6D12 monoclonal antibodies to S. aureus FDA 209 compared to FITC-labeled humanized anti-EGF receptor antibody control (H425) as detected by flow cytometry. Figures 10A-10B show binding of purified human monoclonal antibody 6D12 to

MRSA strain 2058 compared to IgG controls. Figure 10A is a linear graph depicting the binding of two concentrations (1 and 10 μg/ml) of purified human monoclonal antibody 6D12 to MRSA strain 2058, compared to IgG controls. Figure 10B is a histogram showing direct binding of FITC-labeled 6D12 monoclonal antibodies to MRSA strain 2058 compared to FITC-labeled humanized anti-EGF receptor antibody control (H425) as detected by flow cytometry. Figure 11 is a bar graph showing killing of S. aureus after antibody opsonization and PMN phagocytosis using human monoclonal antibodies against S. aureus.

Detailed Description of the Invention

The present invention provides novel antibody-based therapeutics for treating and diagnosing S. aureus infections. Therapeutic and diagnostic reagents of the invention include isolated human monoclonal antibodies, or antigen-binding portions thereof, which bind to an epitope present on at least one, and preferably multiple, strains of S. aureus clinical isolates, including MRS A strains. In one embodiment, the human antibodies are produced in a non-human transgenic animal, e.g., a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies to S. aureus or an S. aureus antigen (e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination and isotype switching. Accordingly, various aspects of the invention include antibodies and antibody fragments, and pharmaceutical compositions thereof, as well as non- human transgenic animals, and B-cells and hybridomas for making such monoclonal antibodies. Methods of using the antibodies of the invention to detect S. aureus or to inhibit S. aureus infectivity, either in vitro or in vivo, are also encompassed by the invention. In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The term "Staphylococcus aureus" (abbreviated herein as "S. aureus"), as used herein, refers to any strain, genotype or isolate of S. aureus, a Grαm-positive bacteria which is among the most common of nosocomial pathogens in hospitals and long-term care facilities (1) today. These organisms are capable of causing symptoms ranging from skin boils to septicemia and death. The term "S. aureus" also includes antibiotic- resistant strains of S. aureus, e.g., penicillin- and methicillin-resistant S. aureus (abbreviated herein as "MRSA"), including, for example, IBERIAN, EMRSA, ONTARIO, BRAZILIAN and COL strains (2,3). MRSA strains are generally resistant to β-lactam antibiotics by expression of a mutated penicillin binding protein (PBP 2a) that has a low binding capacity for these antibiotics (4). As used herein, the term "S. aureus-antigen" includes any S. aureus antigen, including secreted S. aureus antigens and antigens which naturally occur on the surface of S. aureus. In preferred embodiments, the S. aureus-antigen is present in a large percentage of clinical isolates to provide for broad crossreactivity of the antibody that binds to the antigen. In other preferred embodiments, binding of an antibody of the invention to the S. aureus-antigen mediates effector cell phagocytosis and killing of S. aureus. Preferably, the S. aureus antigen can elicit a protective immune response upon immunization of a non-human transgenic animal. Examples of such antigens include capsular polysaccharides (e.g., CP type 5 or CP type 8), fibronectin binding proteins, protein A, toxins (α-, β-, δ, and γ-toxin, enterotoxins, epidemolytic toxin), toxic shock syndrome toxin superantigens, coagulases, staphylokinases, penicillin binding protein 2a (PBP 2a) and adhesins, among others (28). For example, the CP antigens (CP5 and CP8) are present on about 70-80% of clinical isolates.

As used herein, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCNR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl , CDRl , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., S. aureus or an S. aureus antigen). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879- 5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

The term "bispecific molecule" is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has two different binding specificities which bind to, or interact with (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The term "multispecific molecule" or "heterospecific molecule" is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has more than two different binding specificities which bind to, or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Accordingly, the invention includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules which are directed to cell surface antigens, such as S. aureus or an S. aureus antigen, and to Fc receptors on effector cells. The term "bispecific antibodies" further includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444- 6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123).

As used herein, the term "heteroantibodies" refers to two or more antibodies, antibody binding fragments (e.g. , Fab), derivatives therefrom, or antigen binding regions linked together, at least two of which have different specificities. These different specificities include a binding specificity for an Fc receptor on an effector cell, and a binding specificity for an antigen or epitope on a target cell, e.g., S aureus.

The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.

The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (described further in Section I, below); antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. As used herein, a "heterologous antibody" is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal. As used herein, a "heterohybrid antibody" refers to an antibody having a light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody. Examples of heterohybrid antibodies include chimeric and humanized antibodies, discussed supra. An "isolated antibody", as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to S. aureus or an S. aureus antigen is substantially free of antibodies that specifically bind antigens other than S. aureus or an S. aureus antigen). An isolated antibody that specifically binds to one strain or one antigen of S. aureus may, however, have cross-reactivity to other S. aureus strains or antigens. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the invention, a combination of "isolated" monoclonal antibodies having different specificities are combined in a well defined composition. As used herein, "specific binding" refers to antibody binding to a predetermined antigen. Typically, the antibody binds with an affinity of at least about 1 x 10⁷ M^"!, and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The phrases "an antibody recognizing an antigen" and " an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen".

As used herein, the term "high affinity" for an IgG antibody refers to a binding affinity of at least about 1 x 10⁹M^_1, typically at least about 5 x 10⁹M^"', frequently more than about 1 x lO^M^"1, and sometimes 5 x 10¹⁰M^"] to 1 x 10^πM^"' or greater. "High affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype is at least about 1 x 10⁷M^"'.

The term "K_assoc", as used herein, is intended to refer to the association constant of a particular antibody-antigen interaction.

The term "K^", as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction. As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes.

As used herein, "isotype switching" refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes. As used herein, "nonswitched isotype" refers to the isotypic class of heavy chain that is produced when no isotype switching has taken place; the CH gene encoding the nonswitched isotype is typically the first CH gene immediately downstream from the functionally rearranged VDJ gene. Isotype switching has been classified as classical or non-classical isotype switching. Classical isotype switching occurs by recombination events which involve at least one switch sequence region in the transgene. Non- classical isotype switching may occur by, for example, homologous recombination between human σ_μ and human ∑_μ (δ-associated deletion). Alternative non-classical switching mechanisms, such as intertransgene and/or interchromosomal recombination, among others, may occur and effectuate isotype switching.

As used herein, the term "switch sequence" refers to those DNA sequences responsible for switch recombination. A "switch donor" sequence, typically a μ switch region, will be 5' (i.e., upstream) of the construct region to be deleted during the switch recombination. The "switch acceptor" region will be between the construct region to be deleted and the replacement constant region (e.g., γ, ε, etc.). As there is no specific site where recombination always occurs, the final gene sequence will typically not be predictable from the construct.

As used herein, "glycosylation pattern" is defined as the pattern of carbohydrate units that are covalently attached to a protein, more specifically to an immunoglobulin protein. A glycosylation pattern of a heterologous antibody can be characterized as being substantially similar to glycosylation patterns which occur naturally on antibodies produced by the species of the nonhuman transgenic animal, when one of ordinary skill in the art would recognize the glycosylation pattern of the heterologous antibody as being more similar to said pattern of glycosylation in the species of the nonhuman transgenic animal than to the species from which the CH genes of the transgene were derived.

The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.

The term "rearranged" as used herein refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete VH or

VL domain, respectively. A rearranged immunoglobulin gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element.

The term "unrearranged" or "germline configuration" as used herein in reference to a V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.

The term "nucleic acid molecule", as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. The term "isolated nucleic acid molecule", as used herein in reference to nucleic acids encoding antibodies or antibody portions (e.g., VH, VL, CDR3) that bind to S. aureus or an S. aureus antigen, is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than S. aureus or an S. aureus antigen, which other sequences may naturally flank the nucleic acid in human genomic DNA. For nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., eά. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic DNA or mixtures thereof, may be mutated in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, may affect the amino acid sequence as desired. In particular, DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where "derived" indicates that a sequence is identical or modified from another sequence). A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. For switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination. The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Various aspects of the invention are described in further detail in the following subsections. I. Production of Human Antibodies to S. aureus

Human monoclonal antibodies (mAbs) of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murine system. Hybridoma production in the mouse is a very well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

In a preferred embodiment, human monoclonal antibodies directed against S. aureus are generated using transgenic mice carrying the complete human immune system rather than the mouse system, referred to herein as "HuMab" transgenic mice. For example, HuMAb transgenic mice can be used which contain a human immunoblobulin gene miniloci that encodes unrearranged human heavy (μ and γ) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and K chain loci (Lonberg, N. et al. (1994) Nature 368(6474): 856- 859). Accordingly, these mice show reduced expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGκ monoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113 :49- 101 ; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N. Y. Acad. Sci 764:536-546). The preparation of HuMab mice is described in detail Section II below and in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287- 6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:1 17-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.

152:2912-2920; Lonberg et al., (1994) Nature 368(6474): 856-859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101 ; Taylor, L. et al. (1994) International Immunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N. (1995) Ann. N. Y. Acad. Sci 764:536-546; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851, the contents of all of which are hereby incorporated by reference in their entirety. See further, U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;

5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay, and GenPharm International; U.S. Patent No. 5,545,807 to Surani et al.; International Publication Nos. WO 98/24884, published on June 11, 1998; WO 94/25585, published November 10, 1994; WO 93/1227, published June 24, 1993; WO 92/22645, published December 23, 1992; WO 92/03918, published March 19, 1992, the disclosures of all of which are hereby incorporated by reference in their entity.

