JP2003503015A - Human antibodies to Staphylococcus aureus - Google Patents

Abstract

  (57) [Summary] Disclosed are isolated human monoclonal antibodies and antigen-binding portions thereof that specifically bind to one or more strains of Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus strains. These human antibodies can be produced in non-human transgenic animals, such as transgenic mice, which can produce multiple isotypes of a human monoclonal antibody by undergoing V-D-J recombination and isotype switching. Also disclosed are pharmaceutical compositions comprising these human antibodies, non-human transgenic animals and hybridomas producing these human antibodies, and therapeutic and diagnostic methods using these human antibodies. .

Description

Detailed Description of the Invention

[0001] BACKGROUND Staphylococcus aureus invention (Staphylococcus aureus) is a human pathogen that can cause symptoms variety of skin swelling up sepsis and death
(1). Staphylococcus is one of the most common nosocomial pathogens in hospitals and long-term care facilities. In particular, Staphylococcus aureus is an important cause of infections associated with the placement of medical devices such as heart valves and artificial joints. Examples of the particularly susceptible class of patients include elderly and immunocompromised hospital patients. In addition to being a major cause of nosocomial infections, Staphylococcus aureus can also cause disease through community-acquired infections.

Since the common use of antibiotics, antibiotic resistant strains of Staphylococcus aureus have emerged. In addition, hospital strains are often resistant to many antibiotics.
Methicillin resistance was reported shortly after the antibiotic was first introduced in 1959 to treat Penicillin-resistant Staphylococcus aureus, and is becoming a serious problem in many countries ( 2,3). Methicillin-resistant Staphylococcus aureus (MRSA) strains generally have a mutated penicillin-binding protein (PBP 2a) with low binding affinity for all β-lactam antibiotics.
It is resistant to these antibiotics by virtue of its expression (4). Currently MRSA is treated with the glycopeptide antibiotic vancomycin. "Genuine" vancomycin resistance has not been found in clinical cases of Staphylococcus aureus, but the genes for acquiring resistance to vancomycin are known, and vancomycin-resistant Staphylococcus aureus strains were engineered Has been done (5). Therefore,
The emergence of Staphylococcus aureus resistant strains in the clinical setting may simply be a matter of time.

Despite the fact that vancomycin treatment is still generally effective against MRSA infection, this treatment regimen induces significant nephrotoxicity (6). A few effective alternatives to glycopeptides have been developed, including the use of the drugs Synercid and oxazolidinone (7). Although these new types of antibiotics are effective, they can produce resistant organisms after their common use. In addition, some patients cannot take antibiotics because of allergic reactions.

The polysaccharide coat (CP) of Staphylococcus aureus has been a major focus of groups developing immunotherapy for Staphylococcus aureus infections (13,14).
. CP vaccines have been investigated in animal models (14) and given to human volunteers to produce hyperimmune sera (11). Hyperimmune sera from human volunteers immunized with the CP vaccine were tested for Staphylococcus
It is an alternative to passive immunization of individuals at risk of Aureus infection (11). However, in this method, 1) human immunity is typically limited to certain well characterized antigens, 2) human sera must be screened in detail, and still infectious agents , And 3) the relative activity of different batches of serum can be difficult to control. These drawbacks can result in potentially infectious and costly treatments that target a single antigen and are difficult to standardize.

Therefore, there is a need to develop improved strategies that provide safer, less toxic and less likely to result in resistant organisms after common use of current treatments for MRSA. SUMMARY OF THE INVENTIONThe present invention comprises an isolated human monoclonal antibody that specifically binds to Staphylococcus aureus by binding to an antigen of Staphylococcus aureus, as well as one or a combination of such antibodies. A composition is provided. Preferably, human antibodies cross-react with epitopes present on multiple (ie, 2 or more) Staphylococcus aureus clinical isolates. In certain embodiments, the human antibody also binds Staphylococcus aureus or Staphylococcus aureus antigen with high affinity and in the presence of human effector cells (in vitro and in vivo) of Staphylococcus aureus. It is also characterized by inhibiting proliferation and / or causing phagocytosis and cell killing of Staphylococcus aureus. Therefore, the human monoclonal antibody of the present invention can be used as a diagnostic or therapeutic agent in vivo and in vitro.

The isolated human antibody of the present invention comprises IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2.
, IgAsec, IgD and IgE. Typically,
They include IgG1 (eg IgGlκ) and IgM isotypes. Antibodies are full-length (
IgG1 or IgG4 antibody), or an antigen binding moiety (e.g. Fab)
, F (ab ′) ₂ , Fv or single chain Fv fragments). In one aspect, the human antibody is a recombinant human antibody. In another aspect, the human antibody is
Produced by a hybridoma including B cells 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, eg, a transgenic mouse. In a particular embodiment, the antibodies are produced by the hybridomas referred to herein as 2GD12, 2H12, 8.1E5, 8.2C1, 7F1, 6D12 and 5H10.

[0007]   In another embodiment, the human anti-staphylococcus aureus antibody of the present invention is
One or more of the following properties: a) at least one, preferably a plurality of Staphylococcus aureus isolates
Cross-reactivity with strains such as Staphylococcus aureus clinical isolates; b) at least about 10⁷M^-1, Preferably about 10⁸M^-1, And more preferably about 10⁹M^-1~Ten^TenM ^-1 Staphylococcus aureus or strain with higher or higher affinity constants
Affinity for binding to Taphylococcus aureus antigen c) at least about 10³, And more preferably about 10^Four, And most preferably about 10^FiveM^-1S^-1Star of
Binding to Phylococcus aureus or Staphylococcus aureus antigen
Sum constant (K_assoc); d) about 10^-3s^-1, Preferably about 10^-Fours^-1, And more preferably 10^-Fives^-1, Most preferably
Ten^-6s^-1Staphylococcus aureus or Staphylococcus aureus
Dissociation constant (K_dis); e) the ability to opsonize Staphylococcus aureus; or f) human effectors at concentrations of about 10 μg / ml or lower (eg in vitro)
-Inhibits growth and colonization of Staphylococcus aureus in the presence of cells
Ability and / or phagocytosis and lethality of Staphylococcus aureus
Ability Can be characterized.

Examples of Staphylococcus aureus clinical isolates that can be targeted by the human antibodies of the invention include, but are not limited to, IBERIAN, EMRSA, ONTARIO and BRAZILIAN strains. Absent. In a particular embodiment, the antibody binds to at least one methicillin-resistant Staphylococcus aureus (MRSA) strain.

The isolated human antibody of the invention is capable of binding to any Staphylococcus aureus antigen, including for example antigens secreted by or present on the surface of Staphylococcus aureus strains. it can. Examples of such antigens include, inter alia, capsular polysaccharides (e.g. CP type 5 or CP type 8), fibronectin binding proteins, protein A, toxins (α-, β- and γ-toxins, enterotoxins, exfoliative toxins). , Toxic shock syndrome toxin superantigen, coagulase, staphylokinase, penicillin binding protein 2a (PBP2a) and adhesins. In one embodiment, the human antibody has a staphylo with an affinity constant of at least about 10 ⁷ M ^-1 , preferably about 10 ⁸ M ^-1 , more preferably about 10 ⁹ M ^-1 to 10 ¹⁰ M ^-1 or higher. Human effector cells, such as polymorphisms, that bind to Coccus aureus or Staphylococcus aureus antigens and have an IC ₅₀ of about 1 x 10 ^-7 M or lower, or at a concentration of about 10 μg / ml or lower in vitro. It can cause phagocytosis and lethality of Staphylococcus aureus by nuclear cells (PMNs).

In another aspect, the invention provides a nucleic acid molecule encoding the antibody or antigen-binding portion of the invention. Therefore, a recombinant expression vector containing a nucleic acid encoding the antibody of the present invention and a host cell transfected with such a vector are also similar to the method for producing the antibody of the present invention by culturing these host cells. Included by the present invention.

In yet another aspect, the invention expresses various isotypes of human monoclonal antibodies (eg, IgG, IgA and / or IgM) that specifically bind to Staphylococcus aureus or Staphylococcus aureus antigens. be able to,
Provided are isolated B cells from a transgenic non-human animal, eg, a transgenic mouse. Preferably, the isolated B cells are obtained from a transgenic non-human animal, eg, a transgenic mouse, which has been immunized with a purified or enriched preparation of whole Staphylococcus aureus or Staphylococcus aureus antigens. Preferably, the transgenic non-human animal, eg 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 (eg, hybridoma) of human monoclonal antibodies against Staphylococcus aureus or Staphylococcus aureus antigens.

Accordingly, the present invention also provides a hybridoma capable of producing a human monoclonal antibody that specifically binds to Staphylococcus aureus or Staphylococcus aureus antigen. In one embodiment, the hybridoma comprises 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, such as B cells obtained from a transgenic mouse. Is included. The transgenic non-human animal is
Immunization with purified or concentrated preparations of whole Staphylococcus aureus or Staphylococcus aureus antigens can be used to generate antibody-producing hybridomas. Specific hybridomas of the invention include 2GD12, 2H12, 8.1E5, 8.2C.
1, 7F1, 6D12 and 5H10 are included.

In yet another aspect, the invention provides a transgenic mouse (also referred to herein as “HuMAb”) that expresses a human monoclonal antibody that specifically binds to Staphylococcus aureus or Staphylococcus aureus antigen. Call
) Such as a transgenic non-human animal. 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. Transgenic non-human animals include all Staphylococcus aureus or Staphylococcus
It can be immunized with a purified or concentrated preparation of the Aureus antigen. Preferably, the transgenic non-human animal, such as a transgenic mouse, has a VDJ
It is possible to produce multiple isotypes (eg IgG, IgA and / or IgM) of Staphylococcus aureus or human monoclonal antibodies against Staphylococcus aureus antigens by undergoing recombination and isotype switching. Isotype switching can occur, for example, by classical or nonclassical isotype switches.

In another aspect, the present invention provides a method for producing a human monoclonal antibody that specifically reacts with Staphylococcus aureus or Staphylococcus aureus antigen. In one embodiment, the method comprises transfecting a transgenic non-human animal having a genome comprising a human heavy chain transgene and a human light chain transgene, such as transgenic mice, with whole Staphylococcus aureus or Staphylococcus aureus. Immunizing with a purified or concentrated preparation of the antigen. Animal B cells (eg, splenic B cells) are then obtained and fused with myeloma cells to produce immortal hybridoma cells that secrete human monoclonal antibodies against Staphylococcus aureus or Staphylococcus aureus antigens.

The isolated anti-Staphylococcus aureus human monoclonal antibody or antigen binding portion thereof of the invention may be derivatized or another functional molecule, such as another peptide or protein (eg, Fab ′ fragment). Can be connected to. For example, an antibody or antigen-binding portion of the invention may be present in the presence of one or more other molecules such as another antibody (eg, a bispecific or multispecific antibody) (eg, chemical coupling, gene fusion, Functionally linked (via non-covalent bonding or otherwise). Thus, in another aspect, the invention provides at least one first binding specificity for Staphylococcus aureus or a Staphylococcus aureus antigen and an Fc receptor, such as human FcγRI or human Fcα.
Features a bispecific or multispecific molecule comprising a second binding specificity for a receptor.

The multispecific molecules of the invention also include trispecific, tetraspecific and other multispecific molecules. In one embodiment, multispecific molecules include anti-enhancement factors (anti-e
nhancement factor (EF) moieties, such as molecules that bind to surface proteins involved in cytotoxic activity.

In a particular embodiment, the bispecific and multispecific molecules of the invention comprise at least one antibody or fragment thereof (eg Fab, Fab ′, F (ab ′) ₂ , Fv or single chain Fv). Comprises. In certain embodiments, the antibody or fragment thereof is a fully human antibody or portion thereof, or a "chimeric" or "humanized" antibody or portion thereof (e.g., a variable region or at least the complement of a non-human antibody (e.g., mouse)). It has a sex determining region (CDR) and the remaining part (s) is of human origin.

In one embodiment, the at least one antibody or fragment thereof of the bispecific or multispecific molecule comprises an Fc receptor such as the human IgG receptor, eg FcγRI (CD
64), and binds to Fc-γ receptors (FcγR) such as FcγRII (CD32) and FcγRIII (CD16). A preferred Fcγ receptor is the high affinity Fcγ receptor, FcγRI. However,
Other Fc receptors such as the human IgA receptor (eg, FcαRI) can also be targeted. Preferably, the Fc receptor is located on the surface of effector cells such as monocytes, macrophages or activated polymorphonuclear cells. In a preferred embodiment, the bispecific and multispecific molecules bind the Fc receptor at a site that is different from the receptor's immunoglobulin (eg IgG or IgA) binding site. Thus, binding of bispecific and multispecific molecules is not prevented by physiological levels of immunoglobulins.

In another aspect, the invention provides an effector cell that expresses an Fc receptor, such as a macrophage or activated PMN cell, and a target-specific effector comprising a bispecific or multispecific molecule of the invention. Provide cells.

In another aspect, the invention provides a pharmaceutically acceptable carrier and at least one human monoclonal antibody of the invention or an antigen thereof that specifically binds to Staphylococcus aureus or Staphylococcus aureus antigen. Compositions, including pharmaceutical and diagnostic compositions, comprising a binding moiety are provided. In one aspect,
The composition comprises a combination of human antibodies or antigen binding portions thereof, preferably each of which binds to a different epitope. For example, a pharmaceutical composition comprising a human monoclonal antibody which exhibits limited cross-reactivity to Staphylococcus aureus isolates, but which results in highly effective phagocytosis of Staphylococcus aureus. Can be combined with another human monoclonal antibody that exhibits broad cross-reactivity to Staphylococcus aureus strains. Thus, the combination provides multiple treatments tailored to provide the greatest therapeutic benefit. A composition comprising a combination of at least one human monoclonal antibody of the invention or an antigen binding portion thereof and at least one bispecific or multispecific molecule of the invention, eg a pharmaceutical composition, is also a composition of the invention. It is within the range.

In yet another aspect, the present invention provides the antibody of the present invention or an antigen-binding portion thereof (
Or bispecific or multispecific antibodies) to inhibit the growth and / or colonization of Staphylococcus aureus bacteria by human effector cells such as human polymorphonuclear cells (PMNs), and A method for suppressing the infectivity of Staphylococcus aureus by inducing phagocytosis and / or lethality. In one embodiment, the method comprises contacting Staphylococcus aureus with one or a combination of a human monoclonal antibody of the invention or an antigen binding portion thereof, either in vitro or in vivo, in the presence of human effector cells. Comprises. This method can be used in culture, for example in vitro or ex vivo (eg culture comprising Staphylococcus aureus and effector cells). For example, a sample containing Staphylococcus 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, this method
It can be used in a subject, eg, as part of an in vivo (eg, therapeutic or prophylactic) protocol.