HuMab Immunizations

To generate fully human monoclonal antibodies to S. aureus, mice, preferably HuMab transgenic mice, can be immunized, for example, with heat killed whole S aureus (e.g., purified or enriched immunogen containing 5 - 20 μg S. aureus antigen) using, for example, the immunization protocol described in Lonberg, N. et al. (1994) Nαtwre 368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845- 851 and WO 98/24884. Preferably, the mice are 6-16 weeks of age upon the first infusion.

For example, heat killed whole S. aureus from a single clinical isolate that expresses the CP antigen, CP type 5 (CP5), can be used to immunize the HuMab mice intraperitoneally. CP type 5 is a suitable S. aureus antigen for use in the invention because it is a common CP type shared by MRSA isolates. Furthermore, previous studies have demonstrated that antibodies which bind type 5 capsule can mediate protection against experimental S. aureus infections (14, 28). In the event that immunizations using a single S. aureus strain do not result in antibodies cross-reactive with two or more S. aureus strains, mice can be immunized with alternating strains of S. aureus to promote immune responses against shared antigens. Prior to immunization, the bacteria is generally typed, and can be grown either on agar plates to promote expression of capsule (29), or in broth to minimize capsule expression. Cumulative experience with various antigens has shown that the HuMAb transgenic mice respond best when initially immunized intraperitoneally (IP) with antigen in complete Freund's adjuvant, followed by every other week IP immunizations (up to a total of 6) with antigen in incomplete Freund's adjuvant. The immune response can be monitored over the course of the immunization protocol with plasma samples being obtained by retroorbital bleeds. The plasma can be screened by ELISA (as described below), and mice with sufficient titers of anti-S. aureus human immunoglobulin can be used for fusions. Mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions for both the high capsule and the low-capsule immunizations may need to be performed. Six mice will be immunized for each antigen. For example, a total of twelve HuMAb mice of the HC07 and HC012 strains can be immunized.

Generation of Hybridomas Producing Monoclonal Antibodies to S. aureus The mouse splenocytes can be isolated and fused with PEG to a mouse myeloma cell line based upon standard protocols (21, 30). The resulting hybridomas are then screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice are fused to one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2 x 10⁵ in flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L~glutamine, 1 mM sodium pyruvate, 5mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and IX HAT (Sigma; the HAT is added 24 hours after the fusion). After two weeks, cells are cultured in medium in which the HAT is replaced with HT. Individual wells are then screened by ELISA for human anti- S. aureus monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, medium is observed usually after 10-14 days. The antibody secreting hybridomas are replated, screened again, and if still positive for human IgG, anti-S. aureus monoclonal antibodies, can be subcloned at least twice by limiting dilution. The stable subclones are then cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization. Characterization of Binding of Human Monoclonal Antibodies to S. aureus

To characterize binding of human monoclonal anti- S. aureus antibodies of the invention, sera from immunized mice can be tested, for example, by ELISA. Briefly, an S. aureus immunizing strain can be grown overnight in Columbia broth medium or on Columbia agar plates. Bacteria can be recovered from plates by gently resuspending the cells in sterile saline. The bacteria from broth or plates can be washed by centnfugation and diluted to an OD₅ o of 0.2. Bacterial suspensions (50 μl) can be added to 96-well flat bottom plates, and plates are allowed to dry in fume hood. The bacteria can be fixed to the plate with 1% glutaraldehyde for 5 minutes. The plates are blocked with 20% mouse serum (which blocks IgG binding to protein A). These plates can be stored at -80 °C until needed. Plates can then be washed with PBS-tween buffer (Dulbeccos PBS, .05 % Tween 20, 1 mM EDTA, .25% BSA, .05 % NaN₃), and dilutions of plasma from S. αwrews-immunized mice and irrelevant antigen-immunized mice are added, and incubated at 37 °C for 1 hr. Plates are washed and then reacted with anti-human IgG Fc specific alkaline phosphatase and incubated at 37 °C for 1 hour. Plates can be washed again, developed with pNPP substrate (1 mg/ml), and analyzed at OD of 405-650. Preferably, mice which develop the highest titers will be used for fusions.

To screen hybridomas using an ELISA assay, microtiter plates can be prepared as described above using several strains of S. aureus in addition to strain used for immunizations. Exemplary S. aureus strains that can be used include the IBERIAN (BK2058 and BK2709), EMRSA, ONTARIO, and BRAZILIAN isolates and/or purified antigens, e.g., capsular polysaccharides 5 and 8, fibronectin binding protein penicillin binding protein and protein A, which have been demonstrated to spread rapidly in hospital settings. Hybridomas that show positive reactivity with the immunizing strain of S. aureus (by, e.g., ELISA assays as described above) can be tested for cross- reactivity on plates coated with the other strains. Irrelevant human IgG will be used as a negative control. Hybridomas that bind with high avidity to most S. aureus strains will be subcloned and further characterized. One clone from each hybridoma, which retains the reactivity of the parent cells (by ELISA), can be chosen for making a 5-10 vial cell bank stored at -140 °C, and for antibody purification. To purify human anti-S. aureus antibodies, selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, NJ). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD₂₈o using 1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and stored at -80 °C. To determine if the selected human anti-S. aureus monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using S. aureus coated-ELISA plates as described above. Biotinylated mAb binding can be detected with a strep-avidin-alkaline phosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can be performed. Wells of microtiter plates can be coated with 10 μg/ml of anti-human Ig overnight at 4°C. After blocking with 5% BSA, the plates are reacted with 10 μg/ml of monoclonal antibodies or purified isotype controls, at ambient temperature for two hours. The wells can then be reacted with either human IgGl or human IgM-specific alkaline phosphatase-conjugated probes. Plates are developed and analyzed as described above.

In order to demonstrate binding of monoclonal antibodies to live S. aureus, flow cytometry (e.g., as described by Poutrel et at. (31)) can be used. Briefly, bacteria (grown as above, at 10⁷ cells/ml) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% Tween 80 and 20% mouse serum, and incubated at 37°C for 1 hour. After washing, the bacteria are reacted with Fluorescein-labeled anti- human IgG antibody under the same conditions as the primary antibody staining. The samples can be analyzed by F AC Scan instrument using light and side scatter properties to gate on single bacteria. Short sonication steps (which have been shown not to significantly affect cell viability) may be included to reduce clumping of bacteria. An alternative assay using fluorescence microscopy may be used (in addition to or instead of) the flow cytometry assay. Bacteria can be stained exactly as described above and examined by fluorescence microscopy. This method allows visualization of individual cells, but may have diminished sensitivity depending on the density of the antigen.

Anti-S. aureus human IgGs can be further tested for reactivity with specific S. aureus antigens by Western blotting. Briefly, cell wall extracts from selected S. aureus isolates can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked with 20% mouse serum, and probed with the monoclonal antibodies to be tested. Human IgG binding can be detected using anti- human IgG alkaline phosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, MO). The antigens that are specifically bound by the monoclonal antibodies tested can be identified by direct sequencing or through the use of mutants known to lack or produce very low amounts of certain cell wall components.

Phagocytic and Cell Killing Activities of Human Anti-S. aureus Antibodies In addition to demonstrating the ability of selected monoclonal antibodies to bind specifically to S. aureus, the ability of these antibodies to mediate phagocytosis and killing of S. aureus in vitro (i.e., their therapeutic utility) can also be determined. The testing of monoclonal antibody activity in vitro will provide an initial screening prior to testing in vivo models. Briefly, polymorphonuclear cells (PMN) from healthy donors can be purified by Ficoll Hypaque density centrifugation, followed by lysis of contaminating erythrocytes. Washed PMNs, can be suspended in RPMI supplemented with 10% heat-inactivated fetal calf serum and mixed with S. aureus, grown as described above, at various ratios of PMN to bacteria (PMN:bacteria). Purified human anti-S. aureus IgGs can then be added at various concentrations. Irrelevant human IgG can be used as negative control. Assays can be carried out for 0-120 minutes at 37°C. Samples can be diluted in water and then plated on tryptic soy agar plates for overnight incubation before determining colony counts. anti-S. aureus monoclonal can also be tested in combinations with each other to determine whether phagocytosis is enhanced with multiple monoclonal antibodies. Human monoclonal antibodies which show, e.g., high affinity binding and cross- reactivity with one or more of the S. aureus clinical isolates, can be tested in an in vivo challenge model in mice to determine the efficacy against S. aureus infection. These antibodies can be selected, for example, based on the following criteria which are not intended to be exclusive:

1. binding to live S. aureus cells grown on agar or in broth; 2. high affinity of binding to S. aureus;

3. cross-reactivity with a variety of S. aureus clinical isolates by ELISA and/or flow cytometry;

4. binding to a unique epitope on S. aureus (to eliminate the possibility that monoclonal antibodies with complimentary activities when used in combination would compete for binding to the same epitope);

5. opsonization of S. aureus;

4. mediation of growth inhibition, phagocytosis and/or killing of S. aureus in the presence of human effector cells.

Preferred monoclonal antibodies meet one or more, and preferably all, of these criteria.

In a particular embodiment, the human monoclonal antibodies of the present invention are used in combination, e.g., as a pharmaceutical composition comprising two or more anti-S. aureus monoclonal antibodies, or fragments thereof. For example, human anti-S. aureus monoclonal antibodies having different, but complementary activities can be combined in a single therapy to achieve a desired therapeutic or diagnostic effect. In one embodiment, this is achieved by combining a monoclonal antibody having limited cross-reactivity, but mediating efficient phagocytosis, with one or more other human monoclonal antibodies exhibiting broader cross-reactivity, or which exhibit effective inhibition of S. aureus growth and/or colonization.

II. Production of Transgenic Nonhuman Animals Which Generate Human Monoclonal Anti-S. aureus Antibodies

In yet another aspect, the invention provides transgenic non-human animals, e.g., a transgenic mice, which are capable of expressing the aforesaid human monoclonal antibodies that specifically bind to S. aureus or an S. aureus-antigen. In a preferred embodiment, the transgenic non-human animals, e.g., the transgenic mice, have a genome comprising a human heavy chain transgene and a light chain transgene. In one embodiment, the transgenic non-human animals, e.g., the transgenic mice, have been immunized with whole S. aureus or a purified or enriched preparation of an S. aureus- antigen. Preferably, the transgenic non-human animals, e.g., the transgenic mice, are capable of producing multiple isotypes of human monoclonal antibodies to S. aureus or an S. aureus antigen (e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination and isotype switching. Isotype switching may occur by, e.g., classical or non-classical isotype switching.

The design of a transgenic non-human animal that responds to foreign antigen stimulation with a heterologous antibody repertoire, requires that the heterologous immunoglobulin transgenes contain within the transgenic animal function correctly throughout the pathway of B-cell development. In a preferred embodiment, correct function of a heterologous heavy chain transgene includes isotype switching. Accordingly, the transgenes of the invention are constructed so as to produce isotype switching and one or more of the following: (1) high level and cell-type specific expression, (2) functional gene rearrangement, (3) activation of and response to allelic exclusion, (4) expression of a sufficient primary repertoire, (5) signal transduction, (6) somatic hypermutation, and (7) domination of the transgene antibody locus during the immune response. Not all of the foregoing criteria need be met. For example, in those embodiments wherein the endogenous immunoglobulin loci of the transgenic animal are functionally disrupted, the transgene need not activate allelic exclusion. Further, in those embodiments wherein the transgene comprises a functionally rearranged heavy and/or light chain immunoglobulin gene, the second criteria of functional gene rearrangement is unnecessary, at least for that transgene which is already rearranged. For background on molecular immunology, see, Fundamental Immunology, 2nd edition (1989), Paul William E., ed. Raven Press, N.Y., which is incorporated herein by reference.

In certain embodiments, the transgenic non-human animals used to generate the human monoclonal antibodies of the invention contain rearranged, unrearranged or a combination of rearranged and unrearranged heterologous immunoglobulin heavy and light chain transgenes in the germline of the transgenic animal. Each of the heavy chain transgenes comprises at least one C_H gene. In addition, the heavy chain transgene may contain functional isotype switch sequences, which are capable of supporting isotype switching of a heterologous transgene encoding multiple C_H genes in the B-cells of the transgenic animal. Such switch sequences may be those which occur naturally in the germline immunoglobulin locus from the species that serves as the source of the transgene C_H genes, or such switch sequences may be derived from those which occur in the species that is to receive the transgene construct (the transgenic animal). For example, a human transgene construct that is used to produce a transgenic mouse may produce a higher frequency of isotype switching events if it incorporates switch sequences similar to those that occur naturally in the mouse heavy chain locus, as presumably the mouse switch sequences are optimized to function with the mouse switch recombinase enzyme system, whereas the human switch sequences are not. Switch sequences may be isolated and cloned by conventional cloning methods, or may be synthesized de novo from overlapping synthetic oligonucleotides designed on the basis of published sequence information relating to immunoglobulin switch region sequences (Mills et al, Nucl. Acids Res. 15:7305-7316 (1991); Sideras et al, Intl. Immunol. 1 :631-642 (1989), which are incorporated herein by reference). For each of the foregoing transgenic animals, functionally rearranged heterologous heavy and light chain immunoglobulin transgenes are found in a significant fraction of the B-cells of the transgenic animal (at least 10 percent).

The transgenes used to generate the transgenic animals of the invention include a heavy chain transgene comprising DNA encoding at least one variable gene segment, one diversity gene segment, one joining gene segment and at least one constant region gene segment. The immunoglobulin light chain transgene comprises DNA encoding at least one variable gene segment, one joining gene segment and at least one constant region gene segment. The gene segments encoding the light and heavy chain gene segments are heterologous to the transgenic non-human animal in that they are derived from, or correspond to, DNA encoding immunoglobulin heavy and light chain gene segments from a species not consisting of the transgenic non-human animal. In one aspect of the invention, the transgene is constructed such that the individual gene segments are unrearranged, i. e. , not rearranged so as to encode a functional immunoglobulin light or heavy chain. Such unrearranged transgenes support recombination of the V, D, and J gene segments (functional rearrangement) and preferably support incorporation of all or a portion of a D region gene segment in the resultant rearranged immunoglobulin heavy chain within the transgenic non-human animal when exposed to an S. aureus antigen. In an alternate embodiment, the transgenes comprise an unrearranged "mini- locus". Such transgenes typically comprise a substantial portion of the C, D, and J segments as well as a subset of the V gene segments. In such transgene constructs, the various regulatory sequences, e.g. promoters, enhancers, class switch regions, splice- donor and splice-acceptor sequences for RNA processing, recombination signals and the like, comprise corresponding sequences derived from the heterologous DNA. Such regulatory sequences may be incorporated into the transgene from the same or a related species of the non-human animal used in the invention. For example, human immunoglobulin gene segments may be combined in a transgene with a rodent immunoglobulin enhancer sequence for use in a transgenic mouse. Alternatively, synthetic regulatory sequences may be incorporated into the transgene, wherein such synthetic regulatory sequences are not homologous to a functional DNA sequence that is known to occur naturally in the genomes of mammals. Synthetic regulatory sequences are designed according to consensus rules, such as, for example, those specifying the permissible sequences of a splice-acceptor site or a promoter/enhancer motif. For example, a minilocus comprises a portion of the genomic immunoglobulin locus having at least one internal (i.e., not at a terminus of the portion) deletion of a non-essential DNA portion (e.g., intervening sequence; intron or portion thereof) as compared to the naturally-occurring germline Ig locus.

In a preferred embodiment of the invention, the transgenic animal used to generate human antibodies to S. aureus contains at least one, typically 2-10, and sometimes 25-50 or more copies of the transgene described in Example 12 of WO 98/24884 (e.g., pHCl or pHC2) bred with an animal containing a single copy of a light chain transgene described in Examples 5, 6, 8, or 14 of WO 98/24884, and the offspring bred with the J_H deleted animal described in Example 10 of WO 98/24884, the contents of which are hereby expressly incorporated by reference. Animals are bred to homozygosity for each of these three traits. Such animals have the following genotype: a single copy (per haploid set of chromosomes) of a human heavy chain unrearranged mini-locus (described in Example 12 of WO 98/24884), a single copy (per haploid set of chromosomes) of a rearranged human K light chain construct (described in Example 14 of WO 98/24884), and a deletion at each endogenous mouse heavy chain locus that removes all of the functional JH segments (described in Example 10 of WO 98/24884). Such animals are bred with mice that are homozygous for the deletion of the J_H segments (Examples 10 of WO 98/24884) to produce offspring that are homozygous for the J_H deletion and hemizygous for the human heavy and light chain constructs. The resultant animals are injected with antigens and used for production of human monoclonal antibodies against these antigens. B cells isolated from such an animal are monospecific with regard to the human heavy and light chains because they contain only a single copy of each gene. Furthermore, they will be monospecific with regards to human or mouse heavy chains because both endogenous mouse heavy chain gene copies are nonfunctional by virtue of the deletion spanning the J_H region introduced as described in Example 9 and 12 of WO 98/24884. Furthermore, a substantial fraction of the B cells will be monospecific with regards to the human or mouse light chains because expression of the single copy of the rearranged human K light chain gene will allelically and isotypically exclude the rearrangement of the endogenous mouse K and lambda chain genes in a significant fraction of B-cells. The transgenic mouse of the preferred embodiment will exhibit immunoglobulin production with a significant repertoire, ideally substantially similar to that of a native mouse. Thus, for example, in embodiments where the endogenous Ig genes have been inactivated, the total immunoglobulin levels will range from about 0.1 to 10 mg/ml of serum, preferably 0.5 to 5 mg/ml, ideally at least about 1.0 mg/ml. When a transgene capable of effecting a switch to IgG from IgM has been introduced into the transgenic mouse, the adult mouse ratio of serum IgG to IgM is preferably about 10:1. The IgG to IgM ratio will be much lower in the immature mouse. In general, greater than about 10%, preferably 40 to 80% of the spleen and lymph node B cells express exclusively human IgG protein. The repertoire will ideally approximate that shown in a non- transgenic mouse, usually at least about 10% as high, preferably 25 to 50% or more. Generally, at least about a thousand different immunoglobulins (ideally IgG), preferably 10⁴ to 10⁶ or more, will be produced, depending primarily on the number of different V, J and D regions introduced into the mouse genome. These immunoglobulins will typically recognize about one-half or more of highly antigenic proteins, e.g., staphylococcus protein A. Some of the immunoglobulins will exhibit an affinity for preselected antigens of at least about 10⁷M^_1, preferably 10⁸M^_1 to 10⁹M^_1 or greater.