In the in vivo method, the antibody or antigen-binding portion thereof (or the bispecific or multispecific antibody of the invention) is administered to a human subject suffering from a disease caused by Staphylococcus aureus. It can be administered so as to induce growth inhibition, phagocytosis and / or lethality of Coccus aureus. In one embodiment, for example, by treating the subject with a cytokine, the Fc receptor,
The subject can be further treated with an agent that modulates, eg, increases or inhibits, eg, the expression or activity of the Fcα or Fcγ receptor. Preferred cytokines for administration during treatment with 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).

The isolated human monoclonal antibody compositions of the present invention can also be administered in combination with other known antibacterial therapies.

Representative diseases that can be treated (eg, ameliorated) or prevented using the methods and compositions of the invention include, but are not limited to, invasive or toxigenic infections. Not a thing. Such invasive diseases include bacteremia, osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis. Such toxigenic diseases include staphylococcal food poisoning, burn-like skin syndrome and toxic shock syndrome. Treat (eg, prevent or ameliorate) using the methods of the invention.
Further examples of possible diseases include upper and / or lower respiratory tract, heart, gastrointestinal tract, CNS
, Eye, kidney and urinary tract, skin and bone and joint infections.

In yet another aspect, the invention provides the use of Staphylococcus in a sample, eg, to diagnose a disease caused by Staphylococcus aureus.
Methods of detecting the presence of Aureus in vitro or in vivo are provided. In one embodiment, this involves combining the control sample with the sample to be tested with the human monoclonal antibody of the invention or an antigen-binding portion (or bispecific or multispecific molecule) of the antibody and Staphylococcus aureus. It is carried out by bringing them into contact with each other under conditions for forming a complex therebetween. Complex formation in both samples is then detected (e.g. using ELISA) and any statistically significant difference in complex formation between samples indicates the presence of Staphylococcus aureus in the test sample. .

Other features and advantages of the invention will be apparent from the following detailed description and claims. DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel antibody-based therapies for treating and diagnosing Staphylococcus aureus infections. The therapeutic and diagnostic reagents of the present invention include isolated human monoclonal antibodies that bind to an epitope present on at least one, and preferably multiple strains of Staphylococcus aureus clinical isolates, including MRSA strains. The antigen binding portion is included. In one embodiment, the human antibody is V-
By receiving DJ recombination and isotype switching, Staphylococcus
Produced in a non-human transgenic animal, eg, a transgenic mouse, capable of producing multiple isotypes (eg, IgG, IgA and / or IgE) of a human monoclonal antibody against the Aureus or Staphylococcus aureus antigen. 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 producing such monoclonal antibodies. . Also encompassed by the invention is a method of using the antibodies of the invention to detect Staphylococcus aureus or to suppress Staphylococcus aureus infectivity, either in vitro or in vivo.

In order that the invention may be more readily understood, certain terms are first defined. Additional definitions are provided throughout the detailed description of the invention.

The term “Staphylococcus aureus” (abbreviated herein as “S. aureus”), as used herein, currently represents the most common nosocomial pathogen in hospitals and long-term care facilities. It refers to any strain, genotype or isolate of Staphylococcus aureus, one Gram-positive bacterium (1). These organisms can cause a wide range of symptoms, from skin swelling to lung disease and death. The term "Staphylococcus aureus" also includes, for example, IB
Also included are antibiotic resistant strains of Staphylococcus aureus, including ERIAN, EMRSA, ONTARIO, BRAZILIAN and COL strains, such as penicillin- and methicillin-resistant Staphylococcus aureus (abbreviated herein as "MRSA"). Be done
(2,3). MRSA strains are generally resistant to these antibiotics due to the expression of a mutated penicillin binding protein (PBP 2a) that has a low binding capacity for β-lactam antibiotics (4).

As used herein, the term “Staphylococcus aureus antigen” includes secreted Staphylococcus aureus antigens and antigens naturally present on the surface of Staphylococcus aureus, All Staphylococcus aureus antigens are included. In a preferred embodiment, the Staphylococcus aureus antigen is present in a large proportion of clinical isolates to confer broad cross-reactivity of antibodies that bind the antigen. In another preferred embodiment, the binding of the antibody of the invention to the Staphylococcus aureus antigen results in effector cell phagocytosis and lethality of Staphylococcus aureus. Preferably, the Staphylococcus aureus antigen is capable of eliciting a protective immune response upon immunization of a non-human transgenic animal. Examples of such antigens include, among others:
Capsular polysaccharide (e.g. CP type 5 or CP type 8), fibronectin binding protein, protein A, toxins (α-, β- and γ-toxins, enterotoxins, epidermal toxins), toxic shock syndrome toxin superantigen, coagulase, stadium Included are filokinase, penicillin binding protein 2a (PBP2a) and adhesins (28). For example, CP
Antigens (CP5 and CP8) are present on approximately 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 linked together by disulfide bonds. . Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. Heavy chain constant region is three domains, CH1
, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs) flanked by more conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, C
DR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of an antibody can result in the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg effector cells) and the first component of the classical complement system (C1q).

The term “antigen-binding portion” of an antibody (or simply “antibody portion”), as used herein, specifically binds to an antigen (eg Staphylococcus aureus or Staphylococcus aureus antigen). Refers to one or more fragments of an antibody that retain the ability to It has been shown that the antigen binding function of an antibody can be fulfilled by a fragment of a full length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include (i) Fab fragments, VL, V
Monovalent fragment consisting of H, CL and CH1 domains; (ii) F (ab ') ₂ fragment, divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of VL and VH domains of one arm of the antibody; (v) VH
DAb fragment consisting of a domain (Ward et al., (1989) Nature 341 : 544-546)
And (vi) an isolated complementarity determining region (CDR) is included. In addition, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but these have been engineered using recombinant methods to pair the VL and VH regions into a monovalent molecule (single chain). Fv (scFv)
Known as; Bird et al. (1988) Science 242 : 423-426; and H, for example.
uston et al. (1988) Proc. Natl. Acad. Sci. USA 85 : 5879-5883) can be joined by synthetic linkers that can make them as a single protein chain. Such single chain antibodies are also "antigen-binding portions" of antibodies.
Shall be included in the term. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for utility in the same manner as whole antibodies.

The term “bispecific molecule” has two different binding specificities that bind or interact with (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. It is intended to include any factor such as a protein, peptide, or protein or peptide complex. The term "multispecific molecule" or "heterospecific molecule" refers to (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) binding to at least one other component. Any factor that interacts with or has more than two different binding specificities, such as a protein, a peptide, or a protein or peptide complex, shall be included. Accordingly, the present invention provides cell surface antigens such as Staphylococcus aureus or Staphylococcus aureus antigens and bispecific, trispecific, tetraspecific and directed to Fc receptors on effector cells. Other multispecific molecules are included, but are not limited to. The term "bispecific antibody" also includes diabodies. Diabodies have VH and VL domains expressed on one polypeptide chain, but with a linker that is too short to allow pairing between two domains on the same chain, thereby allowing the domains to be linked to another chain. Is a bivalent, bispecific antibody that pairs with the complementary domain of E. coli and produces two antigen-binding sites (eg Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90 : 6444-6448
; Poljak, RJ, et al. (1994) Structure 2 : 1121-1123).

The term “heteroantibody”, as used herein, refers to two or more antibodies, antibody binding fragments (eg, Fab), derivatives thereof, or linked together, of which at least two have different specificities. Antigen binding region. These different specificities include binding specificity for the Fc receptor on effector cells,
And binding specificity for an antigen or epitope on a target cell, eg Staphylococcus aureus.

As used herein, the term “human antibody” is meant to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention comprise amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo). You can However, as used herein, the term "human antibody" does not include antibodies in which CDR sequences from the germ line of another mammalian species, such as mouse, have been grafted onto human skeletal sequences. To do.

The term “monoclonal antibody” or “monoclonal antibody composition” refers to
As used herein, refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Thus, 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 antibody is a B cell obtained from a transgenic non-human animal having a genome comprising a human heavy chain transgene and a light chain transgene, such as a transgenic mouse, fused to an immortalized cell. Are produced by hybridomas including.

As used herein, the term “recombinant human antibody” is prepared by recombinant means, such as an antibody isolated from an animal (eg, mouse) that is transgenic for human immunoglobulin genes, All human antibodies expressed, produced or isolated (further described in Section I below); antibodies expressed using recombinant expression vectors transfected into host cells, recombinant random sequence human antibody libraries Or an antibody prepared, expressed, produced or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when using animals that are transgenic for human Ig sequences) and are thus recombinant. The amino acid sequences of the VH and VL regions of antibodies are sequences derived from and related to human germline VH and VL sequences, but which are not naturally present in vivo within the human antibody germline repertoire.

As used herein, “heterologous antibody” is defined with respect to the transgenic non-human organism producing such antibody. The term refers to an antibody present in an organism that does not consist of the transgenic non-human animal, generally having an amino acid sequence from a species other than that of the transgenic non-human animal, or encoding a nucleic acid sequence corresponding thereto.

As used herein, “heterohybrid antibody” refers to an antibody that has light and heavy chains from different organisms. For example, an antibody having a human heavy chain associated with a mouse light chain is a heterohybrid antibody. Examples of heterohybrid antibodies include the chimeric and humanized antibodies described above.

“Isolated antibody” as used herein shall refer to an antibody that is substantially free of other antibodies having different antigenic specificities (eg, Staphylococcus aureus or Staphylococcus aureus). (An isolated antibody that specifically binds to a Phylococcus aureus antigen does not substantially include an antibody that specifically binds to Staphylococcus aureus or an antigen other than Staphylococcus aureus antigen).
. However, an isolated antibody that specifically binds to one strain or antigen of Staphylococcus aureus can have cross-reactivity to other strains of Staphylococcus aureus or antigen. In addition, the isolated antibody may be substantially free of other cellular material and / or chemicals. In one aspect of the invention, combinations of "isolated" monoclonal antibodies with different specificities are combined in a well-specified composition.

As used herein, "specific binding" refers to antibody binding to a previously determined antigen. Typically, the antibody binds with an affinity of at least about 1 x 10 ⁷ M ^-1 , and a non-specific antigen other than the predetermined or closely related antigen (e.g., B
It binds to a predetermined antigen with an affinity that is at least 2 times greater than its binding affinity for SA, casein). "Antibody that recognizes an antigen" and "Antigen-specific antibody"
The term is used interchangeably herein with the term "antibody that specifically binds an antigen".

As used herein, the term “high affinity” for IgG antibodies refers to
At least about 1 x 10 ⁹ M ^-1 , typically at least about 5 x 10 ⁹ M ^-1 , often about 1 x
A binding affinity of greater than 10 ¹⁰ M ^-1 , and sometimes 5 x 10 ¹⁰ M ^-1 to about 1 x 10 ¹¹ M ^-1 or greater. "High affinity" binding can differ for other antibody isotypes. For example, IgM isotype "high affinity" binding is at least about 1.
x 10 ⁷ M ^-1 .

The term “K _assoc ” as used herein shall refer to the binding constant of a particular antibody-antigen interaction.

The term “K _dis ”, as used herein, shall refer to the dissociation constant of a particular antibody-antigen interaction.

As used herein, “isotype” refers to the antibody class (eg, IgM or IgG1) encoded by the heavy chain constant region genes.

As used herein, “isotype switch” refers to the phenomenon where the class or isotype of an antibody changes from one Ig class to another Ig class.

As used herein, “non-switched isotype” refers to the isotype class of heavy chain produced in the absence of any isotype switch; a CH gene encoding a non-switched isotype. Is typically the first CH gene immediately downstream from the functionally rearranged VDJ gene.
Isotype switches are classified as classical or non-classical isotype switches. Classical isotype switching occurs by recombination events involving at least one switch sequence region in the transgene. Non-classical isotype switches are, for example, homologous recombination between human σ _μ and human Σ _μ (δ-related deletion (δ-as
Sociated deletion)). Among other things, between transgenes and /
Alternatively, another non-classical switch mechanism, such as interchromosomal recombination, can occur resulting in an isotype switch.

As used herein, the term “switch sequence” refers to a DNA sequence that results in switch recombination. The “switch-donor” sequence, typically the μ-switch region, is 5 ′ (ie, upstream) of the construct region that is deleted during switch recombination. The "switch acceptor" region is between the deleted construct region and the replacement construct region (eg, γ, ε, etc.). The final gene sequence is typically not predictable from the construct, since there is no specific site where recombination will necessarily occur.

As used herein, “glycosylation pattern” is defined as the pattern of carbohydrate units that are covalently linked to proteins, more particularly immunoglobulin proteins. The glycosylation pattern of a heterologous antibody is recognized by those of skill in the art as being more similar to the glycosylation pattern of the heterologous antibody in the species of the non-human transgenic animal than in the species from which the CH gene of the transgene is derived. , Can be considered to be substantially similar to the glycosylation pattern naturally present on the antibodies produced by the non-human transgenic animal species. The term “natively existing” as used herein when applied to an object refers to the fact that an object can exist in nature. For example, there are naturally occurring polypeptide or polynucleotide sequences in organisms (including viruses) that can be isolated from sources in nature and have not been intentionally modified by researchers.

The term “reorganized” as used herein is essentially complete.
Refers to the arrangement of the heavy or light chain immunoglobulin loci in which the V segment is located immediately adjacent to the DJ or J segment, in an arrangement that encodes a VH or VL domain, respectively. The rearranged immunoglobulin loci can be identified by comparison with germline DNA; the rearranged loci are composed of at least one recombined heptamer.
/ Has a nonamar homology element.

The terms “unrearranged” or “germline arrangement” as used herein with respect to V segments are arrangements that have not been recombined so that the V segment is in close proximity to the D or J segment. Point

As used herein, the term “nucleic acid molecule” includes DNA molecules and RNA
Molecules shall be included. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.

The term “isolated nucleic acid molecule” refers to a nucleic acid that encodes an antibody or portion of an antibody (eg, VH, VL, CDR3) that binds to Staphylococcus aureus or Staphylococcus aureus antigens. As used herein, the nucleotide sequence encoding the antibody or portion of the antibody is another nucleotide sequence encoding an antibody or portion of the antibody that binds to an antigen other than Staphylococcus aureus or Staphylococcus aureus antigen. Refers to nucleic acid molecules that do not contain any of these other sequences, which may naturally flank the nucleic acid in human genomic DNA.

For nucleic acids, the term “substantial homology” includes at least about 80, including the appropriate nucleotide insertions or deletions, when two nucleic acids or designated sequences thereof are compared in an optimal alignment. % Nucleotides, usually at least about 90% to 95%, more preferably at least about 98% to 99.5% nucleotides. Alternatively, there is substantial homology if the segment hybridizes to the complement of the strands under selective hybridization conditions.

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

The percent identity between the two nucleotide sequences is determined by the NWSgapdna.CMP matrix and the 4
In GCG software package (available at http://www.gcg.com) with gap weights of 0, 50, 60, 70 or 80 and length weights of 1, 2, 3, 4, 5 or 6. GAP
It can be determined using a program. The percent identity between two nucleotide or amino acid sequences was also determined using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 for E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1
It can also be determined using the algorithm of 989)). In addition, the percent identity between the two amino acid sequences is either Blossum 62 matrix or PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and 1, 2, 3, 4, 5 or Needleman and Wunsch (J. Mol.) Incorporated into the GAP program of the GCG software package (available at http://www.gcg.com) using a length weight of 6.
Biol. (48): 444-453 (1970)) algorithm.

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, for example to identify related sequences. Such a search is described in Altschul, et al. (1990) J. Mol. Biol.
215: 403-10 NBLAST and XBLAST programs (version 2.0). BLAST nucleotide searches can be performed using 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 using the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gap-aligned alignments for comparison purposes, Gapped BLAST was used in Altschul et al. (1997) Nucleic Acids Res. 25 (17): 338.
It can be used as described in 9-3402. BLAST and Gapped BLAST
When using the programs, the respective programs (for example, XBLAST and NBLAST
) Default parameters can be used. http://www.ncbi.nlm.ni
See h.gov.

The nucleic acid molecule can be present in whole cells, in cell lysates, or in partially purified or substantially pure form. Nucleic acid may be treated with alkali / SDS, CsCl banding, column chromatography, which are well known in the art.
"Isolated" or "substantially pure" when purified separately from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including agarose gel electrophoresis and the like. Will be defeated. " F. Ausubel, et al., Edit
.Current Protocols in Molecular Biology , Greene Publishing and Wiley In
See terscience, New York (1987).

Nucleic acid compositions of the present invention that remain in their native sequence (except for modified restriction sites, etc.) from either cDNA, genomic DNA or mixtures thereof will be abruptly modified according to standard techniques for providing gene sequences. It can be mutated. In the coding sequence, these mutations can result in changes in the amino acid sequence as desired. In particular, DNA sequences that are substantially homologous to or derived from the native V, D, J, constants, switches and other such sequences described herein are contemplated (where "derived from").
Indicates that the sequence is identical to, or modified from, another sequence).

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcriptional regulatory sequences, operably linked means that the DNA sequences being linked are contiguous, and contiguous and open reading frame when it is necessary to join the two protein coding regions. means. In switch sequences, operably linked indicates that the sequence is capable of effecting switch recombination.

As used herein, the term “vector” refers to a nucleic acid molecule capable of carrying another nucleic acid to which it has been linked. One type of vector is
“Plasmid”, which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which case additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors, such as non-episomal mammalian vectors, integrate into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such a vector is referred to herein as a "recombinant expression vector" (or simply "expression vector"). Generally, recombination
Expression vectors useful in DNA technology are often in the form of plasmids.
In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to encompass other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses) that serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as used herein, refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms refer not only to the particular cell but to the progeny of such a cell. Such progeny may not be identical to the parental cell in nature, as certain alterations may occur in later generations, either due to mutations or environmental influences, although the present specification As used in the text, it is nevertheless included within the term "host cell".

Various aspects of the invention are described in further detail in the following subsections. I. Manufacture of Human Antibodies Against Staphylococcus aureus The human monoclonal antibodies (mAbs) of the present invention may be prepared using conventional monoclonal antibody methodologies, such as the standard somatic cell hybridization techniques of Kohler and Milstein, Nature 256: 495 (1975). Can be manufactured by a variety of techniques including. Somatic cell hybridization methods are preferred in principle, but other techniques for producing monoclonal antibodies, such as B lymphocyte virus or oncogenic transformation can be used.

The preferred animal system for producing hybridomas is the mouse system. Hybridoma production in the mouse is a very established method. Immunization protocols and techniques for isolation of immunized spleen cells for fusion are known in the art. Fusion partners (eg mouse myeloma cells) and fusion methods are also known.

In a preferred embodiment, a human monoclonal antibody directed against Staphylococcus aureus is referred to herein as a "HuMAb" transgenic mouse, a transgenic mouse carrying a complete human immune system rather than a mouse line. It is made by using. For example, human immunoglobulin genes encoding unrearranged human heavy chain (μ and γ) and kappa light chain immunoglobulin sequences, with selective mutations that inactivate the endogenous μ and κ chain loci. Contains a mini locus
HuMAb transgenic mice can be used (Lonberg, N. et al. (19
94) Nature 368 (6474): 856-859). Thus, these mice show reduced expression of mouse IgM or kappa, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and It undergoes somatic mutation (Lonberg, N. et al. (1994), supra; 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. NY Acad. Sci 764: 536-546).
. The production of HuMAb mice is described in Section II below and Taylor, L. et al. (1992) Nucleic Ac
ids Research 20: 6287-6295; Chen, J. et al. (1993) International Immunolo
gy 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci USA 90: 3720-3.
724; Choi et al. (1993) Nature Genetics 4: 117-123; Chen J. et al. (1993)
EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152: 2912-2920; Lo.
nberg et al., (1994) Nature 368 (6474): 856-859; Lonberg, N. (1994) Handbo
ok of Experimental Pharmacology 113: 49-101; Taylor, L. et al. (1994) Int
ernational Immunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Inter
n. Rev. Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N. (1995) Ann.
NY Acad. Sci 764: 536-546; Fishwild, D. et al. (1996) Nature Biotechn
ology 14: 845-851, the entire contents of which are incorporated herein by reference in their entireties. In addition, all Lonberg and Kay,
And US Pat. No. 5,545,806 to GenPharm International; 5,569,825;
No. 5,625,126; No. 5,633,425; No. 5,789,650; No. 5,877,397; No. 5,661,016;
5,814,318; 5,874,299; and 5,770,429; U.S. Patent No. 5,545,807 to Surani et al .; International Publication No. WO 98/24884 published June 11, 1998; 1994 1
WO 94/25585 published on January 10; WO 93/122 published on June 24, 1993
No. 7; WO 92/22645, published 23 December 1992; see WO 92/03918, published 19 March 1992, all disclosures of which are incorporated by reference. Are incorporated herein by reference in their entirety. To generate fully human monoclonal antibodies against HuMAb immunostaphylococcus aureus, for example, Lonberg, N. et al. (1994) Nature 368 (6474): 856-859;
Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 and WO 98/2488.
Whole Staphylococcus aureus (e.g., 5-20 μg of Staphylococcus aureus antigens containing heat-killed mice, preferably HuMAb transgenic mice, using the immunization protocol described in 4. Immunogen). Preferably, the mice are 6-during the first injection.
16 weeks of age.

For example, it is possible to use heat-killed whole Staphylococcus aureus from a single clinical isolate expressing the CP antigen, CP type 5 (CP5), to immunize HuMAb mice intraperitoneally. it can. CP type 5 is a common CP type shared by MRSA isolates and is therefore a suitable Staphylococcus aureus antigen for use in the present invention. Furthermore, previous studies have shown that antibodies that bind to the type 5 capsule can provide protection against experimental Staphylococcus aureus infection (14,28). Where immunization with a single Staphylococcus aureus strain does not result in antibodies that cross-react with two or more Staphylococcus aureus strains, Staphylococcus Mice can be immunized with an alternative strain of Aureus.
Prior to immunization, bacteria can generally be typed and expanded on agar plates to promote capsular expression (29) or in media to minimize capsular expression.

Due to cumulative experience with various antigens, HuMAb transgenic mice were first immunized intraperitoneally (IP) with antigen in complete Freund's adjuvant, followed by biweekly IP with antigen in incomplete Freund's adjuvant. It has been shown to respond best when immunized (up to 6 total). The immune response can be monitored over the course of the immunization protocol using plasma samples obtained by posterior orbital bleeding. Plasma can be screened by ELISA (as described below) and mice with sufficient titers of anti-staphylococcus aureus human immunoglobulin can be used for fusions. Kill the mouse and remove the spleen 3
The antigen can be boosted intravenously prior to the day. It is anticipated that 2-3 fusions may need to be performed for both high and low capsule immunity. Six mice are immunized against each antigen. For example, a total of 12 HuMAb mice of HC07 and HC012 strains can be immunized. Preparation mouse spleen cells hybridoma producing a monoclonal antibody against Staphylococcus aureus 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 production of antigen-specific antibodies. For example, a single cell suspension of splenic lymphocytes from immunized mice is fused to 1/6 number P3X63-Ag8.653 non-secreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cells were plated at approximately 2 x 10 ⁵ in flat bottom microtiter plates, followed by 20% fetal cloned serum, 18% "
653 "conditioned medium, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units / ml penicillin, 50 mg / ml Incubate for 2 weeks in selection medium containing streptomycin, 50 mg / ml gentamicin and 1X HAT (Sigma; HAT is added 24 hours after fusion) .After 2 weeks, culture cells in medium with HAT replaced with HT. Individual wells are then screened by ELISA for human anti-Staphylococcus aureus monoclonal IgM and IgG antibodies, once extensive hybridoma growth occurs, medium is usually measured after 10-14 days. Re-plated, re-screened and tested for human IgG, anti-Staphylococcus aureus monoclonal antibody If still positive, they can be subcloned at least twice by limiting dilution, then the stable subclones are cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization. Characterization of Binding of Human Monoclonal Antibodies to Staphylococcus aureus To characterize binding of the human monoclonal anti-staphylococcus aureus antibodies of the invention, sera from immunized mice can be tested, for example by ELISA. Yes, briefly, the Staphylococcus aureus immune strain can be expanded overnight in Columbia Broth medium or on Columbia agar plates.
Bacteria can be harvested from the plates by gently resuspending the cells in sterile saline. Bacteria from the medium or plate were washed by centrifugation and 0.
It can be diluted to an OD ₅₉₀ of 2. Bacterial suspension (50 μl) can be added to 96-well flat bottom plates and the plates are dried in a fume hood. Bacteria can be fixed to the plate with 1% glutaraldehyde for 5 minutes. Plates are blocked with 20% mouse serum (which blocks IgG binding to protein A). These plates can be stored at -80 ° C until needed. Then plate the plate in PBS-Tween buffer (Dulbecco PBS, .05% Tween 20, 1 mM EDTA, .25% BSA, .05% N
aN ₃ ) and can be washed with Staphylococcus aureus immunized mice and dilutions of plasma from mice immunized with irrelevant antigens and incubated at 37 ° C. for 1 hour. The plates are washed, then reacted with anti-human IgG Fc-specific alkaline phosphatase and incubated at 37 ° C for 1 hour. The plates can be washed again, developed with pNPP substrate (1 mg / ml) and analyzed at an OD of 405-650. Preferably, the mouse showing the highest titer is used for fusion.

To screen hybridomas using an ELISA assay, microtiter plates can be prepared as described above with several strains of Staphylococcus aureus in addition to the strain used for immunization. Representative Staphylococcus aureus strains that can be used have been shown to spread rapidly in a hospital setting, IBERIAN (BK2058 and BK2709), EMRSA, ONTARIO and BRAZILIAN isolates and / or purified Antigens such as capsular polysaccharides 5 and 8, fibronectin binding protein, penicillin binding protein and protein A. Hybridomas that are positively reactive with an immune strain of Staphylococcus aureus (eg, by an ELISA assay as described above) can be tested for cross-reactivity on plates coated with other strains. Irrelevant human IgG is used as a negative control. High-gliomas that bind with high affinity to most Staphylococcus aureus strains are subcloned and further characterized.
Store at -140 ° C for 5-10 vial cell bank and antibody purification (E
One clone can be selected from each hybridoma that retains the reactivity of the parental cells (by LISA).

To purify human anti-Staphylococcus aureus antibodies, selected hybridomas can be expanded in 2 liter spinner flasks for monoclonal antibody purification. Protein A-Sepharose (Pharmacia, Piscataway,
The supernatant can be filtered and concentrated before affinity chromatography on NJ). Eluted IgG can be examined 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 OD ₂₈₀ using an extinction coefficient of 1.43. The monoclonal antibody can be aliquoted and stored at -80 ° C.

To determine whether selected human anti-Staphylococcus aureus monoclonal antibodies bind to a unique epitope, commercially available reagents (Pierc
e, Rockford, IL) can be used to biotinylate each antibody. An ELISA plate coated with Staphylococcus aureus as described above can be used to perform competition studies with unlabeled and biotinylated monoclonal antibodies. Biotinylated mAb binding can be detected with a strep-avidin-alkaline phosphatase probe.

An isotype ELISA can be performed to determine the isotype of the purified antibody. Wells of microtiter plates can be coated with 10 μg / ml anti-human Ig at 4 ° C overnight. After blocking with 5% BSA, plates are reacted with 10 μg / ml monoclonal antibody or purified isotype control for 2 hours at ambient temperature. The wells can then be reacted with either human IgG1 or human IgM-specific alkaline phosphatase-coupled probes. Plates are developed as above and analyzed.

Flow cytometry (eg, as described by Poutrel et al. (31)) can be used to demonstrate binding of monoclonal antibodies to live Staphylococcus aureus. Briefly, bacteria (increased as above, at 10 ⁷ cells / ml) were mixed with various concentrations of monoclonal antibody in PBS containing 0.1% Tween 80 and 20% mouse serum and incubated at 37 ° C. Incubate for 1 hour. After washing, bacteria were labeled with fluorescein under the same conditions as for primary antibody staining.
-React with human IgG antibody. Samples can be analyzed on a FACScan instrument that uses light and side scatter properties to gate against a single bacterium.
A short sonication step (shown to have no significant effect on cell viability) can be included to reduce bacterial aggregation. Another assay using fluorescence microscopy (in addition to or instead of the flow cytometry assay) can be used. Bacteria can be stained exactly as described above and examined by fluorescence microscopy. This method allows visualization of individual cells, but may be less sensitive due to antigen density.

Anti-Staphylococcus aureus human IgG can be further tested by Western blotting for reactivity with specific Staphylococcus aureus antigens. Briefly, cell wall extracts from selected Staphylococcus aureus isolates can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked with 20% mouse serum and probed with the monoclonal antibody to be tested. Human IgG binding was detected using anti-human IgG alkaline phosphatase and B
Color can be developed with CIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, MO). Antigens specifically bound by the tested monoclonal antibodies may be identified by direct sequencing or by the use of mutants known to lack certain cell wall components or to produce very small amounts. it can. Phagocytosis and cytotoxic activity of human anti-Staphylococcus aureus antibodies In addition to showing the ability of selected monoclonal antibodies to specifically bind Staphylococcus aureus, phagocytosis of Staphylococcus aureus in vitro And the ability of these antibodies to cause lethality (ie their therapeutic utility) can also be determined. Testing for monoclonal antibody activity in vitro provides an initial screen prior to testing in an in vivo model. Briefly, polymorphonuclear cells (PMN) from healthy donors can be purified by Ficoll Hypaque density centrifugation followed by lysis of contaminating red blood cells. Washed PMNs were suspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum and mixed with Staphylococcus aureus augmented as described above at various ratios of PMN: bacteria (PMN: bacteria). can do. Purified human anti-Staphylococcus aureus IgG can then be added at various concentrations. Irrelevant human IgG can be used as a negative control. The assay can be performed at 37 ° C. for 0-120 minutes. Samples can be diluted in water and then plated on tryptic soy agar plates for overnight incubation before colony numbers can be determined. Anti-Staphylococcus aureus monoclonals can also be tested in combination with each other to determine if phagocytosis is enhanced by multiple monoclonal antibodies.