In some embodiments, it may be preferable to generate mice with predetermined repertoires to limit the selection of V genes represented in the antibody response to a predetermined antigen type. A heavy chain transgene having a predetermined repertoire may comprise, for example, human VH genes which are preferentially used in antibody responses to the predetermined antigen type in humans. Alternatively, some VH genes may be excluded from a defined repertoire for various reasons (e.g., have a low likelihood of encoding high affinity V regions for the predetermined antigen; have a low propensity to undergo somatic mutation and affinity sharpening; or are immunogenic to certain humans). Thus, prior to rearrangement of a transgene containing various heavy or light chain gene segments, such gene segments may be readily identified, e.g. by hybridization or DNA sequencing, as being from a species of organism other than the transgenic animal.

The transgenic mice of the present invention can be immunized with whole S. aureus or an S. aureus antigen as described in Section I, supra. The mice will produce B cells which undergo class-switching via intratransgene switch recombination (cis- switching) and express immunoglobulins reactive with whole S. aureus or an S. aureus antigen. The immunoglobulins can be human sequence antibodies, wherein the heavy and light chain polypeptides are encoded by human transgene sequences, which may include sequences derived by somatic mutation and V region recombinatorial joints, as well as germline-encoded sequences; these human sequence immunoglobulins can be referred to as being substantially identical to a polypeptide sequence encoded by a human V_L or V_H gene segment and a human J_L or J_L segment, even though other non- germline sequences may be present as a result of somatic mutation and differential V-J and V-D-J recombination joints. With respect to such human sequence antibodies, the variable regions of each chain are typically at least 80 percent encoded by human germline V, J, and, in the case of heavy chains, D, gene segments; frequently at least 85 percent of the variable regions are encoded by human germline sequences present on the transgene; often 90 or 95 percent or more of the variable region sequences are encoded by human germline sequences present on the transgene. However, since non-germline sequences are introduced by somatic mutation and VJ and VDJ joining, the human sequence antibodies will frequently have some variable region sequences (and less frequently constant region sequences) which are not encoded by human V, D, or J gene segments as found in the human transgene(s) in the germline of the mice. Typically, such non-germline sequences (or individual nucleotide positions) will cluster in or near CDRs, or in regions where somatic mutations are known to cluster.

The human sequence antibodies which bind to the predetermined antigen can result from isotype switching, such that human antibodies comprising a human sequence γ chain (such as γl, γ2a, γ2B, or γ3) and a human sequence light chain (such as K) are produced. Such isotype-switched human sequence antibodies often contain one or more somatic mutation(s), typically in the variable region and often in or within about 10 residues of a CDR) as a result of affinity maturation and selection of B cells by antigen, particularly subsequent to secondary (or subsequent) antigen challenge. These high affinity human sequence antibodies may have binding affinities of at least 1 x 10⁹ M^"1, typically at least 5 x 10⁹ M^"1, frequently more than 1 x 10¹⁰ M^"1, and sometimes 5 x 10¹⁰ M^"1 to 1 x 10^{1 1} M^"! or greater.

Another aspect of the invention pertains to the B cells from such mice which can be used to generate hybridomas expressing monoclonal high affinity (greater than 2 x 10 M^" ) human sequence antibodies against whole S. aureus or an S. aureus antigen. These hybridomas can be used to generate a composition comprising an immunoglobulin having an affinity constant (Ka) of at least 2 x 10 M^"1 for binding whole S. aureus or an S. aureus antigen, wherein said immunoglobulin comprises: a human sequence light chain composed of (1) a light chain variable region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human V gene segment and a human J_L segment, and (2) a light chain constant region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human C_L gene segment; and a human sequence heavy chain composed of a (1 ) a heavy chain variable region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human V_H gene segment, optionally a D region, and a human J_H segment, and (2) a constant region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human C_H gene segment.

The development of high affinity human sequence antibodies against whole S. aureus or an S. aureus antigen is facilitated by a method for expanding the repertoire of human variable region gene segments in a transgenic mouse having a genome comprising an integrated human immunoglobulin transgene, said method comprising introducing into the genome a V gene transgene comprising V region gene segments which are not present in said integrated human immunoglobulin transgene. Often, the V region transgene is a yeast artificial chromosome comprising a portion of a human V_H or V (V_K) gene segment array, as may naturally occur in a human genome or as may be spliced together separately by recombinant methods, which may include out-of-order or omitted V gene segments. Often at least five or more functional V gene segments are contained on the YAC. In this variation, it is possible to make a transgenic mouse produced by the V repertoire expansion method, wherein the mouse expresses an immunoglobulin chain comprising a variable region sequence encoded by a V region gene segment present on the V region transgene and a C region encoded on the human Ig transgene. By means of the V repertoire expansion method, transgenic mice having at least 5 distinct V genes can be generated; as can mice containing at least about 24 V genes or more. Some V gene segments may be non-functional (e.g., pseudogenes and the like); these segments may be retained or may be selectively deleted by recombinant methods available to the skilled artisan, if desired.

Once the mouse germline has been engineered to contain a functional YAC having an expanded V segment repertoire, substantially not present in the human Ig transgene containing the J and C gene segments, the trait can be propagated and bred into other genetic backgrounds, including backgrounds where the functional YAC having an expanded V segment repertoire is bred into a mouse germline having a different human Ig transgene. Multiple functional YACs having an expanded V segment repertoire may be bred into a germline to work with a human Ig transgene (or multiple human Ig transgenes). Although referred to herein as YAC transgenes, such transgenes when integrated into the genome may substantially lack yeast sequences, such as sequences required for autonomous replication in yeast; such sequences may optionally be removed by genetic engineering (e.g., restriction digestion and pulsed-field gel electrophoresis or other suitable method) after replication in yeast in no longer necessary (i.e., prior to introduction into a mouse ES cell or mouse prozygote). Methods of propagating the trait of human sequence immunoglobulin expression, include breeding a transgenic mouse having the human Ig transgene(s), and optionally also having a functional YAC having an expanded V segment repertoire. Both V_H and V_L gene segments may be present on the YAC. The transgenic mouse may be bred into any background desired by the practitioner, including backgrounds harboring other human transgenes, including human Ig transgenes and/or transgenes encoding other human lymphocyte proteins. The invention also provides a high affinity human sequence immunoglobulin produced by a transgenic mouse having an expanded V region repertoire YAC transgene. Although the foregoing describes a preferred embodiment of the transgenic animal of the invention, other embodiments are contemplated which have been classified in four categories:

I. Transgenic animals containing an unrearranged heavy and rearranged light immunoglobulin transgene;

II. Transgenic animals containing an unrearranged heavy and unrearranged light immunoglobulin transgene;

III. Transgenic animal containing rearranged heavy and an unrearranged light immunoglobulin transgene; and IV. Transgenic animals containing rearranged heavy and rearranged light immunoglobulin transgenes.

Of these categories of transgenic animal, the preferred order of preference is as follows II > I > III > IV where the endogenous light chain genes (or at least the K gene) have been knocked out by homologous recombination (or other method) and I > II > III >IV where the endogenous light chain genes have not been knocked out and must be dominated by allelic exclusion.

III. Production of Bispecific/ Multispecific Molecules Which Bind to S. aureus

In yet another embodiment of the invention, human monoclonal antibodies to S. aureus or an S. aureus-antigen, or antigen-binding portions thereof, of the invention are derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., an Fab' fragment). For example, an antibody or antigen-binding portion of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody (e.g., a bispecific or a multispecific antibody), antibody fragment, peptide, or binding mimetic. Accordingly, in another aspect, the present invention features bispecific and multispecific molecules comprising at least one first binding specificity for S. aureus or an S. aureus-antigen and a second binding specificity an Fc receptor, e.g., human FcγRI or a human Fcα receptor. These bispecific and multispecific molecules are capable of binding both to FcγRI -or FcαR-expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and to target cells, including S. aureus or an S. aureus antigen. When binding in this manner, the bispecific and multispecific molecules trigger Fc receptor-mediated effector cell activities, such as phagocytosis of an S. aureus cell, antibody dependent cellular cytoxicity (ADCC), cytokine release, or generation of superoxide anion. Multispecific molecules of the invention can further include a third binding specificity, in addition to an anti-Fc binding specificity and an anti-target cell antigen binding specificity, such as S. aureus or an S. aureus antigen. In one embodiment, the third binding specificity is an anti-enhancement factor (EF) portion, e.g., a molecule which binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell. The "anti-enhancement factor portion" can be an antibody, functional antibody fragment or a ligand that binds to a given molecule, e.g., an antigen or a receptor, and thereby results in an enhancement of the effect of the binding determinants for the F_c receptor or target cell antigen. The "anti-enhancement factor portion" can bind an F_c receptor or a target cell antigen. Alternatively, the anti- enhancement factor portion can bind to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti- enhancement factor portion can bind a cytotoxic T-cell (e.g. via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that results in an increased immune response against the target cell). In one embodiment, the bispecific and multispecific molecules of the invention comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an Fab, Fab', F(ab')2, Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. U.S. Patent No. 4,946,778, issued August 7, 1990, the contents of which is expressly incorporated by reference.