Human monoclonal antibodies exhibiting, for example, high affinity binding and cross-reactivity with one or more Staphylococcus aureus clinical isolates have been tested in mice to determine their efficacy against Staphylococcus aureus infections. It can be tested in an in vivo challenge model. These antibodies may be selected, for example, based on the following criteria, which are not limiting: 1. Binding to live Staphylococcus aureus increased on agar or in medium; High affinity for binding to Phylococcus aureus; 3. Cross-reactivity with various Staphylococcus aureus clinical isolates by ELISA and / or flow cytometry; 4. Unique on Staphylococcus aureus Binding to an epitope (to eliminate the possibility that monoclonal antibodies having complementary activities when used in combination compete for binding to the same epitope); 5. Optonization of Staphylococcus aureus; 4. Human effector cells Inhibition of growth inhibition, phagocytosis and / or lethality of Staphylococcus aureus in the presence of To take a break.

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

In a particular embodiment, the human monoclonal antibodies of the invention are used in combination as a pharmaceutical composition comprising, for example, two or more anti-staphylococcus aureus monoclonal antibodies or fragments thereof. For example, human anti-Staphylococcus aureus monoclonal antibodies with different but complementary activities can be combined in a single treatment to obtain the desired therapeutic or diagnostic result. In one embodiment, it exhibits broader cross-reactivity with monoclonal antibodies that have limited cross-reactivity but provides efficient phagocytosis or effects on the growth and / or colonization of Staphylococcus aureus. It is obtained by combining with one or more other human monoclonal antibodies that show specific inhibition. II. Generating human monoclonal anti-staphylococcus aureus antibody
Production of Lancegenic Non-Human Animals In yet another aspect, the present invention provides a transgenic non-human capable of expressing Staphylococcus aureus or a human monoclonal antibody as described above that specifically binds to Staphylococcus aureus antigens. An animal, eg, a transgenic mouse, is provided. In a preferred embodiment, the transgenic non-human animal, eg transgenic mouse, has a genome comprising a human heavy chain transgene and a light chain transgene. In one embodiment, transgenic non-human animals, such as transgenic mice, are immunized with whole Staphylococcus aureus or a purified or concentrated preparation of Staphylococcus aureus antigens. Preferably, the transgenic non-human animal, e.g., transgenic mouse, has multiple isotypes (e.g., IgG, IgA) of a human monoclonal antibody against Staphylococcus aureus or Staphylococcus aureus antigen by undergoing VDJ recombination and isotype switching. And / or
IgE) can be produced. Isotype switching can occur, for example, by classical or nonclassical isotype switches.

The design of transgenic non-human animals that respond to foreign antigenic stimuli with a heterologous antibody repertoire is such that It needs to function properly. In a preferred embodiment, the correct function of the heterologous heavy chain transgene includes isotype switching. Therefore, the transgene of the present invention is
An isotype switch 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, It is constructed to cause (4) expression of a sufficient primary repertoire, (5) signal transduction, (6) somatic hypermutation and (7) control of the transgene antibody locus during the immune response.

Not all of the above criteria need to be met. For example, in the embodiment where the endogenous immunoglobulin loci of the transgenic animal are functionally disrupted, the transgene need not activate allelic exclusion. Further, in the embodiment wherein the transgene comprises a functionally rearranged heavy and / or light chain immunoglobulin gene, at least the already rearranged transgene has a second criterion for functional gene rearrangement. Is unnecessary. For background on molecular immunology, see Fundamentals Immunology, Second Edition (1989), Paul Willi, which is incorporated herein by reference.
See am E. Editor, Raven Press, NY.

In some embodiments, the transgenic non-human animals used to make the human monoclonal antibodies of the invention are heterologous of rearranged, unrearranged, or rearranged and unrearranged combinations. Containing the immunoglobulin heavy and light chain transgenes in the germ line of transgenic animals. Each heavy chain transgene comprises at least one C _H gene. In addition, the heavy chain transgene may contain a functional isotype switch sequence, which may aid in isotype switching of a heterologous transgene encoding multiple C _H genes in B cells of transgenic animals. it can. Such a switch sequence can be native to the germline immunoglobulin loci from the species that can serve as the source of the transgene C _H gene, or such a switch sequence will receive the transgene construct. It can be obtained from what is present in the species (transgenic animal). Presumably, the mouse switch sequence has been optimized to function in the mouse switch recombinase enzyme system, while the human switch sequence is not so that, for example, the human transgene construct used to produce transgenic mice. Can cause a higher frequency of isotype switches if they contain switch sequences similar to those naturally present at the mouse heavy chain locus. The switch sequence can be isolated and cloned by conventional cloning methods, or can be newly synthesized from overlapping synthetic oligonucleotides designed based on published sequence information regarding immunoglobulin switch region sequences. (Mills
et al., Nucl. Acids Res. 15: 7305-7316 (1991); Sideras et al., Intl. Imm.
unol. 1: 631-642 (1989), which are incorporated herein by reference.
). In each of the transgenic animals described above, a functionally rearranged, heterologous, heavy and light chain immunoglobulin transgene is present in a significant proportion of the B cells of the transgenic animal (at least 10%).

The transgenes used to produce the transgenic animals of the invention include
Included is a heavy chain transgene comprising DNA encoding at least one variable gene segment, one diversity gene segment, one connecting 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 connecting gene segment and at least one constant region gene segment. Gene segments encoding light chain and heavy chain gene segments are obtained or corresponding to DNAs encoding immunoglobulin heavy chain and light chain gene segments from species that do not consist of transgenic non-human animals, so transgenic non-human Heterogeneous to animals. In one aspect of the invention, the transgene is constructed so that the individual gene segments are not rearranged, ie, rearranged to encode a functional immunoglobulin light or heavy chain. Such unrearranged transgenes aid in the recombination (functional rearrangement) of the V, D and J gene segments, and preferably transgenic non-human when exposed to Staphylococcus aureus antigens. Helps incorporate all or part of the D region gene segment in the rearranged immunoglobulin heavy chain produced in the animal.

In another embodiment, the transgene comprises an unrearranged “minilocus”. Such transgenes typically comprise a significant portion of the C, D and J segments and a subset of the V gene segments. In such transgene constructs, various regulatory sequences, such as promoters, enhancers, class switch regions, splice donor and splice acceptor sequences for RNA processing, recombination signals, etc., include the corresponding sequences derived from heterologous DNA. It consists of Such regulatory sequences can be introduced into the transgene from the same or similar species of non-human animals used in the present invention. For example, the human immunoglobulin gene segment can be combined in a transgene with a rodent immunoglobulin enhancer for use in transgenic mice. Alternatively, synthetic regulatory sequences may be introduced into the transgene, where such synthetic regulatory sequences are functionally known to be naturally present in the mammalian genome.
It is not homologous to the DNA sequence. Synthetic regulatory sequences are designed according to common rules, such as those that specify permissive sequences for splice acceptor or promoter / enhancer motifs. For example, a mini-locus is at least one internal (ie, not at the end of that portion) of a nonessential DNA portion (eg, intervening sequence; intron or portion thereof) when compared to the naturally occurring germline Ig locus. ) Comprises a portion of a genomic immunoglobulin locus having a deletion.

In a preferred embodiment of the present invention, the transgenic animals used to produce human antibodies against Staphylococcus aureus are those described in Example 1 of WO 98/24884.
2, containing at least one copy, typically 2-10 copies, and sometimes 25-50 copies or more of the transgene (eg pHC1 or pHC2), WO 98/2488
The light chain transgene single copy described in Example 5, 6, 8 or 14 of the 4 bred with an animal containing and J are described progeny in Example 10 of WO 98/24884 _H
The deletion animals are bred and their contents are expressly incorporated herein by reference. Animals are bred to homozygosity for each of these three traits. Such animals have the following genotypes: (per haploid set of chromosomes) single copy unrearranged human heavy chain minilocus (Example 1 of WO 98/24884).
2), a single copy (per haploid set of chromosomes) unrearranged human kappa light chain construct (described in Example 14 of WO 98/24884) and a functional J _H.
Deletion at each endogenous mouse heavy chain locus that removes all of the segments (WO 98/24884
Described in Example 10). Mice homozygous for the deletion of the _JH segment to produce progeny that are homozygous for the _JH deletion and hemizygous for the human heavy and light chain constructs ( Cross with Example 10) of WO 98/24884. Antigens are injected into the obtained animals and used for the production of human monoclonal antibodies against these antigens.

B cells isolated from such animals are monospecific for human heavy and light chains because they contain only a single copy of each gene. Furthermore, both endogenous mouse heavy chain gene copies were introduced as described in Example 9 of WO 98/24884.
They are monospecific for human or mouse heavy chains as they do not function due to deletions spanning the _JH region. In addition, expression of a single copy of the rearranged human kappa light chain gene allelically and isotypically eliminates rearrangement of the endogenous mouse kappa and lambda chain genes in a significant proportion of B cells, thus Of B cells are monospecific for human or mouse light chains.

The transgenic mice of the preferred embodiment exhibit immunoglobulin production with a substantial repertoire, which is ideally substantially similar to that of the native mouse. Thus, for example, in an embodiment in which the endogenous Ig gene is inactivated, total immunoglobulin levels will be about 0.1-10 mg / ml serum, preferably 0.5-5 mg / ml, ideally at least about 1.0 mg / ml. Reach When a transgene capable of effecting a switch from IgM to IgG has been introduced into the transgenic mouse, the serum IgG: IgM adult mouse ratio is preferably about 10: 1. The IgG: IgM ratio is much lower in immature mice. Generally, greater than about 10%, preferably 40-80% of spleen and lymph node B cells only express human IgG protein.

The repertoire is ideally close to that exhibited in non-transgenic mice, usually on the order of at least about 10%, preferably 25-50% or more. In general, the number of different V, J and D regions primarily introduced into the mouse genome produces at least about 1000 different immunoglobulins (ideally IgG), preferably 10 ⁴ to 10 ⁶ or more. These immunoglobulins typically recognize about 1/2 or more of highly antigenic proteins, such as Staphylococcus coccus protein A. Some immunoglobulins exhibit an affinity for the preselected antigen of at least about 10 ⁷ M ^-1 , preferably 10 ⁸ M ^-1 to 10 ⁹ M ^-1 or greater.

In certain embodiments, it may be preferable to generate mice with a predetermined repertoire to limit the selection of V genes displayed in antibody responses to the predetermined antigen types. A heavy chain transgene with a predetermined repertoire can comprise, for example, the human VH gene which is preferentially used in the antibody response to a predetermined antigen type in humans. Alternatively, for a variety of reasons (eg, less likely to encode a high affinity V region for a predetermined antigen; less prone to somatic mutation and affinity enhancement; or to some humans VH genes can be excluded from a particular repertoire. Thus, prior to rearrangement of a transgene containing various heavy or light chain gene segments, such gene segments may be derived from a species of organism other than the transgenic animal, such as hybridization or DNA bases. It can be easily identified by sequencing.

The transgenic mice of the invention can be immunized with whole Staphylococcus aureus or Staphylococcus aureus antigens as described in Section I above. In mice, transgene switch recombination (cis-switch
) To give B cells expressing immunoglobulins that react with whole Staphylococcus aureus or Staphylococcus aureus antigens. The immunoglobulin can be a human sequence antibody, wherein the heavy and light chain polypeptides are encoded by human transgene sequences, which include sequences obtained by somatic mutation and V region recombination and germline. These human sequence immunoglobulins may be present as a result of other non-germline sequences being present as a result of somatic mutations and specific VJ and VDJ recombination ligations. Human V _L or V _H gene segment and human J _L or
It can be said that it is substantially identical to the polypeptide sequence encoded by the J _L gene segment. For such human sequence antibodies, the variable region of each chain is typically encoded by at least 80% of the human germline V, J and, in the case of heavy chains, the D gene segment; frequently at least 85 of the variable regions. % Are encoded by human germline sequences present on the transgene; often 90 or 95% or more of the variable region sequences are encoded by human germline sequences present on the transgene. However, since non-germ sequence sequences are introduced by somatic mutation and VJ and VDJ ligation, human sequence antibodies are used in human V, D as present in the human transgene (s) in the mouse germline. Or frequently some variable region sequences not encoded by the J gene segment (and less frequently constant region sequences)
Have. Typically, such non-germline sequences (or individual nucleotide positions)
Cluster in or near the CDRs or in regions where somatic mutations are known to cluster.

Human sequence antibodies that bind to a predetermined antigen are human sequence γ chains (γ1, γ2a,
Isotype switching can occur as a result of the production of human antibodies comprising a human sequence light chain (such as κ2) and a human sequence light chain (such as γ2B or γ3). Such isotype-switched human sequence antibodies are typically in the variable region, and often as a result of affinity maturation and selection of B cells by the antigen, especially after a secondary (or subsequent) antigen challenge, and often around the CDRs. Often contains one or more somatic mutations in or within 10 residues. These high affinity human sequence antibodies are at least about
1 x 10 ⁹ M ^-1 , typically at least about 5 x 10 ⁹ M ^-1 , often greater than about 1 x 10 ¹⁰ M ^-1 , and sometimes 5 x 10 ¹⁰ M ^-1 to about 1 x 10 It can have a binding affinity of ¹¹ M ^-1 or greater.

Another aspect of the invention is to generate hybridomas expressing monoclonal high affinity (greater than 2 × 10 ⁹ M ⁻¹ ) human sequence antibodies to whole Staphylococcus aureus or Staphylococcus aureus antigens. B cells from a mouse as can be used for. These hybridomas are for producing a composition comprising an immunoglobulin having an affinity constant (Ka) of at least 2 x 10 ⁹ M ^-1 , which binds to whole Staphylococcus aureus or Staphylococcus aureus antigens. Wherein the immunoglobulin is: (1) a light chain variable region having a polypeptide sequence that is substantially identical to the polypeptide sequence encoded by the human V _L gene segment and the human J _L segment. And (2
) A human sequence light chain consisting of a light chain constant region having a polypeptide sequence that is substantially identical to the polypeptide sequence encoded by the human _CL gene segment; and (1) a human _VH gene segment, optionally a D region And a heavy chain variable region having a polypeptide sequence that is substantially identical to the polypeptide sequence encoded by the human J _H segment and (2) substantially identical to the polypeptide sequence encoded by the human C _H gene segment. It comprises a human sequence heavy chain consisting of a constant region having a polypeptide sequence.