In one embodiment bispecific and multispecific molecules of the invention comprise a binding specificity for an FcγR or an FcαR present on the surface of an effector cell, and a second binding specificity for a target cell antigen, e.g., an S. aureus or an S. aureus antigen.

In one embodiment, the binding specificity for an Fc receptor is provided by a human monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG). As used herein, the term "IgG receptor" refers to any of the eight γ-chain genes located on chromosome 1. These genes encode a total of twelve transmembrane or soluble receptor isoforms which are grouped into three Fcγ receptor classes: FcγRI (CD64), FcγRII(CD32), and FcγRIII (CD16). In one preferred embodiment, the Fcγ receptor a human high affinity FcγRI. The human FcγRI is a 72 kDa molecule, which shows high affinity for monomeric IgG (10⁸ - 10⁹ M->).

The production and characterization of these preferred monoclonal antibodies are described by Fanger et al. in PCT application WO 88/00052 and in U.S. Patent No. 4,954,617, the teachings of which are fully incorporated by reference herein. These antibodies bind to an epitope of FcγRI, FcγRII or FcγRIII at a site which is distinct from the Fcγ binding site of the receptor and, thus, their binding is not blocked substantially by physiological levels of IgG. Specific anti-FcγRI antibodies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. The hybridoma producing mAb 32 is available from the American Type Culture Collection, ATCC Accession No. HB9469. Anti-FcγRI mAb 22, F(ab')2 fragments of mAb 22, and can be obtained from Medarex, Inc. (Annandale, N.J.). In other embodiments, the anti-Fcγ receptor antibody is a humanized form of monoclonal antibody 22 (H22). The production and characterization of the H22 antibody is described in Graziano, R.F. et al. (1995) J. Immunol 155 (10): 4996-5002 and PCT/US93/10384. The H22 antibody producing cell line was deposited at the American Type Culture Collection on November 4, 1992 under the designation HA022CL1 and has the accession no. CRL 11177. In still other preferred embodiments, the binding specificity for an Fc receptor is provided by an antibody that binds to a human IgA receptor, e.g., an Fc-alpha receptor (FcαR (CD89)). Preferably, the antibody binds to a human IgA receptor at a site that is not blocked by endogenous IgA. The term "IgA receptor" is intended to include the gene product of one α-gene (FcαRI) located on chromosome 19. This gene is known to encode several alternatively spliced transmembrane isoforms of 55 to 110 kDa. FcαRI (CD89) is constitutively expressed on monocytes/macrophages, eosinophilic and neutrophilic granulocytes, but not on non-effector cell populations. FcαRI has medium affinity (« 5 x 10⁷ M-¹) for both IgAl and IgA2, which is increased upon exposure to cytokines such as G-CSF or GM-CSF (Morton, H.C. et al. (1996) Critical Reviews in Immunology 16:423-440). Four FcαRI-specific monoclonal antibodies, identified as A3, A59, A62 and A77, which bind FcαRI outside the IgA ligand binding domain, have been described (Monteiro, R.C. et al., 1992, J Immunol. 148:1764).

FcαRI and FcγRI are preferred trigger receptors for use in the invention because they are (1) expressed primarily on immune effector cells, e.g., monocytes, PMNs, macrophages and dendritic cells; (2) expressed at high levels (e.g., 5,000-100,000 per cell); (3) mediators of cytotoxic activities (e.g., ADCC, phagocytosis); (4) mediate enhanced antigen presentation of antigens, including self-antigens, targeted to them. In other embodiments, bispecific and multispecific molecules of the invention further comprise a binding specificity which recognizes, e.g. , binds to, a target cell antigen, e.g., S. aureus antigen. In a preferred embodiment, the binding specificity is provided by a human monoclonal antibody of the present invention.

An "effector cell specific antibody" as used herein refers to an antibody or functional antibody fragment that binds the Fc receptor of effector cells. Preferred antibodies for use in the subject invention bind the Fc receptor of effector cells at a site which is not bound by endogenous immunoglobulin.

As used herein, the term "effector cell" refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, e.g., lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and carry out specific immune functions. In preferred embodiments, an effector cell is capable of inducing antibody- dependent cellular toxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes which express FcαR are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In other embodiments, an effector cell can phagocytose a target antigen, target cell, or microorganism. The expression of a particular FcR on an effector cell can be regulated by humoral factors such as cytokines. For example, expression of FcγRI has been found to be up-regulated by interferon gamma (IFN-γ). This enhanced expression increases the cytotoxic activity of FcγRI -bearing cells against targets. An effector cell can phagocytose or lyse a target antigen or a target cell.

"Target cell" shall mean any undesirable cell in a subject (e.g., a human or animal) that can be targeted by a composition (e.g. , a human monoclonal antibody, a bispecific or a multispecific molecule) of the invention. In preferred embodiments, the target cell is an S. aureus cell. For example, an S. aureus cell can be an antibiotic - resistant strains of S. aureus, e.g., penicillin- and methicillin-resistant S. aureus, including, for example, IBERIAN, EMRSA, ONTARIO, BRAZILIAN and COL strains (2,3). MRSA strains are generally resistant to β-lactam antibiotics by expression of a mutated penicillin binding protein (PBP 2a) that has a low binding capacity for these antibiotics (4).

In certain embodiments, the antibodies used in the bispecific or multispecific molecules of the invention can be human, chimeric or humanized antibody to an Fc receptor. Chimeric mouse-human monoclonal antibodies (i.e., chimeric antibodies) can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted, (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al, 1987, J. Immunol. 139:3521-3526; Sun et α/. (1987) PNAS 84:214-218; Nishimura et α/., 1987, Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al, 1988, J. Natl Cancer Inst. 80:1553-1559).

The chimeric antibody can be further humanized by replacing sequences of the Fv variable region which are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General reviews of humanized chimeric antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207 and by Oi et al, 1986, BioTechniques 4:214. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from 7E3, an anti-GPIIbIII_a antibody producing hybridoma. The recombinant DNA encoding the chimeric antibody, or fragment thereof, can then be cloned into an appropriate expression vector. Suitable humanized antibodies can alternatively be produced by CDR substitution U.S. Patent 5,225,539; Jones et al. 1986 Nature 321 :552-525; Verhoeyan et al. 1988 Science 239:1534; and Beidler et al. 1988 J. Immunol. 141 :4053- 4060.

All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non- human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to the Fc receptor. An antibody can be humanized by any method, which is capable of replacing at least a portion of a CDR of a human antibody with a CDR derived from a non-human antibody. Winter describes a method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on March 26, 1987), the contents of which is expressly incorporated by reference. The human CDRs may be replaced with non-human CDRs using ohgonucleotide site- directed mutagenesis as described in International Application WO 94/10332 entitled, Humanized Antibodies to Fc Receptors for Immunoglobulin G on Human Mononuclear Phagocytes.

Also within the scope of the invention are chimeric and humanized antibodies in which specific amino acids have been substituted, deleted or added. In particular, preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, in a humanized antibody having mouse CDRs, amino acids located in the human framework region can be replaced with the amino acids located at the corresponding positions in the mouse antibody. Such substitutions are known to improve binding of humanized antibodies to the antigen in some instances. Antibodies in which amino acids have been added, deleted, or substituted are referred to herein as modified antibodies or altered antibodies.

The term modified antibody is also intended to include antibodies, such as monoclonal antibodies, chimeric antibodies, and humanized antibodies which have been modified by, e.g., deleting, adding, or substituting portions of the antibody. For example, an antibody can be modified by deleting the constant region and replacing it with a constant region meant to increase half-life, e.g., serum half-life, stability or affinity of the antibody. Any modification is within the scope of the invention so long as the bispecific and multispecific molecule has at least one antigen binding region specific for an FcγR and triggers at least one effector function. Bispecific and multispecific molecules of the present invention can be made using chemical techniques (see e.g., D. M. Kranz et al. (1981) Proc. Natl. Acad. Sci. USA 78:5807), "polydoma" techniques (See U.S. Patent 4,474,893, to Reading), or recombinant DNA techniques.

In particular, bispecific and multispecific molecules of the present invention can be prepared by conjugating the constituent binding specificities, e.g., the anti-FcR and anti-S. aureus binding specificities, using methods known in the art and described in the examples provided herein. For example, each binding specificity of the bispecific and multispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross- linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), N- succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N- maleimidomethyl) cyclohaxane-1-carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (Behring Ins. Mitt. (1985) No. 78, 118-132); Brennan et al. (Science (1985) 229:81-83), and Glennie et al. (J. Immunol. (1987) 139: 2367-2375). Preferred conjugating agents are SATA and sulfo- SMCC, both available from Pierce Chemical Co. (Rockford, IL). When the binding specificities are antibodies (e.g., two humanized antibodies), they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific and multispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2 or ligand x Fab fusion protein. A bispecific and multispecific molecule of the invention, e ., a bispecific molecule can be a single chain molecule, such as a single chain bispecific antibody, a single chain bispecific molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific and multispecific molecules can also be single chain molecules or may comprise at least two single chain molecules. Methods for preparing bi- and multispecific molecules are described for example in U.S. Patent Number 5,260,203; U.S. Patent Number 5,455,030; U.S. Patent Number 4,881,175; U.S. Patent Number 5,132,405; U.S. Patent Number 5,091,513; U.S. Patent Number 5,476,786; U.S. Patent Number 5,013,653; U.S. Patent Number 5,258,498; and U.S. Patent Number 5,482,858. Binding of the bispecific and multispecific molecules to their specific targets can be confirmed by enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), or a Western Blot Assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the FcR-antibody complexes can be detected using e.g., an enzyme-linked antibody or antibody fragment which recognizes and specifically binds to the antibody-FcR complexes. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a γ counter or a scintillation counter or by autoradiography.

IV. Antibody Conjugates/Immunotoxins

In another aspect, the present invention features a human anti-PSMA monoclonal antibody, or a fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug or a radioisotope. When conjugated to a cytotoxin, these antibody conjugates are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fiuorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). An antibody of the present invention can be conjugated to a radioisotope, e.g., radioactive iodine, to generate cytotoxic radiopharmaceuticals for treating a PSMA-related disorder, such as a cancer.

The antibody conjugates of the invention can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-γ; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al, "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).

V. Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, containing one or a combination of human monoclonal antibodies, or antigen-binding portion(s) thereof, of the present invention, formulated together with a pharmaceutically acceptable carrier. In a preferred embodiment, the compositions include a combination of multiple (e.g., two or more) isolated human antibodies or antigen-binding portions thereof of the invention. Preferably, each of the antibodies or antigen-binding portions thereof of the composition binds to a distinct, preselected epitope of an S. aureus or S. αwreus-antigen.

In one embodiment, human anti-S. aureus monoclonal antibodies having complementary activities are used in combination, e.g., as a pharmaceutical composition, comprising two or more human anti-S. aureus monoclonal antibodies. For example, a human monoclonal antibody having limited cross-reactivity among S. aureus strains, but mediating very efficient phagocytosis can be used in combination with other human monoclonal antibodies to S. aureus exhibiting broader specificity.

In another embodiment, the composition comprises one or a combination of bispecific or multispecific molecules of the invention (e.g., which contains at least one binding specificity for an Fc receptor and at least one binding specificity for an S. aureus or an S. aureus antigen).

Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include a composition of the present invention with at least one anti- infectious agent (e.g. , an antibiotic), or other conventional therapy.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, bispecific and multispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N- methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like. A composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent.

Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol 7:27).

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze- drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about 1 per cent to about 30 per cent.

Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of compositions of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.01 to 99.5% (more preferably. 0.1 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a compositions of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic compositions may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4. ,486, 194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., VN. Ranade (1989) J. Clin. Pharmacol. 29:685).

Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al); mannosides (Umezawa et al, (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol 1233:134), different species of which may comprise the formulations of the inventions, as well as components of the invented molecules; pi 20 (Schreier et al. (1994) J. Biol Chem. 269:9090); see also K. Keinanen; ML. Laukkanen (1994) FEBS Lett. 346:123; J.J. Killion; I.J. Fidler (1994) Immunomethods 4:273. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety. In a most preferred embodiment, the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the tumor or infection. The composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

A "therapeutically effective dosage" preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to inhibit cancer can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. The composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. When the active compound is suitably protected, as described above, the compound may be orally administered, for example, with an inert diluent or an assimilable edible carrier.

VI. Uses and Methods of the Invention The compositions (e.g., human antibodies and derivatives thereof) of the present invention have in vitro and in vivo diagnostic and therapeutic utilities. For example, these molecules can be administered to cells in culture, e.g. in vitro or ex vivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose a variety of disorders. As used herein, the term "subject" is intended to include human and non-human animals. Preferred human animals include a human patient having an S. αwrews-mediated disorder. For example, the methods and compositions of the present invention can be used to treat a subject at risk of acquiring a nosocomial infection. Examples of particularly susceptible classes of subjects include the elderly and immunocompromised hospital patients. The term "non-human animals" of the invention includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc. The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can be initially tested for binding activity associated with therapeutic or diagnostic use in vitro. For example, compositions of the invention can be tested using the ELISA and flow cytometric assays described in the Examples below. Moreover, the activity of these molecules in triggering at least one effector-mediated effector cell activity, including phagocytosis of S. aureus can be assayed. Protocols for assaying for effector cell-mediated phagocytosis are described in the Examples below.

The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention have additional utility in therapy and diagnosis of S. aureus- mediated diseases. For example, the human monoclonal antibodies, the multispecific or bispecific molecules can be used, for example, to elicit in vivo or in vitro one or more of the following biological activities: to opsonize S. aureus; to mediate phagocytosis or to inhibit growth of S. aureus in the presence of human effector cells; or to inhibit S. aureus growth. For example, human antibodies and derivatives thereof of the invention can be used in vivo to treat, prevent or diagnose a variety of S. αwrews-mediated diseases. Examples of S. αwrews-mediated diseases include, for example, invasive and toxigenic infectious diseases. Exemplary invasive diseases include: Bacteremia, osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis. Exemplary toxigenic diseases include: Staphylococcol food poisoning, scalded skin syndrome and toxic shock syndrome. Additional examples of S. aureus-mediated diseases that can be treated using the methods and compositions of the invention include infections of the upper respiratory tract (e.g., Otis media, bacterial trachetis, acute epiglottitis, thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac (e.g., infective endocarditis), gastrointestinal (e.g., secretory diarrhea, splenic abscess , retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis , keratitis, endophthalmitis, preseptal and orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g., epididymitis, intrarenal and perinephric abscess , toxic shock system), skin (e.g., impetigo, folloculitis, cutaneuos abscesses, cellulitis, wound infection, bacterial myositis), bone and joint (e.g., septic arthritis, osteomyelitis). Methods of administering the compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention are known in the art. Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used. The molecules can be coupled to radionuclides, such as 1311, 90Y, 105Rh, etc., as described in Goldenberg, D.M. et al. (1981) Cancer Res. 41 : 4354-4360, and in EP 0365 997. The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be coupled to anti-infectious agents. Target-specific effector cells, e.g., effector cells linked to compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be used as therapeutic agents. Effector cells for targeting can be human leukocytes such as macrophages, neutrophils or monocytes. Other cells include and other IgG- or IgA- receptor bearing cells. If desired, effector cells can be obtained from the subject to be treated. The target-specific effector cells, can be administered as a suspension of cells in a physiologically acceptable solution. The number of cells administered can be in the order of 10^- 10^ but will vary depending on the therapeutic purpose. In general, the amount will be sufficient to obtain localization at the target cell, e.g., an S. aureus cell, and to effect cell killing by, e.g., phagocytosis. Routes of administration can also vary. Therapy with target-specific effector cells can be performed in conjunction with other techniques for removal of targeted cells. For example, anti-bacterial therapy using the compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention and/or effector cells armed with these compositions can be used in conjunction with antibiotic therapy. Additionally, combination immunotherapy may be used to direct two distinct cytotoxic effector populations toward tumor cell rejection. For example, anti-S. aureus antibodies linked to anti-Fc-gammaRI or anti-T3 may be used in conjunction with IgG- or IgA-receptor specific binding agents.

Bispecific and multispecific molecules of the invention can also be used to modulate FcγR or FcαR levels on effector cells, such as by capping and elimination of receptors on the cell surface. Mixtures of anti-Fc receptors can also be used for this purpose. The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention which have complement binding sites, such as portions from IgGl, -2, or -3 or IgM which bind complement can also be used in the presence of complement. In one embodiment, ex vivo treatment of a population of cells comprising target cells with a binding agent of the invention and appropriate effector cells can be supplemented by the addition of complement or serum containing complement. Phagocytosis of target cells coated with a binding agent of the invention can be improved by binding of complement proteins. In another embodiment target cells coated with the compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be lysed by complement.

The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be administered together with complement. Accordingly, within the scope of the invention are compositions comprising human antibodies, multispecific or bispecific molecules and serum or complement. These compositions are advantageous in that the complement is located in close proximity to the human antibodies, multispecific or bispecific molecules. Alternatively, the human antibodies, multispecific or bispecific molecules of the invention and the complement or serum can be administered separately.

Also within the scope of the invention are kits comprising the compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention and instructions for use. The kit can further contain a least one additional reagent, such as complement, or one or more additional human antibodies of the invention (e.g., a human antibody having a complementary activity which binds to an epitope in S. aureus or an S. aureus antigen distinct from the first human antibody).

In other embodiments, the subject can be additionally treated with an agent that modulates, e.g., enhances or inhibits, the expression or activity of Fcγ or Fcα receptors, by for example, treating the subject with a cytokine. Preferred cytokines for administration during treatment with the multispecific molecule include of granulocyte colony-stimulating factor (G-CSF), granulocyte- macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumor necrosis factor (TNF).