Development of high affinity human sequence antibodies against whole Staphylococcus aureus or Staphylococcus aureus antigens has been accomplished by developing human variable region genes in transgenic mice having a genome comprising an integrated human immunoglobulin transgene. Facilitated by a method of increasing the repertoire of a segment, the method comprising:
Comprising introducing into the genome a V gene transgene comprising a V region gene segment that is not present in the integrated human immunoglobulin transgene. The V region transgene may include a damaged or omitted V gene segment, may be naturally present in the human genome, or may be joined together separately by recombinant methods. Often a yeast artificial chromosome comprising a portion of the _H or V _L (V _κ ) gene segment sequence. Usually at least 5 or more functional V gene segments are contained on the YAC. In this variation, it is possible to produce transgenic mice by the V repertoire expansion method, wherein the mice are characterized by the variable region sequences encoded by the V region gene segments present on the V region transgene and human Ig. It expresses an immunoglobulin chain that comprises the C region encoded on the transgene. The V repertoire expansion method can be used to generate transgenic mice with at least 5 different V genes; as well, mice containing at least about 24 V genes or more can be generated. Certain V gene segments may not function (eg pseudogenes); these segments may be retained or, if desired, selectively deleted by recombinant methods available to those of skill in the art. ..

Functional YACs Once the Mouse Germline Has an Increased V Segment Repertoire that Is Substantially Absent in the Human Ig Transgene Containing the J and C Gene Segments
Engineered to contain this trait in other gene backgrounds, including the background of crossing functional YACs with an increased V segment repertoire to mouse germlines with different human Ig transgenes. Can be inherited and bred. Multiple functional YAs with an increased V segment repertoire to work with the human Ig transgene (or multiple human Ig transgenes)
C can be bred to the germline. Although referred to herein as YAC transgenes, such transgenes, when integrated into the genome, may be substantially devoid of yeast sequences such as those required for autonomous replication in yeast. Yes; such a sequence may be present after replication in yeast is no longer necessary (ie,
It may optionally be removed by genetic engineering (eg prior to introduction into mouse ES cells or mouse prozygote) (eg restriction digestion and pulsed field gel electrophoresis or other suitable method). Methods for increasing the trait of human sequence immunoglobulin expression include mating transgenic mice with a human Ig transgene (s) and optionally with a functional YAC containing an increased V segment repertoire. It Both V _H and V _L gene segments can be present on YAC. Transgenic mice should be bred by a professional to any desired background, including backgrounds carrying human Ig transgenes and / or other human transgenes, including transgenes encoding other human lymphocyte proteins. You can The invention also provides high affinity human sequence immunoglobulins produced by transgenic mice that have an increased V region repertoire YAC transgene. Although the foregoing describes preferred embodiments of the transgenic animals of the present invention, there are four types: I. Transgenic animals containing unrearranged heavy chain and rearranged light chain immunoglobulin transgenes; II. Transgenic animals containing unrearranged heavy chain and unrearranged light chain immunoglobulin transgenes; III. Containing rearranged heavy chain and unrearranged light chain immunoglobulin transgenes Transgenic animals; and IV. Transgenic animals containing rearranged heavy chain and rearranged light chain immunoglobulin transgenes.

Among these types of transgenic animals, the preferred order of preference is as follows: the endogenous light chain gene (or at least the kappa gene) has been knocked out by homologous recombination (or other method). II>I>III> IV in some cases, and I>II> III if the endogenous light chain gene is not knocked out and must be governed by allelic exclusion. III. Production of Bispecific / Multispecific Molecules that Bind Staphylococcus aureus In yet another aspect of the invention, a human monoclonal antibody against Staphylococcus aureus or a Staphylococcus aureus antigen of the invention or The antigen-binding portion is derivatized or linked to another functional molecule, such as another peptide or protein (eg Fab 'fragment). For example, an antibody or antigen-binding portion of the invention may be attached to one or more other binding molecules such as another antibody (e.g., a bispecific or multispecific antibody), antibody fragment, peptide or binding mimetic ( Functionally linked (by chemical coupling, gene fusion, non-covalent binding or otherwise).

Therefore, in another aspect, the invention relates to at least one first binding specificity for Staphylococcus aureus or a Staphylococcus aureus antigen and an Fc receptor, such as a human FcγRI or human Fcα receptor. Featuring bispecific and multispecific molecules comprising a second binding specificity. These bispecific and multispecific molecules are capable of expressing FcγR1- or FcαR-expressing effector cells (
For example, it can bind both monocytes, macrophages or polymorphonuclear cells (PMNs) and target cells including Staphylococcus aureus or Staphylococcus aureus antigens. When bound in this way, bispecific and multispecific molecules can bind Fc, such as phagocytosis of Staphylococcus aureus cells, antibody-dependent cellular cytotoxicity (ADCC), cytokine release or superoxide anion production.
Causes effector cell activity mediated by the receptor.

The multispecific molecules of the present invention further include, in addition to anti-Fc binding specificity and anti-target cell antigen binding specificity, such as Staphylococcus aureus or Staphylococcus aureus antigens, a third Binding specificity can be included. In one embodiment, the third binding specificity is a molecule that binds to an anti-enhancement factor (EF) moiety, such as a surface protein involved in cytotoxic activity, thereby increasing the immune response to target cells. An "anti-enhancement factor portion" is an antibody, functional antibody fragment or ligand that binds to a defined molecule, such as an antigen or receptor, and thereby results in enhanced action of the binding determinants of the Fc receptor or target cell antigen. Can be
The "anti-enhancement factor portion" is capable of binding the Fc receptor or 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 moiety can be applied to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1) or other immune cells that result in an increased immune response to target cells. Can be combined.

In one embodiment, the bispecific and multispecific molecules of the invention have at least one antibody as binding specificity, or for example Fab, Fab ′, F (ab ′) ₂ , Fv or single chain. It comprises an antibody fragment thereof which comprises Fv. Antibodies can also be found in light chain or heavy chain dimers, or in Fv or Ladner et al. US Pat. No. 4,946,778 issued Aug. 7, 1990, the contents of which are expressly incorporated herein by reference. It can also be any minimal fragment thereof such as the single-stranded construct described.

In one embodiment, the bispecific and multispecific molecules of the invention have binding specificity for FcγR or FcαR present on the surface of effector cells, and a target cell antigen such as Staphylococcus aureus or It comprises a second binding property for the Staphylococcus aureus antigen.

In one embodiment, the binding specificity for the Fc receptor is conferred by a human monoclonal antibody and this binding is not hindered by human immunoglobulin G (IgG).
As used herein, the term "IgG receptor" refers to any of the eight gamma chain genes located on chromosome 1. These genes encode a total of 12 transmembrane or soluble receptor isoforms that fall into three Fcγ receptor classes: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). In one preferred embodiment, the Fcγ receptor is human high affinity FcγRI. Human FcγRI is a 72 kDa molecule that exhibits high affinity (10 ⁸ -10 ⁹ M ^-1 ) for monomeric IgG.

The preparation and characterization of these preferred monoclonal antibodies is described by Fanger et al. In PCT application WO 88/00052 and in US Pat. No. 4,954,617,
These teachings are fully incorporated herein by reference. These antibodies bind to an epitope of FcγRI, FcγRII or FcγRIII at a site different from the Fcγ binding site of the receptor, thus their binding is not substantially prevented by physiological levels of IgG. Specific anti-FcγRI antibodies useful in the present invention include mAb22, mAb32, mAb44
, MAb62 and mAb197. The hybridoma producing mAb32 is an American Type
ATCC deposit number HB9469, available from the Culture Collection. Anti-FcγRI mAb22, m
The F (ab ') ₂ fragment of Ab22 is available from Medarex, Inc. (Annandale, NJ). In another embodiment, the anti-Fcγ receptor antibody is monoclonal antibody 22 (H2
It is a humanized form of 2). Production and characterization of the H22 antibody is described by Graziano, RF et al.
1995) J. Immunol 155 (10): 4996-5002 and PCT / US93 / 10384. H2
2 The antibody-producing cell line is HA022CL1 under the name American Type Culture Co on November 4, 1992.
It has been deposited with llection and has accession number CRL 11177.

In yet another preferred embodiment, the binding specificity for the Fc receptor is conferred by an antibody that binds to the human IgA receptor, eg the Fc-α receptor (FcαR (CD89)). Preferably, the antibody binds to the human IgA receptor at a site that is not interfered with by endogenous IgA.
The term "IgA receptor" includes a single α gene (FcαRI) located on chromosome 19.
The gene product of This gene is known to encode several alternatively spliced transmembrane isoforms of 55-110 kDa. FcαRI (CD89) is constitutively expressed on monocytes / macrophages, eosinophils and neutrophilic granulocytes, but not on non-effector cell populations. FcαRI

[0099]

[Outer 1]

Increased upon exposure to cytokines such as G-CSF or GM-CSF (Morton, HC
et al. (1996) Critical Reviews in Immunology 16: 423-440). Four FcαRI-specific monoclonal antibodies identified as A3, A59, A62 and A77 that bind FcαRI outside the IgA ligand binding domain have been described (Monteiro, RC et a
I., 1992, J. Immunol. 148: 1764).

FcαRI and FcγRI are (1) primarily immune effector cells such as monocytes, PMNs
Expressed on macrophages and dendritic cells; (2) expressed at high levels (eg 5,000-100,000 per cell); (3) mediators of cytotoxic activity (eg ADCC, phagocytosis); (4 And) are preferred trigger receptors for use in the present invention as they result in increased antigen presentation of antigens directed against them, including self-antigens.

In another aspect, the bispecific and multispecific molecules of the invention are targeted cell antigens,
For example, it further comprises a binding affinity that recognizes, eg, binds to, the Staphylococcus aureus antigen. In a preferred embodiment, binding affinity is conferred by the human monoclonal antibody of the invention.

“Effector cell-specific antibody” as used herein refers to an antibody or functional antibody fragment that binds to the Fc receptor of effector cells. Preferred antibodies for use in the present invention bind to the Fc receptor of effector cells at sites that are not bound by endogenous immunoglobulins.

As used herein, the term “effector cell” refers to an immune cell involved in the effector phase of an immune response, as opposed to the recognition and activation phases of the immune response. Representative immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., T cells including B cells and cytotoxic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes. Included are spheres, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells and basophils. Effector cells express Fc receptors and carry out specific immune functions. In a preferred embodiment, the effector cells are
Antibody-dependent cellular cytotoxicity (ADCC) can be induced, eg, neutrophils can induce ADCC. For example, monocytes expressing FcαR, macrophages, neutrophils,
Eosinophils and lymphocytes are involved in specifically killing target cells and presenting antigens to other components of the immune system or binding to antigen presenting cells. In another aspect, the effector cells can phagocytose the target antigen, target cell or microorganism. Expression of specific FcRs on effector cells can be regulated by humoral factors such as cytokines. For example, FcγRI expression has been found to be upregulated by interferon gamma (IFN-γ). This increased expression enhances the cytotoxic activity of FcγRI-bearing cells against the target. Effector cells can phagocytose or lyse target antigens or target cells.

A “target cell” is any harmful cell in a subject (eg, human or animal) that can be targeted by a composition of the invention (eg, human monoclonal antibody, bispecific or multispecific molecule). Shall mean. In a preferred embodiment, the target cells are Staphylococcus aureus cells. For example, Staphylococcus aureus cells are, for example, IBERIAN, EMRSA, ONTRARIO, BRAZILIA
There can be antibiotic resistant strains of Staphylococcus aureus, including N and COL strains, such as penicillin- and methicillin resistant Staphylococcus aureus (2,3). MRSA strains are generally resistant to these antibiotics due to the expression of a mutated penicillin binding protein (PBP 2a) that has a low binding capacity for β-lactam antibiotics.

In certain embodiments, the antibodies used in the bispecific or multispecific molecules of the invention can be human, chimeric or humanized antibodies to the Fc receptor.

Chimeric mouse-human monoclonal antibodies (ie, chimeric antibodies) can be produced by recombinant DNA technology known in the art. For example, the gene encoding the Fc constant region of a mouse (or other species) monoclonal antibody molecule is digested with a restriction enzyme to remove the region encoding mouse Fc and the corresponding portion of the gene encoding the human Fc constant region removed. Use instead. (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; Neuberge
r et al., International Application WO 86/01533; Cabilly et al. US Pat. No. 4,816,567; Cab
illy et al. European patent application 125,023; Better et al. (1988 Science 240: 1041-10
43); Liu et al. (1987) PNAS 84: 3439-3443; Liu et al., 1987, J. Immunol.
139: 3521-3526; Sun et al. (1987) PNAS 84: 214-218; Nishimura et al., 1987.
, Canc. Res. 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw.
et al., 1988, J. Natl Cancer Inst. 80: 1553-1559).

Chimeric antibodies can be further humanized by replacing sequences of the Fv variable region that are not directly involved in antigen binding with the corresponding sequences from the human Fv variable region. For a general review of humanized chimeric antibodies, see Morrison, SL, 1985, Science 229: 1202-120.
7 and Oi et al., 1986, Bio Techniques 4: 214. These methods include isolating, manipulating and expressing a nucleic acid sequence encoding all or part of an immunoglobulin Fv variable region from at least one of the heavy or light chains. Sources of such nucleic acids are well known to those of skill in the art, eg, 7E3.
, An anti-GPII _b III _a antibody-producing hybridoma. The recombinant DNA encoding the chimeric antibody or fragment thereof can then be cloned into a suitable expression vector. Alternatively, a suitable humanized antibody can be produced by CDR substitution, US Pat. No. 5,225,539; Jones et al. 1986 Nature 321: 55.
2-525; 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 can be replaced with at least a portion of the non-human CDRs, or only some of the CDRs can be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for the binding of the humanized antibody to the Fc receptor.

Antibodies can be humanized by any method that can replace at least a portion of the CDRs of a human antibody with CDRs from a non-human antibody. Winter describes methods that can be used to produce humanized antibodies of the invention (1987
UK patent application GB 2188638A, filed Mar. 26, 2014, the content of which is expressly incorporated herein by reference. Human CDR is a humanized antibody to the Fc receptor of immunoglobulin G on human mononuclear phagocytes.
or Immunoglobulin G on Human Mononuclear Phagocytes)
Oligonucleotide site-directed mutagenesis as described in WO 94/10332 can be used to replace non-human CDRs.

Also within the scope of the invention are chimeric and humanized antibodies in which certain amino acids have been substituted, deleted or added. In particular, preferred humanized antibodies have amino acid substitutions in the backbone region, such as to enhance binding to antigen. For example,
In humanized antibodies with mouse CDRs, amino acids located in the human skeletal region can be replaced with amino acids located in corresponding positions in the mouse antibody. Such substitutions are known to enhance binding of humanized antibodies to the antigen in some cases. Antibodies with amino acids added, deleted or substituted are referred to herein as modified or remodeled antibodies.

The term modified antibody is also meant to include antibodies such as monoclonal antibodies, chimeric antibodies and humanized antibodies which have been modified by, for example, deleting, adding or substituting a portion of the antibody. To do. For example, an antibody can be modified by deleting the constant region and replacing it with a constant region intended to increase its half-life, eg serum half-life, stability or antibody affinity. All modifications are within the scope of the invention as long as the bispecific and multispecific molecule has at least one antigen binding region specific for FcγR and causes at least one effector function.

Bispecific and multispecific molecules of the present invention can be synthesized using chemical techniques (eg DM Kranz et al.
al. (1981) Proc. Natl. Acad. Sci. USA 78: 5807), `` polydoma (polyd
oma) technology (see US Pat. No. 4,474,893 to Reading) or recombinant DNA technology.

In particular, the bispecific and multispecific molecules of the present invention may be made up of their constituent binding specificities, eg Anti-FcR and anti-staphylococcus aureus binding specificities can be combined to produce. For example, each binding specificity of the bispecific and multispecific molecule can be made separately and then bound to each other. When the binding specificity is a protein or peptide, various coupling or cross-linking agents can be used for covalent attachment. Examples of crosslinkers include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SA
TA), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) and sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) (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 Paulus (Behring Ins. Mitt. (1985) No. 78, 118-132); Brenn
an et al. (Science (1985) 229: 81-83) and Glennie et al. (J. Immunol. (198
7) Includes those described by 139: 2367-2375). The preferred binder is S
ATA and Sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL). If the binding specificity is an antibody (eg two humanized antibodies), these can be linked by a sulfhydryl bond in the C-terminal hinge region 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, expressed and assembled in the same host cell. This method is particularly useful when the bispecific and multispecific molecule is a mAb x mAb, mAb x Fab, Fab x F (ab ') ₂ or ligand x Fab fusion protein. Bispecific and multispecific molecules of the invention, such as bispecific molecules, include single chain molecules such as single chain bispecific antibodies, one single chain antibody and a binding determinant. Can be a single-stranded bispecific molecule consisting of or a single-stranded bispecific molecule comprising two binding determinants. Bispecific and multispecific molecules can also be multiple single-stranded molecules or can comprise at least two single-stranded molecules. Methods for producing bi- and multi-specific molecules are described, for example, in US Pat. No. 5,260,203; US Pat. No. 5,455,030; US Pat.
4,881,175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,4
76,786; U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent No. 5,4.
82,858.

Binding of bispecific and multispecific molecules to their specific targets can be confirmed by solid phase enzyme immunoassay (ELISA), radioimmunoassay (RIA) or Western blot assay. . Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a labeled reagent (eg, an antibody) specific for the complex of interest. For example, the FcR-antibody complex can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds the antibody-FcR complex. Alternatively, the complex can be detected using any of a variety of other immunoassays. For example, the antibody can be radiolabeled and used in a radioimmunoassay (RIA) (e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh, incorporated herein by reference).
Training Course on Radioligand Assay Techniques, The Endocrine Society,
See March 1986). Radioisotopes can be detected by such means as the use of gamma counters or scintillation counters, or by autoradiography. IV. Antibody conjugates / immunotoxins In another aspect, the invention provides a human anti-PSMA monoclonal antibody or fragment thereof conjugated to a therapeutic component such as a cytotoxin, drug or radioisotope. Is characterized by. These antibody conjugates are “immunotoxins” when conjugated to a cytotoxin.
Called. Cytotoxic or cytotoxic agents include any agent that is detrimental to (eg kills) cells. Examples are taxol, cytochalasin B, gramicidin D
, Ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin,
Dihydroxy anthracin dione, mitozantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol and puromycin and analogs or homologues thereof are included. Therapeutic agents include antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g. mechlorethamine, thioepaclorambucil, melphalan, carmustine (BSNU)) And lomustine (CCNU), cyclotosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g. daunorubicin (formerly daunomycin) and doxorubicin), antibiotics. Substances such as, but not limited to, substances (e.g. dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)) and anti-mitotic drugs (e.g. vincristine and vinblastine). The antibodies of the invention can be conjugated to radioisotopes, such as radioiodine, to produce cytotoxic radiopharmaceuticals for treating PSMS-related diseases such as cancer.

The antibody conjugates of the invention can be used to modify a defined biological response, and the drug component should not be construed as limited to classical chemotherapeutic agents. For example, the drug component can be a protein or polypeptide that possesses the desired biological activity. Such proteins include, for example, enzymatically active toxins or active fragments thereof such as abrin, ricin A, Pseudomonas exotoxin or diphtheria toxin; proteins such as tumor necrosis factor or interferon-γ; or, 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 biological response regulators such as growth factors be able to.

Techniques for coupling such therapeutic moieties to antibodies are well known, eg Arnon et a
l., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Thera
py ”, Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (edit)
, pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies F
or Drug Delivery ”, Controlled Drug Delivery (2nd edition), Rovinson et al.
(Edit), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carrie
rs Of Cytotoxic Agents In Cancer Therapy: A Review ”, Monoclonal Antibod
ies '84: Biological And Clinical Applications, Pinchera et al. (Editor)
, pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The
Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy ”, Monoclona
l Antibodies For Cancer Detection And Therapy, Baldwin et al. (Editor),
pp. 303-16 (Academic Press 1985) and Thorpe et al., “The Preparation And
Cytotoxic Properties Of Antibody-Toxin Conjugates ”, Immunol. Rev .. 62:
See 119-58 (1982). V. Pharmaceutical Compositions In another aspect, the invention relates to one or a combination of human monoclonal antibodies of the invention or antigen binding portion (s) thereof, formulated with a pharmaceutically acceptable carrier. There is provided a composition containing, for example, a pharmaceutical composition. In a preferred embodiment, the composition comprises a combination of more than one (eg, two or more) isolated human antibodies of the invention or antigen binding portion thereof. Preferably, each of the antibodies of the composition or antigen-binding portion thereof binds to a different preselected epitope of Staphylococcus aureus or a Staphylococcus aureus antigen.

In one embodiment, a pharmaceutical combination comprising human anti-Staphylococcus aureus monoclonal antibodies having complementary activities, eg, comprising two or more human anti-staphylococcus aureus monoclonal antibodies Used as a composition. For example, human monoclonal antibodies that have limited cross-reactivity between Staphylococcus aureus strains, but that result in highly efficient phagocytosis, can be expressed by other humans against Staphylococcus aureus that exhibit broader specificity. It can be used in combination with a monoclonal antibody.

In another embodiment, the composition comprises (eg, contains at least one binding specificity for the Fc receptor and at least one binding specificity for Staphylococcus aureus or Staphylococcus aureus antigens) of the invention. Of one or a combination of bispecific or multispecific molecules.

The pharmaceutical compositions of this invention may also be administered in combination therapy, ie, in combination with other agents. For example, the combination therapy can include at least one anti-infective (eg, antibiotic) or other conventional treatment and the composition of the invention.

As used herein, "pharmaceutically acceptable carrier" includes all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. Agents and the like are included. Preferably the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg by injection or infusion). Depending on the route of administration, the active compounds, ie antibodies, bispecific and multispecific molecules, are
The material can be coated to protect the compound from the action of acids and other natural conditions that can inactivate the compound.

“Pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the compounds of the invention and does not exert any undesired toxicological effects (eg, Berge, S. et al. 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 non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, and aliphatic monoacids.
-And dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids,
Those derived from non-toxic organic acids such as aromatic acids, aliphatic and aromatic sulfonic acids and the like are included. Base addition salts include alkaline earth metals such as sodium, potassium, magnesium, calcium and N, N'-dibenzylethylenediamine,
Included are those derived from non-toxic organic amines such as N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

The compositions of the present invention can be administered by a variety of methods known in the art. As will be appreciated by those in the art, the route and / or form of administration will depend on the desired result. 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 such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Many methods of making such formulations are either patented or generally known to those skilled in the art. For example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson
See Edit, Marcel Dekker, Inc., New York, 1978.

In order to administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with a substance which prevents its inactivation or co-administer the compound with it. For example, the compound can be administered to the subject in a suitable carrier, such as liposomes or diluents. 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. Neur.
oimmunol. 7:27).

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the optional preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Any conventional medium or agent is contemplated for its use in the pharmaceutical compositions of the present invention, unless it is contraindicated with the active compound. 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 such as glycerol, propylene glycol and liquid polyethylene glycol, 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, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol,
Alternatively, it is preferable to include sodium chloride in the composition. Prolonged absorption of 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 are prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. be able to. 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 (l
yophilization)), whereby a powder of the active ingredient is obtained from the previously sterile-filtered solution plus any additional desired ingredients.

Dosage regimens are adjusted to provide the optimum desired response (eg, 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. be able to. It is especially beneficial to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically divided units suitable as unit dosages for the subject to be treated; each unit being in association with the required pharmaceutical carrier as desired. It contains a predetermined quantity of active compound calculated to produce a therapeutic effect. The specifications for the unit dosage forms of the present invention are specific to the art of formulating (a) the unique properties of the active compound and the particular therapeutic effect obtained, and (b) the formulation of such active compound for the treatment of susceptibility in an individual. It depends on and is directly dependent on them.

Examples of pharmaceutically acceptable antioxidants are: (1) Water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc .; 2) Oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol; and (3) citric acid, ethylenediamine tetra Metal chelating agents such as acetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like are included.

In therapeutic compositions, formulations of the present invention include oral, nasal, topical (including buccal and sublingual)
Suitable for rectal, vaginal and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the subject 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 100%,
This amount ranges from about 0.01% to about 99% of active ingredient, preferably about 0.1% to about 70%, most preferably about 1% to about 30%.

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

The terms “parenteral administration” and “administered parenterally” as used herein refer to modes of administration other than enteral and topical administration, usually by infusion,
And without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, Intraspinal, epidural and intrasternal injections and infusions are included.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and their suitable. Various mixtures, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. The proper fluidity can be maintained, for example, by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

These compositions may also contain additives such as preservatives, wetting agents, emulsifying agents and dispersing aids. The prevention of the presence of microorganisms can be ensured both by the above-mentioned sterilization methods and the inclusion of various antibacterial and antifungal agents such as parabens, 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 composition of the present invention is administered as a pharmaceutical to humans and animals, they may be used alone or in combination with a pharmaceutically acceptable carrier, for example 0.01-99.5% (more preferably 0.1-90%). It can be provided as a pharmaceutical composition containing the active ingredient.

Regardless of the route of administration chosen, the compounds of the invention and / or the pharmaceutical compositions of the invention which can be used in suitable hydrated form are prepared by conventional methods known to those skilled in the art. Formulated into a pharmaceutically acceptable dosage form.

The actual dosage level of active ingredient in the pharmaceutical compositions of the present invention is not toxic to the patient and is effective to obtain the desired therapeutic response for the particular patient, composition and mode of administration. Varying the amount of active ingredient may be varied. The dosage level chosen will be the activity of the particular composition of the invention or its ester, salt or amide used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, the particular composition used. Other drugs, compounds and / or substances used in combination with
It depends on a variety of pharmacokinetic factors, including age, sex, weight, symptoms of the patient being treated, general health and previous medical history, and similar 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, a physician or veterinarian may start the dose of a compound of the invention used in a pharmaceutical composition at a level lower than that required to obtain the desired therapeutic effect, and until the desired effect is obtained. It should be possible to gradually increase the dose. In general, a suitable daily dose of a composition of the invention is that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose generally depends on the factors mentioned above. It is preferred that administration be intravenous, intramuscular, intraperitoneal or subcutaneous, preferably administered near the target site. If desired, the effective daily amount of the therapeutic composition optionally comprises 2, 3, 4, 5, 6 or more sub-doses administered separately in appropriate dosage units throughout the day. (sub-doses). 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, the therapeutic compositions of the present invention are disclosed in US Pat.
No. 163, No. 5,383,851, No. 5,312,335, No. 5,064,413, No. 4,941,880, No. 4,7
It can be administered with a needleless hypodermic injection device such as the devices disclosed in 90,824 or 4,596,556. Examples of well known implants and modules useful in the present invention include: US Pat. No. 4,487,603, which discloses an implantable microinfusion pump for delivering a drug at a controlled rate; US Pat. No. 4,486,194, which discloses a therapeutic device; US Pat. No. 4,447,233, which discloses a drug infusion pump for delivering a drug at a precise infusion rate; variable flow implantable for continuous drug delivery. Patent No. 4,447,224 which discloses an injection device
U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having a multi-chamber compartment;
And US Pat. No. 4,475,196, which discloses osmotic drug delivery systems. These patents are incorporated herein by reference. Many other such implants, delivery systems and modules are known to those skilled in the art.

In one aspect, 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. See, eg, US Pat. Nos. 4,522,811; 5,374,548; and 5,399,331 for methods of making liposomes. Liposomes are selectively delivered to specific cells or organs 1
Or more components and thus enhance selective drug delivery (see, eg, VV Ranade (1989) J. Clin. Pharmacol. 29: 685).
. Typical targeting components include folate or biotin (e.g., L
US Patent 5,416,016 to ow et al.); Mannoside (Umezawa et al., (1988).
Biochem. Biophys. Res. Commun. 153: 1038); antibody (PG Bloeman et al. (199
5) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemoth
er. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Ph.
ysiol. 1233: 134), different species of which may comprise the formulation of the invention as well as components of the invented molecule; p120 (Schreier et al. (1994) J. Biol. Chem. 269: 9.
090); K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346: 123; JJ Killio
n; IJ Fidler (1994) Immunomethods 4: 273 is also incorporated by reference. In one aspect of the invention, the therapeutic compounds of the invention are formulated in liposomes; in a more preferred aspect, the liposomes include a targeting component. In the most preferred embodiment, the therapeutic compound in liposomes is delivered by bolus injection at the site near 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 dose” is preferably at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably to an untreated subject. Preferably, it inhibits tumor growth by at least about 80%. The ability of compounds to suppress cancer can be evaluated in animal model systems that predict efficacy in human tumors. Alternatively, this property of the composition can be assessed by examining the ability of the compound to inhibit, such as in vitro inhibition by assays known to those of skill in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size, or otherwise improve symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's condition, and the particular composition or route of administration chosen.

The composition must be sterile and must be fluid to the extent that the composition can be delivered by syringe. In addition to water, the carrier can be isotonic buffered saline solution, ethanol, polyols such as glycerol, propylene glycol and liquid polyethylene glycol, 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 or sorbitol, and 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, aluminum monostearate or gelatin.

When the active compound is suitably protected as described above, the compound may be administered orally, for example with an inert diluent or an assimilable edible carrier. VI. Uses and Methods of the Invention The compositions of the invention (eg, human antibodies and derivatives thereof) have in vitro and in vivo diagnostic and therapeutic utility. For example, these molecules treat various diseases,
It can be administered to cells in culture for prevention or diagnosis, eg in vitro or ex vivo, or to a subject, eg in vivo. As used herein, the term “subject” shall include humans and non-human animals. Preferred human animals include human patients suffering from the disease caused by Staphylococcus aureus. For example, the methods and compositions of the invention can be used to treat a subject at risk of nosocomial infection. Examples of particularly vulnerable types of subjects include elderly and immunocompromised hospital patients. The term "non-human animal" of the present invention includes non-human primates,
All vertebrates such as sheep, dogs, cows, chickens, amphibians, reptiles etc. are included, eg mammals and non-mammals.