The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be used to target cells expressing FcγR or S. αwrews-antigens, for example for labeling such cells. For such use, the binding agent can be linked to a molecule that can be detected. Thus, the invention provides methods for localizing ex vivo or in vitro cells expressing FcγR or S. αwrews-antigens. The detectable label can be, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. In one embodiment, the invention provides methods for detecting the presence of S. aureus in a sample, comprising: contacting the sample, and a control sample, with a human monoclonal antibody, or an antigen binding portion thereof, which specifically binds to S. aureus or an

S. aureus-antigen, under conditions that allow for formation of a complex between the antibody or portion thereof and the S. ureus or an S. aureus- antigen, and detecting the formation of a complex, wherein a difference complex formation between the sample compared to the control sample is indicative the presence of S. aureus in the sample.

In still another embodiment, the invention provides a method for detecting the presence of an Fc-expressing cell in vivo or in vitro. The method comprises (i) administering to a subject a composition (e.g., a multi- or bispecific molecule) of the invention or a fragment thereof, conjugated to a detectable marker; (ii) exposing the subject to a means for detecting said detectable marker to identify areas containing Fc- expressing cells.

The present invention is further illustrated by the following examples, which should not be construed as further limiting.

EXAMPLES

Example 1 Production of Human Monoclonal Antibodies Against S. aureus

Human anti-S. aureus monoclonal antibodies were generated by immunizing two strains of HC07 HuMAb mice with heat killed S. aureus strain FDA 209. The HC07 HuMAb mice used in the study were generated as described in U.S. Patent Nos. 5,545,806, 5,625,825, and 5,545,807, the entire disclosures of which are hereby incorporated by reference. In particular, HC07 mice were immunized for 12 weeks using heat killed FDA

209 grown in broth and emulsified in CFA. Immunized mice were boosted twice at two week intervals with the same bacteria in IFA. At 8 and 10 weeks, serum was collected from these mice. S. aureus-specific titers were detected in the immunized animals after the third immunization. Figure 1 shows the optical density (OD (405-650)) for the indicated dilution of pooled plasma from S. aureus immunized mice (hatched bar) and non-immunized mice (solid bar) as analyzed by ELISA using S. aureus-coated microtiter plates and detected with an alkaline phosphatase-conjugated anti-human IgG probe. When the mice showed a titer against S. aureus 209 as measured by ELISA assay, they were boosted with an i.v. injection of heat killed S. aureus 209. Three days later the mice were sacrificed and spleens were harvested.

To generate hybridomas producing anti-S. aureus antibodies, splenocytes from mice showing plasma containing anti-S. aureus antibodies were fused with

P3X63-Ag8.653 cells (deposited with the ATCC under designation ATCC CRL 1580 nonsecreting mouse myeloma cells) and PEG. Approximately 800 mixed hybridoma cultures were generated. Supernatants from these hybridoma cultures were screened for production of human IgGl and for binding to S. aureus 209 strain. Figure 2 depicts the binding of six supernatants from the hybridoma cultures termed 2G12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12 to S. aureus 209 strain as measured by ELISA. Briefly, bacteria were dried onto a 96-well plate and fixed with .25% glutaraldehyde. Supernatants were added were added and incubated for 1 hour. Human IgG was detected using goat anti- human IgG alkaline phosphatase and pNPP. Binding to human IgG of the anti-FcγRI (H22 antibody) was used as a control. Significant binding was detected with the six hybridoma supernatants shown as compared to the H22 control.

Example 2 Characterization of Human Monoclonal Antibodies Against S. aureus

The mixed hybridoma supernatants generated in Example 1 were tested for binding to Methicillin resistant S. aureus (MRSA) strains as follows.

Binding of the supernatants to the MRSA strains BK2058 and BK2709, were tested using an ELISA assay. These two strains are Iberian MRSA clones isolated from a hospital outbreak and they both overexpress a Penicillin binding protein. BK2058 also overexpresses a fibronectin binding protein. BK2058 and BK2709 strains were fixed to wells of a 96 well plate and the antibody in the supernatant was detected using the goat anti-human IgG alkaline phosphatase described in Example 1. Media alone was used as a control. Figure 3 shows the binding of the mixed hybridoma supernatants 2G12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12 to S. aureus MRS A strain 2058. No significant binding of the supernatant from the mixed hybridoma cultures (2G12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12) to mixed bacteria (Escherichia coli, Pseudomonas aeroginosa, or Micrococcus luteus) was detected, as measured by ELISA (Figure 4).

The specificity of the human anti-S. aureus monoclonal antibodies was confirmed by comparing the binding of the supernatants from mixed hybridoma cultures (2G12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12) to S. aureus (speckled bar) or E. coli (solid bar), as measured by flow cytometry (Figure 5). As shown in Figure 5, significant levels of supernatant binding were detected only upon incubation with S. aureus.

Figure 6 is a bar graph showing the binding of the supernatants from subcloned hybridoma cultures (2G12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12) to S. aureus FDA 209, as measured be ELISA. As before, S. aureus cells were fixed to wells of a 96-well plate and the monoclonal antibody present on the supernatant was detected using anti-human IgG (kappa chain) alkaline phosphatase. The second antibody confirmed that the supernatants contain a human antibody that binds to S. aureus. Binding to human IgG of the anti-FcγRI (H22 antibody) was used as a control.

In addition, supernatants from subclones of the hybridoma cultures 6D12 and 5H10 were assayed by ELISA for binding to S. aureus strain ATCC 27661. Briefly, S. aureus bacteria were grown overnight in trypticase soy agar broth, resuspended in 1% gelatin in PBS, dried onto a 96-well plate at 40°C overnight, and fixed with 0.1% glutaraldehyde in PBS. The S. aureus coated plates were blocked with 20% mouse sera, 0.5% EDTA, 0.25% BSA, 0.5% NaN₃, washed, and hybridoma supernatants (or antibodies (25 μg/ml)) were added at 100 μl/well and incubated for 2 hours at 37°C or overnight at 4°C. Human antibody was detected by incubation with anti-human IgG Fab'2 conjugated to alkaline phosphatase for 1 hour at 37°C, followed by the addition of pNPP. The 6D12 and 5H10 subcloned hybridoma supernatants demonstrated significant binding to S. aureus as compared to the isotype control antibody (Figure 7). Selected hybridoma supernatants that showed significant, specific binding to S. aureus (e.g., 2G12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12) were then tested for the ability to mediate S. aureus phagocytosis (Figures 8A-8C). Briefly, PMNs were incubated for 30 minutes at 37°C with FITC labeled S. aureus, with and without supernatant from mixed hybridoma cultures. Changes in fluorescence were detected using a FACSscan. Figures 8A-8C are histograms showing neutrophil-mediated phagocytosis of S. aureus upon incubation of polymorphonuclear cells (PMNs) with a mixed hybridoma supernatant. Figures 8A-8C show the results from one representative supernatant of 6 supernatants that had previously been shown to contain anti-S. aureus human IgG. Figures 8A-8C show FACScan analyses of control PMNs alone (Figure 8 A), PMNs and S. aureus, with or without supernatant from mixed hybridoma cultures (Figure 8B), and PMNs and control E. coli, with or without supernatant from mixed hybridoma cultures (Figure 8C). A shift in the fluorescence was detected when PMNs and S. aureus were incubated with the supernatant from the mixed hybridoma cultures, which indicates that there was phagocytosis of FITC-labeled S. aureus (Figure 8B). No significant changes in bacterial phagocytosis were detected in the following conditions: PMNs alone (Figure 8 A); upon incubation of PMNs with S. aureus without supernatant from the mixed hybridoma cultures (Figure 8B); and upon incubation of PMNs with E. coli with supernatant from the mixed hybridoma cultures (Figure 8C). Selected hybridomas that showed specific binding and phagocytotic activities

(e.g.,, 2G12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12) were also subcloned to develop purified cultures that produced human monoclonal antibodies against S. aureus. The resulting purified antibodies were again tested for binding to S. aureus FDA 209 (Figures 9 A and 9B) and MRS A strain 2058 (Figures 10A and 10B) and for their ability to mediate S. aureus phagocytosis as described above.