The compositions of the invention (eg human antibodies, multispecific and bispecific molecules) can be initially tested in vitro for binding activity associated with therapeutic or diagnostic applications. For example, the compositions of the present invention can be tested using the ELISA and flow cytometric assays described in the Examples below. In addition, the activity of these molecules can be assayed to cause effector cell activity brought about by at least one effector, including Staphylococcus aureus phagocytosis. A protocol for assaying the phagocytosis effected by effector cells is described in the Examples below.

The compositions of the invention (eg human antibodies, multispecific and bispecific molecules) have additional utility in the treatment and diagnosis of diseases caused by Staphylococcus aureus. For example, human monoclonal antibodies, multispecific or bispecific molecules, eg, in vivo or in vitro, opsonize one or more of the following biological activities: Staphylococcus aureus; It can be used to induce phagocytosis or inhibition of growth of Staphylococcus aureus in the presence; or to inhibit growth of Staphylococcus aureus.

For example, the human antibodies and derivatives thereof of the present invention can be used in vivo to treat, prevent or diagnose various diseases caused by Staphylococcus aureus. Examples of diseases caused by Staphylococcus aureus include, for example, invasive and toxigenic infections. Representative invasive diseases include: bacteremia, osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis. Representative toxigenic diseases include: Staphylococcal food poisoning, burn-like skin syndrome and toxic shock syndrome. Further examples of diseases caused by Staphylococcus aureus that can be treated using the methods and compositions of the present invention include the upper respiratory tract (e.g., otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis), lower Respiratory tract (e.g. pyometra, lung abscess), heart (e.g. infective endocarditis)
), Gastrointestinal (e.g. secretory diarrhea, spleen abscess, retroperitoneal abscess), CNS (e.g. cerebral abscess), eye
(E.g. blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal and orbital cellulitis,
Dacryocystitis), kidney and urinary tract (e.g. epididymitis, intrarenal and perirenal abscess, toxic shock system), skin (e.g. impetigo, folliculitis, skin abscess, cellulitis, wound infection, bacterial infection) (Myositis)
, Bone and joint (eg, septic arthritis, osteomyelitis) infections.

Methods of administering the compositions of the present invention (eg, human antibodies, multispecific and bispecific molecules) 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 numerator is Goldenberg, D.
It can be linked to radionuclides such as 131I, 90Y, 105Rh, etc. as described in M. et al. (1981) Cancer Res. 41: 4354-4360 and EP 0365 997. The compositions of the invention (eg, human antibodies, multispecific and bispecific molecules) can also be linked to anti-infective agents.

Target-specific effector cells, such as effector cells linked to the compositions of the invention (eg, human antibodies, multispecific and bispecific molecules), can also be used as therapeutic agents. Effector cells for targeting can be human leukocytes such as macrophages, neutrophils or monocytes. Other cells include other IgG- or IgA-receptor bearing cells. If desired, effector cells can be obtained from the subject to be treated. 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 ⁸ -10 ⁹ , but will depend on the therapeutic purpose. Generally, the amount is sufficient to obtain localization in target cells, eg Staphylococcus aureus cells, and to result in cell killing, eg by phagocytosis. The route of administration can also vary.

Treatment with target-specific effector cells can be performed in conjunction with other techniques for removal of target cells. For example, antimicrobial therapy using the compositions of the invention (eg, human antibodies, multispecific and bispecific molecules) and / or effector cells with these compositions can be used in conjunction with antibiotic therapy. Furthermore, combination immunotherapy can be used to guide two different populations of cytotoxic effector towards tumor cell clearance. For example, anti-Fc-γRI or anti-T3 linked anti-staphylococcus aureus antibodies can be used with IgG- or IgA-receptor specific binding agents.

The bispecific and multispecific molecules of the invention can also be used to modulate FcγR or FcαR levels on effector cells, such as by covering and eliminating receptors on the cell surface. . Mixtures of anti-Fc receptors can also be used for this purpose.

Also, compositions of the invention having complement binding sites, such as moieties from IgG1, -2 or -3 or IgM that bind complement (eg human antibodies, multispecific and bispecific). Molecule) in the presence of complement. In one embodiment, ex vivo treatment of a population of cells comprising target cells having a binding agent of the invention and appropriate effector cells can be supplemented by the addition of complement or serum containing complement. The phagocytosis of target cells coated with the binding agent of the present invention can be enhanced by binding of complement proteins. In another embodiment, target cells coated with the compositions of the invention (eg, human antibodies, multispecific and bispecific molecules) can also be lysed by complement.

The compositions of the invention (eg, human antibodies, multispecific and bispecific molecules) can also be administered with complement. Thus, within the scope of the invention is a composition comprising a human antibody, a multispecific or bispecific molecule and serum or complement. These compositions are advantageous because complement is located in close proximity to human antibodies, multispecific or bispecific molecules. Alternatively, the human antibody, multispecific or bispecific molecule of the invention and complement or serum can be administered separately.

Also within the scope of the invention is a kit comprising a composition of the invention (eg, human antibodies, multispecific and bispecific molecules) and instructions for use. The kit comprises at least one additional reagent, such as complement, or one or more additional human antibodies of the invention (eg in Staphylococcus aureus or Staphylococcus aureus antigens different from the first human antibody). Human antibody having complementary activity that binds to the epitope of.

In one embodiment, the subject can be further treated with an agent that modulates, eg, increases or inhibits, the expression or activity of the Fcγ or Fcα receptor, eg, by treating the subject with a cytokine. Preferred cytokines for administration during treatment with 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) are included.

The compositions of the invention (eg, human antibodies, multispecific and bispecific molecules) also include Fc
It can also be used to target, eg, label, cells that express γR or Staphylococcus aureus antigens. In such applications, the binding agent can be linked to a molecule that can be detected. Accordingly, the invention provides methods for locating cells expressing FcγR or Staphylococcus aureus antigens ex vivo or in vitro. The detectable label can be, for example, a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor. In one embodiment, the present invention provides a human monoclonal antibody that specifically binds to Staphylococcus aureus or a Staphylococcus aureus antigen or an antigen-binding portion thereof and a sample and a control sample, an antibody or a portion thereof and a staphylococcus. The presence of Staphylococcus aureus in the sample, comprising contacting under conditions that form a complex between Coccus aureus or Staphylococcus aureus antigens and detecting the formation of the complex. A method of detecting is provided, wherein the difference in complex formation of the sample compared to the control sample indicates the presence of Staphylococcus aureus in the sample.

In yet another aspect, the invention provides methods for detecting the presence of Fc-expressing cells in vivo or in vitro. This method comprises: (i) administering to the subject a composition of the invention (eg, a multi- or bispecific molecule) or fragment thereof conjugated to a detectable marker; (ii) cells expressing Fc. Exposing the subject to means for detecting the detectable marker to identify the region containing.

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

[0159] Human anti-by immunizing HC07 HuMAb mice of two systems in Staphylococcus aureus strain FDA209 killed in manufacturing heat of human monoclonal antibodies against EXAMPLES Example 1 Staphylococcus aureus - Staphylococcus aureus A monoclonal antibody was produced. The HC07 HuMAb mouse used in this study was obtained from US Pat.
Made as described in Nos. 806, 5,625,825 and 5,545,807, the entire disclosures of which are incorporated herein by reference.

Specifically, HC07 was prepared using heat-killed FDA209 that had been expanded in medium and emulsified in CFA.
Mice were immunized for 12 weeks. The immunized mice were boosted twice with the same bacteria in IFA at 2-week intervals. Serum was collected from these mice at 8 and 10 weeks. Staphylococcus aureus-specific titers were detected in the immunized animals after the third immunization.
FIG. 1 shows the analysis of Staphylococcus aureus coated microtiter plates by ELISA and anti-human binding to alkaline phosphatase.
Optical density of the indicated dilutions of pooled plasma from mice immunized with Staphylococcus aureus (shaded bars) and non-immunized mice (solid bars) as detected by IgG probe (OD (405 -650)). If mice showed a titer against Staphylococcus aureus 209 as determined by an ELISA assay, they were boosted with an iv injection of heat killed Staphylococcus aureus 209. After 3 days, the mouse was killed and the spleen was removed.

To produce hybridomas that produce anti-Staphylococcus aureus antibodies, spleen cells from mice showing plasma containing anti-Staphylococcus aureus antibodies were treated with P3X63-Ag8.653 cells (name ATCC). CRL 1580 non-secretory mouse myeloma cells deposited with ATCC) and fused with PEG. About 800 mixed hybridoma cultures were generated. Supernatants from these hybridoma cultures were screened for human IgG1 production and binding to Staphylococcus aureus strain 209. Figure 2
2GD1 to Staphylococcus aureus strain 209 as measured by ELISA
Shown is the binding of 6 supernatants from hybridoma cultures designated 2, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12. Briefly, bacteria were dried on 96-well plates and fixed with 0.25% glutaraldehyde. The supernatant was added and incubated for 1 hour.
Human IgG was detected using goat anti-human IgG alkaline phosphatase and pNPP.
Binding of anti-FcγRI to human IgG (H22 antibody) was used as a control. Significant binding was detected in the 6 hybridoma supernatants shown when compared to the H22 control. Example 2 Characterization of Human Monoclonal Antibodies Against Staphylococcus aureus The mixed hybridoma supernatants produced in Example 1 were tested for binding to methicillin-resistant Staphylococcus aureus (MRSA) strains as follows.

Supernatant binding to MRSA strains BK2058 and BK2709 was tested using an ELISA assay. These two strains are Iberian MRSA clones isolated from a hospital outbreak, both of which overexpress the penicillin binding protein. BK2058 also overexpresses fibronectin binding protein.

Strains BK2058 and BK2709 were fixed in wells of a 96-well plate and the antibodies in the supernatant were detected using the goat anti-human IgG alkaline phosphatase described in Example 1. Only the medium was used as a control. Figure 3 shows mixed hybridoma supernatants 2GD12, 2H12, 8.1E5, 8.2C1, 7 to Staphylococcus aureus MRSA strain 2058.
The binding of F1 and 6D12 is shown. Mixed bacteria (Escherichia coli, Pseudomonas aeruginosa) as determined by ELISA.
oginosa) or Micrococcus luteus), no significant binding of supernatants (2GD12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12) from mixed hybridoma cultures was detected (Fig. 4). ).

Supernatants (2GD12, 2H12, 8.1E5, 8.2 from mixed hybridoma cultures to Staphylococcus aureus (mottled bars) or Escherichia coli (plain bars) as determined by flow cytometry. The specificity of the human anti-Staphylococcus aureus monoclonal antibody was confirmed by comparing the binding of C1, 7F1 and 6D12) (Fig. 5). As shown in FIG. 5, significant levels of supernatant binding were detected only upon incubation with Staphylococcus aureus.

FIG. 6 shows Staphylococcus aureus FDA209 as measured by ELISA.
Supernatant from subcloned hybridoma cultures (2GD12, 2H12, 8.1E5, 8.2
3 is a bar graph showing binding of C1, 7F1 and 6D12). As before, Staphylococcus aureus cells were fixed to the wells of a 96-well plate, and anti-human IgG (κ chain) alkaline phosphatase was used to detect the monoclonal antibody present in the supernatant. A secondary antibody confirmed that the supernatant contained human antibodies that bind Staphylococcus aureus. Binding of anti-FcγRI to human IgG (H22 antibody) was used as a control.

In addition, supernatants from subclones of hybridoma cultures 6D12 and 5H10 were ELIS.
Assayed by A for binding to Staphylococcus aureus strain ATCC 27661. Briefly, trypticase for Staphylococcus aureus bacteria
(trypticase) Growth in soy agar overnight, resuspend in 1% gelatin in PBS,
Dry on 6-well plates at 40 ° C. overnight and fix with 0.1% glutaraldehyde in PBS. Staphylococcus aureus coated plates were blocked with 20% mouse serum, 0.5% EDTA, 0.25% BSA, 0.5% NaN ₃ , washed, and 100 μl / ml of hybridoma supernatant (or antibody (25 μg / ml)). Add wells at 37 ° C for 2 hours or
Incubated overnight at 4 ° C. Human antibodies were 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 were
It showed significant binding to Staphylococcus aureus when compared to the isotype control antibody (Figure 7).

Next, selected hybridoma supernatants that showed significant specific binding to Staphylococcus aureus (eg 2GD12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12) were placed on Staphylococcus aureus diet. Tested for ability to effect (FIGS. 8A-8C)
). Briefly, FITC-labeled Staphylococcus aureus and PMN were incubated at 37 ° C. for 30 minutes with and without supernatant from mixed hybridoma cultures. Changes in fluorescence were detected using FACSscan. 8A-8C are histograms showing phagocytosis produced by Staphylococcus aureus neutrophils upon incubation of mixed hybridoma supernatants with polymorphonuclear cells (PMNs). Figures 8A-8C show results from one representative supernatant of the six supernatants previously shown to contain anti- Staphylococcus aureus human IgG. Figure 8
A-8C includes control PMNs only (Figure 8A), with or without supernatant from mixed hybridoma cultures, PMNs and Staphylococcus aureus (Figure 8B) and supernatants from mixed hybridoma cultures. FACScan analysis of PMN and control Escherichia coli (Figure 8C) with or without. Fluorescence transfer was detected when PMN and Staphylococcus aureus were incubated with supernatant from mixed hybridoma cultures, indicating that there was phagocytosis of FITC-labeled Staphylococcus aureus (Figure 8B). ). Under the following conditions: PMN only (Fig. 8A); when Staphylococcus aureus was incubated with PMN without the supernatant from the mixed hybridoma culture (Fig. 8B); and the supernatant from the mixed hybridoma culture. When Escherichia coli was incubated with PMN containing
No significant change in bacterial phagocytosis was detected in 8C).

Also, in order to develop a purified culture producing human monoclonal antibodies against Staphylococcus aureus, selected hybridomas (eg, 2GD12, 2H12, 8.1E5, which showed specific binding and phagocytic activity, 8.2C1, 7F1 and 6D12) were subcloned. The resulting purified antibody is associated with Staphylococcus aureus FDA209 (FIGS. 9A and 9B) and MRSA strain 2058 (FIGS. 10A and 10B) as described above, and results in phagocytosis of Staphylococcus aureus. Tested again for their ability.

9A-9B show Staphylococcus aureus FDA2 compared to IgG control.
Shows binding of purified human monoclonal antibody 6D12 to 09. Specifically, Figure 9A shows two concentrations (1 and 10 μg / ml) of purified human to Staphylococcus aureus FDA209 as compared to an IgG control (mottled bar) when measured using ELISA. 6 is a bar graph showing binding of monoclonal antibody 6D12 (open bar). Purified 6D12 human monoclonal antibody was compared to control human IgG (anti-FcγRI monoclonal antibody H22) for binding to Staphylococcus aureus FDA209. Bacteria dried on microtiter plates were blocked with 20% mouse serum and then reacted with a monoclonal antibody preparation. Monoclonal antibody binding was detected with an anti-human IgG Fc specific probe conjugated with alkaline phosphatase.
Figure 9B shows FITC-labeled humanized anti-EGF as detected by flow cytometry.
Staphylococcus aureus FDA2 compared to receptor antibody control (H425)
FIG. 6 is a histogram showing direct binding of FITC-labeled 6D12 monoclonal antibody to 09. FIG. Briefly, 25 μg / ml FITC-labeled 6D12 monoclonal antibody and FITC-labeled H425 control were incubated with Staphylococcus aureus FDA209 and 2.8 mg / ml irrelevant human IgG to block protein A. Staphylococcus aureus cells were fixed with paraformaldehyde and BD
Analyzed on a flow cytometer.

10A-10B show binding of purified human monoclonal antibody 6D12 to MRSA strain 2058 compared to IgG control. Specifically, FIG. 10A is a linear graph showing binding of two concentrations (1 and 10 μg / ml) of purified human monoclonal antibody 6D12 to MRSA strain 2058 compared to IgG control. Figure 10B is a FITC-labeled humanized anti-EGF receptor antibody control (H425) as detected by flow cytometry.
2 is a histogram showing the direct binding of FITC-labeled 6D12 monoclonal antibody to MRSA strain 2058 compared to. Briefly, 25 μg / ml FITC labeled 6D12 monoclonal antibody and FITC labeled H425 control were incubated with MRSA strain 2058 and 2.8 mg / ml irrelevant human IgG to block protein A. Staphylococcus aureus cells were fixed with paraformaldehyde and analyzed on a BD flow cytometer.

Next, the survival of Staphylococcus aureus strain FDA-209 after PMN phagocytosis was evaluated as follows. Bacteria were grown overnight in trypticase soy agar, collected and resuspended in PBS at a concentration of 10 ⁶ cfu / ml. PMNs were collected from whole blood from healthy volunteers. Briefly, 120 ml whole blood was diluted 1: 1 with RPMI cell medium and
Layered on ll-paque and centrifuged at 500 xg for 30 minutes. The PMN layer was collected and contaminating red blood cells were lysed with a KCO ₃ lysing solution. PMNs were washed twice, counted and adjusted to a concentration of 10 ⁶ cells / ml. All reagents were prepared in 2% human serum pre-absorbed with live Staphylococcus aureus. Bacteria (200 μl) were opsonized with 10 μl of 6D12 antibody and isotype control antibody (100 μg / ml) for 10 minutes at room temperature and then incubated with human PMN (200 μl) at 37 ° C. for 1 hour with constant rotation. The bacteria and PMNs were then microfuged for 5 minutes and the cell pellet resuspended in 1 ml distilled water. Serial dilutions were made in distilled water, 50 μl of each dilution was plated on trypsinized agar plates and viable Staphylococcus aureus was counted.

As shown in FIG. 11, human anti-Staphylococcus aureus antibody 6D12 was found to bind to CD3
It increased the lethality of Staphylococcus aureus caused by PMNs by 60% when compared to the human isotype control antibody to 0. CONCLUSION The above examples show that at least two strains of Staphylococcus aureus (FDA
209 and MRSA strain 2058) shows the production of human monoclonal antibodies that specifically react with high affinity. Furthermore, the human monoclonal anti-Staphylococcus aureus antibody effectively engenders phagocytic activity against at least two strains of Staphylococcus aureus (FDA209 and MRSA strain 2058) using PMNs as effector cells. Thus, we demonstrated the phagocytic activity of these human antibodies against methicillin-resistant Staphylococcus aureus. These results confirm that the fully human monoclonal antibodies against Staphylococcus aureus of the present invention are useful in the treatment of nosocomial Staphylococcus aureus infections.

[0173]

[Table 1]

[0174]

[Table 2]

[0175]

[Table 3]

[0176]

[Table 4]

Equivalents One of ordinary skill in the art will recognize or be able to ascertain using no more than routine experimentation numerous equivalents of the specific embodiments of the invention described herein. Such an equivalent is
It is intended to be covered by the following claims. All references cited incorporation herein by reference, the entire contents of the patent applications and issued patents pending are expressly incorporated herein by reference to the disclosure of the present invention.

[Brief description of drawings]

FIG. 1 is a bar graph showing the levels of anti-Staphylococcus aureus human antibodies present in plasma of HuMAb mice immunized with heat-killed Staphylococcus aureus when compared to non-immunized controls. is there. Shown are mice immunized with Staphylococcus aureus analyzed by ELISA (shaded bars).
And optical density results for the indicated dilutions of pooled plasma from non-immunized mice (blank bars).

FIG. 2: Supernatants from 6 mixed hybridoma cultures to Staphylococcus aureus strain 209, 2GD12, 2H12, 8.1E5, 8.2C1, 7F1 compared to human IgG control.
3 is a bar graph showing the binding of 6D12 and 6D12. The anti-FcγRI antibody H22 was used as a control.

FIG. 3: Supernatant from mixed hybridoma cultures to methicillin-resistant Staphylococcus aureus strain BK2058 as compared to medium alone (bkd) as measured by ELISA.
3 is a bar graph showing the binding of (2GD12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12).

FIG. 4: Supernatant (2GD12, 2H12, 8.1E5, 8.2C1, 7F1 and 2G12, 2H12, 8.1E5, 8.2C1, 7F1 and 6D12) is a bar graph showing binding.

FIG. 5: Supernatants (2GD12, 2H12, 8.1E5, from mixed hybridoma cultures to Staphylococcus aureus (mottled sticks) or Escherichia coli (plain sticks) as measured by flow cytometry. 8C is a bar graph comparing bindings of 8.2C1, 7F1 and 6D12).

FIG. 6 is a bar graph showing the binding of supernatant from the indicated subcloned hybridoma cultures to Staphylococcus aureus FDA209 as measured by ELISA.

FIG. 7 is a bar graph showing binding of supernatants from subcloned hybridoma cultures to Staphylococcus aureus ATCC 27661 as measured by ELISA.

8A-8C are histograms showing phagocytosis produced by Staphylococcus aureus neutrophils upon incubation of mixed hybridoma supernatants with polymorphonuclear cells (PMNs). Control PMNs only (Figure 8A), with or without supernatant from mixed hybridoma cultures, PMNs with or without Staphylococcus aureus (Figure 8B) and supernatants from mixed hybridoma cultures, PMNs And FACScan analysis of control Escherichia coli (Figure 8C).

9A-9B show binding of purified human monoclonal antibody 6D12 to Staphylococcus aureus FDA209 compared to IgG control. FIG. 9A shows two concentrations (1 and 10 μg / ml) of purified human monoclonal antibody 6D12 (1 and 10 μg / ml) against Staphylococcus aureus FDA209 compared to an IgG control (mottled bar) as measured using ELISA. 3 is a bar graph showing a combination of open bars). Figure 9B shows a FITC-labeled humanized anti-EGF receptor antibody control (H) as detected by flow cytometry.
425) is a histogram showing direct binding of FITC-labeled 6D12 monoclonal antibody to Staphylococcus aureus FDA209 compared to 425).

Figures 10A-10B show binding of purified human monoclonal antibody 6D12 to MRSA strain 2058 compared to IgG control. Figure 10A shows 2 for MRSA strain 2058 compared to IgG control.
Figure 3 is a linear graph showing binding of purified human monoclonal antibody 6D12 at two concentrations (1 and 10 μg / ml). Figure 10B shows FIT as detected by flow cytometry.
FIG. 6 is a histogram showing direct binding of FITC-labeled 6D12 monoclonal antibody to MRSA strain 2058 compared to C-labeled humanized anti-EGF receptor antibody control (H425).

FIG. 11 is a bar graph showing lethality of Staphylococcus aureus following antibody opsonization with a human monoclonal antibody against Staphylococcus aureus and PMN phagocytosis.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 7/00 A61P 9/00 9/00 17/02 17/02 19/00 19/00 19/02 19 / 02 31/04 31/04 39/02 39/02 C07K 16/12 C07K 16/12 16/46 16/46 C12P 21/08 C12N 5/10 G01N 33/569 E C12P 21/08 33/577 B G01N 33/569 C12R 1: 445 33/577 C12N 15/00 C // (C12P 21/08 5/00 B C12R 1: 445) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, M , SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA , BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO , NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW F terms ( Reference) 4B024 AA01 AA11 BA41 DA02 GA05 GA18 HA15 4B064 AG27 CA10 CA20 CC24 DA01 DA13 4B065 AA91X AA92X AA93Y AB01 AB05 AC14 CA25 CA44 CA46 4C085 AA03 AA14 AA16 BA13 BB33 BB34 BB35 BB36 BB37 BB41 BB43 CC07 EE01 EE05 4H045 AA10 AA11 AA20 AA30 BA10 BA41 CA11 CA40 DA76 EA29 EA52 FA74

Claims (26)

[Claims] 1. One or more of the following properties: a) reactivity with at least one Staphylococcus aureus isolate; b) at least about 10 ⁷ M ^-1 Staphylococcus aureus or staphylo. Binding affinity constant for Coccus aureus antigen; c) ability to opsonize Staphylococcus aureus; or d) Staphylococcus aureus in the presence of human effector cells at a concentration of about 10 μg / ml or lower in vitro. An isolated human monoclonal antibody or antigen-binding portion thereof that specifically binds to Staphylococcus aureus or a Staphylococcus aureus antigen, having the ability to effect phagocytosis or inhibit growth. 2. IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD and
The isolated human antibody of claim 1 having an isotype selected from the group consisting of IgE or an antigen-binding portion thereof. 3. The isolated human antibody or antigen-binding portion thereof according to claim 1, which is IgG1κ. 4. The isolated human antibody or antigen-binding portion thereof of claim 1, wherein the at least one Staphylococcus aureus isolate is a methicillin-resistant Staphylococcus aureus strain. 5. At least one Staphylococcus aureus isolate
The isolated human antibody or antigen-binding portion thereof according to claim 1, which is selected from the group consisting of IBERIAN, EMRSA, ONTARIO and BRAZILIAN strains. 6. A hybridoma comprising B cells 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. Item 1. The isolated human antibody or an antigen-binding portion thereof according to Item 1. 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, 8.2C1, 7F1, 6D12 and 5H10. 8. The isolated human antibody of claim 1 or an antigen thereof, wherein the Staphylococcus aureus antigen is selected from the group consisting of secreted antigens and antigens present on the surface of Staphylococcus aureus. Join part. 9. A Staphylococcus aureus antigen is a capsular polysaccharide, fibronectin binding protein, protein A, toxin, toxic shock syndrome toxin superantigen, coagulase, staphylokinase, penicillin binding protein.
9. The isolated human antibody of claim 8 or an antigen binding portion thereof selected from the group consisting of 2a and an adhesin. 10. An isolated human monoclonal antibody or antigen-binding portion thereof that produces the phagocytosis of Staphylococcus aureus in the presence of human effector cells. 11. The isolated human antibody of claim 10, or an antigen thereof, which is capable of causing phagocytosis of Staphylococcus aureus by human effector cells with an IC ₅₀ of 1 × 10 ⁻⁷ M or lower in vitro. Join part. 12. A human heavy chain transgene and human light fused to an immortalized cell that produces a detectable amount of a human monoclonal antibody that specifically binds to Staphylococcus aureus or Staphylococcus aureus antigen. A hybridoma comprising B cells obtained from a transgenic non-human animal having a genome comprising a chain transgene. 13. A human monoclonal antibody having one or more of the following properties: a) reactivity with at least one Staphylococcus aureus isolate; b) at least about 10 ⁷ M ^-1 Staphylococcus. Binding affinity constants for Aureus or Staphylococcus aureus antigens; c) ability to opsonize Staphylococcus aureus; and d) in vitro in the presence of human effector cells at a concentration of about 10 μg / ml or less. 13. The hybridoma of claim 12, which has the ability to cause phagocytosis or inhibit growth of Phylococcus aureus. 14. The hybridoma of claim 13, which is selected from the group consisting of 2GD12, 2H12, 8.1E5, 8.2C1, 7F1, 6D12 and 5H10. 15. Staphylococcus aureus or Staphylococcus has a genome comprising a human heavy chain transgene and a human light chain transgene.
A transgenic non-human animal capable of expressing a human monoclonal antibody that specifically binds to an aureus antigen. 16. A transgenic non-human animal having a genome comprising a human heavy chain transgene and a human light chain transgene is a whole Staphylococcus aureus or Staphylococcus aureus antigen, and the antibody is an animal B cell. Human B monoclonal antibody specific for Staphylococcus aureus or Staphylococcus aureus antigen by fusing the B cells with myeloma cells. A method for producing a human monoclonal antibody that specifically binds to Staphylococcus aureus or Staphylococcus aureus antigen, comprising producing immortal hybridoma cells that secrete S. 17. A bispecific molecule comprising at least one first binding specificity for Staphylococcus aureus or Staphylococcus aureus antigen and a second binding specificity for Fc receptor. 18. The Fc receptor is human FcγRI or human Fcα receptor.
17 bispecific molecules. 19. The bispecific molecule of claim 17, which binds to the Fc receptor at a site different from the immunoglobulin binding site of the receptor. 20. A composition comprising the isolated human monoclonal antibody of claim 1 or an antigen binding portion thereof 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, each of the antibodies or antigen-binding portions thereof being a star. A composition that binds to a different epitope of a Phylococcus aureus or Staphylococcus aureus antigen. 22. A single substance which specifically binds Staphylococcus aureus to Staphylococcus aureus or Staphylococcus aureus antigen in the presence of effector cells so that Staphylococcus aureus phagocytosis occurs. A method of inhibiting the growth or inducing phagocytosis of Staphylococcus aureus in the presence of human effector cells, comprising contacting with a released human monoclonal antibody or antigen binding portion thereof. 23. An isolated human antibody that specifically binds to an amount of Staphylococcus aureus or Staphylococcus aureus antigen effective to treat or prevent a disease caused by Staphylococcus aureus or A method of treating or preventing a disease caused by Staphylococcus aureus, comprising administering the antigen-binding portion thereof to a subject. 24. The method of claim 23, wherein the disease caused by Staphylococcus aureus is an invasive or toxigenic infection. 25. The disease caused by Staphylococcus aureus is bacteremia, osteomyelitis, septic arthritis, septic thrombophlebitis, acute bacterial endocarditis, staphylococcal food poisoning, burn-like skin syndrome and toxic 24. The method of claim 23, selected from the group consisting of shock syndrome. 26. A human monoclonal antibody or an antigen-binding portion thereof which specifically binds to Staphylococcus aureus or a Staphylococcus aureus antigen, a sample and a control sample, and the antibody or a part thereof and Staphylococcus aureus. Or contacting under conditions that form a complex between Staphylococcus aureus antigens and detecting the formation of the complex, where the difference in complex formation of the sample compared to the control sample is A method of detecting the presence of Staphylococcus aureus in a sample comprising: indicating the presence of Staphylococcus aureus.