Figures 9A-9B show binding of purified human monoclonal antibody 6D12 to S. aureus FDA 209 compared to IgG controls. In particular, Figure 9A is a bar graph depicting the binding of two concentrations (1 and 10 μg/ml) of purified human monoclonal antibody 6D12 (open bars) to S. aureus FDA 209, compared to IgG controls (speckled bars) as measured using ELISA. The purified 6D12 human monoclonal antibody was compared with a control human IgG (anti-FcγRI monoclonal antibody H22) for binding to S. aureus FDA 209. Bacteria dried onto microtiter plates were blocked with 20% mouse serum, and then reacted with monoclonal antibody preparations. Monoclonal antibody binding was detected with an alkaline phosphatase- conjugated anti-human IgG Fc specific probe. Figure 9B is a histogram showing direct binding of FITC-labeled 6D12 monoclonal antibody to S. aureus FDA 209 compared to FITC-labeled humanized anti-EGF receptor antibody control (H425) as detected by flow cytometry. Briefly, 25 μg/ml of FITC-labeled 6D12 monoclonal antibody and FITC- labeled H425 control were incubated with S. aureus FDA 209 and 2.8 mg/ml of irrelevant human IgG to block protein A. S. aureus cells were fixed with paraformaldehyde and analyzed on a BD flow cytometer. Figures 10A-10B show binding of purified human monoclonal antibody 6D12 to

MRS A strain 2058 compared to IgG controls. In particular, Figure 10A is a linear graph depicting the binding of two concentrations (1 and 10 μg/ml) of purified human monoclonal antibody 6D12 to MRSA strain 2058, compared to IgG controls. Figure 10B is a histogram showing direct binding of FITC-labeled 6D12 monoclonal antibodies to MRSA strain 2058 compared to FITC-labeled humanized anti-EGF receptor antibody control (H425) as detected by flow cytometry. Briefly, 25 μg/ml of FITC-labeled 6D12 monoclonal antibody and FITC-labeled H425 control were incubated with MRSA strain 2058 and 2.8 mg/ml of irrelevant human IgG to block protein A. S. aureus cells were fixed with paraformaldehyde and analyzed on a BD flow cytometer. The survival of S. aureus strain FDA-209 after PMN phagocytosis was then assessed as follows. Bacteria were grown overnight in trypicase soy agar broth, harvested and resuspended in PBS at a concentration of 10 cfu/ml. PMNs were collected from whole blood from a healthy volunteer. Briefly, 120 ml of whole blood was diluted 1 :1 with RPMI cell media, layered over Ficoll-paque, and centrifuged at 500 x g for 30 minutes. The PMN layer was collected and contaminating red blood cells were lysed with KCO₃ lysing solution. PMNs were washed twice, counted , and adjusted to a concentration of 10⁶ cells/ml. All reagents were prepared in 2% human sera which was preabsorbed to live S. aureus. Bacteria (200 μl) were opsonized with 10 μl of the 6D12 antibody and an isotype control antibody (100 μg/ml) for 10 minutes at room temperature, and then incubated with human PMNs (200 μl) for 90 minutes at 37°C with constant rotation. The bacteria and PMNs were then microfuged for 5 minutes and the cell pellet was resuspended in 1 ml of distilled water. Serial dilutions were made in distilled water and 50 μl of each dilution was plated on tryptic soy agar plates and surviving S. aureus was counted.

As shown in Figure 11, the human anti-S. aureus antibody 6D12 increased the PMN mediated killing of S. aureus by 60% as compared to a human isotype control antibody against CD30.

Conclusion

The foregoing Examples demonstrate the generation of human monoclonal antibodies that specifically react with high affinity to at least two strains of S. aureus (FDA 209 and MRSA strain 2058). In addition, the human monoclonal anti-S. aureus antibodies effectively mediate phagocytic activity against at least two strains of S. aureus (FDA 209 and MRSA strain 2058) using PMNs as effector cells. Thus, the phagocytic activities of these human antibodies against Methicillin-resistant S. aureus was established. These results support the conclusion that the fully human monoclonal antibodies against S. aureus of the present invention are useful for the treatment of S. aureus nosocomial infections.

VII. References

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Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Incorporation by Reference

The entire contents of all references, pending patent applications and issued patents cited herein are hereby expressly incorporated by reference into the present disclosure.

Claims

We claim:

1. An isolated human monoclonal antibody, or an antigen binding portion thereof, that specifically binds to S. aureus or an S. aureus-antigen, wherein the antibody or antigen binding portion thereof has one or more of the following characteristics: a) reactivity with at least one S. aureus isolate; b) a binding affinity constant to S. aureus or an S. aureus-antigen of at least about 10⁷ M-¹; c) the ability to opsonize S. aureus; or d) the ability to mediate phagocytosis or to inhibit growth of S. aureus in the presence of human effector cells at a concentration of about 10 μg/ml or less in vitro.

2. The isolated human antibody of claim 1, or an antigen binding portion thereof, having an isotype selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD, and IgE.

3. The isolated human antibody of claim 1, or an antigen binding portion thereof, which is an IgG Ik.

4. The isolated human antibody of claim 1, or an antigen binding portion thereof, wherein the at least one S. aureus isolate is a methicillin-resistant S. aureus strain.

5. The isolated human antibody of claim 1, or an antigen binding portion thereof, wherein the at least one S. aureus isolate is selected from the group consisting of IBERIAN, EMRSA, ONTARIO and BRAZILIAN strains.

6. The isolated human antibody of claim 1, or an antigen binding portion thereof, produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal having a genome comprising a human heavy chain transgene and a human light chain transgene fused to an immortalized cell.

7. The isolated human antibody of claim 1 , or an antigen binding portion thereof, produced by a hybridoma selected from the group consisting of 2GD12, 2H12, 8.1E5, 8.2Cl, 7Fl, 6D12 and 5H10.

8. The isolated human antibody of claim 1 , or an antigen binding portion thereof, wherein the S. aureus antigen is selected from the group consisting of a secreted antigen and an antigen present on the surface of S. aureus.

9. The isolated human antibody of claim 8, or an antigen binding portion thereof, wherein the S. aureus antigen is selected from the group consisting of a capsular polysaccharide, a fibronectin binding protein, protein A, a toxin, a toxic shock syndrome toxin superantigen, a coagulase, a staphylokinase, a penicillin binding protein 2a and an adhesin.

10. An isolated human monoclonal antibody, or antigen-binding portion thereof, which mediates phagocytosis of S. aureus in the presence of human effector cells.

11. The isolated human antibody of claim 10, or an antigen binding portion thereof, which is capable of mediating phagocytosis of S. aureus by human effector cells at an IC50 of 1 x 10" M or less in vitro.

12. A hybridoma comprising a B cell obtained from a transgenic non-human animal having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell, wherein the hybridoma produces a detectable amount of a human monoclonal antibody that specifically binds to S. aureus or an S. aureus antigen.

13. The hybridoma of claim 12, wherein the human monoclonal antibody has one or more of the following characteristics: a) reactivity with at least one S. aureus isolate; b) a binding affinity constant to S. aureus or an S. aureus-antigen of at least about 10⁷ M-¹; c) the ability to opsonize S. aureus; and d) the ability to mediate phagocytosis or to inhibit growth of S. aureus in the presence of human effector cells at a concentration of about 10 μg/ml or less in vitro.

14. The hybridoma of claim 13, selected from the group consisting of 2GD12, 2H12, 8.1E5, 8.2C1, 7F1, 6D12 and 5H10.

15. A transgenic non-human animal capable of expressing a human monoclonal antibody that specifically binds to S. aureus or an S. αwrews-antigen, wherein the transgenic non-human animal has a genome comprising a human heavy chain transgene and a human light chain transgene.

16. A method of producing a human monoclonal antibody that specifically bind to S. aureus or an S. aureus-antigen, comprising: immunizing a transgenic non-human animal having a genome comprising a human heavy chain transgene and a human light chain transgene with whole S. aureus or an S. aureus-antigen, such that antibodies are produced by B cells of the animal; isolating B cells of the animal; and fusing the B cells with myeloma cells to form immortal, hybridoma cells that secrete human monoclonal antibodies specific for S. aureus or an S. aureus- antigen.

17. A bispecific molecule comprising at least one first binding specificity for S. aureus or an S. aureus-antigen and a second binding specificity an Fc receptor.

18. The bispecific molecule of claim 17, wherein the Fc receptor is a human FcγRI or a human Fcα receptor.

19. The bispecific molecule of claim 17, which binds to the Fc receptor at a site which is distinct from the immunoglobulin binding site of the receptor.

20. A composition comprising an isolated human monoclonal antibody or antigen- binding portion thereof of claim 1 , and a pharmaceutically acceptable carrier.

21. A composition comprising a combination of two or more isolated human antibodies or antigen-binding portions thereof according to claim 1 , wherein each of said antibodies or antigen-binding portions thereof binds to a distinct epitope of an S. aureus or S. αwrerø-antigen.

22. A method of inhibiting growth or inducing phagocytosis of S. aureus in the presence of human effector cells, comprising contacting S. aureus with an isolated human monoclonal antibody, or an antigen binding portion thereof, that specifically binds to S. aureus or an S. aureus-antigen, in the presence of an effector cell, such that phagocytosis of the S. aureus occurs.

23. A method of treating or preventing an S. αwrews-mediated disease, comprising administering to a subject an isolated human antibody, or an antigen binding portion thereof, that specifically binds to S. aureus or an S. aureus-antigen in an amount effective to treat or prevent the S. αureus-meάiateά disease.

24. The method of claim 23, wherein the S. αwrews-mediated disease is an invasive or a toxigenic infectious disease.

25. The method of claim 23, wherein the S. αurews-mediated disease is selected from the group consisting of bacteremia, osteomyelitis, septic arthritis, septic thrombophlebitis, acute bacterial endocarditis, Staphylococcol food poisoning, scalded skin syndrome and toxic shock syndrome.

26. A method for detecting the presence of S. aureus in a sample, comprising: contacting the sample, and a control sample, with a human monoclonal antibody, or an antigen binding portion thereof, which specifically binds to S. aureus or an S. aureus- antigen, under conditions that allow for formation of a complex between the antibody or portion thereof and the S. aureus or an S. aureus-antigen, and detecting the formation of a complex, wherein a difference complex formation between the sample compared to the control sample is indicative the presence of S. aureus in the sample.