JP2005524624A - Multifunctional monoclonal antibody against peptidoglycan of gram-positive bacteria - Google Patents

Abstract

The present invention includes protective monoclonal antibodies that bind to peptidoglycans of Gram positive bacteria. This antibody also binds to whole bacteria, enhances phagocytosis and killing effects on bacteria in vitro, and prevents nasal colonization by gram-positive bacteria in vivo. The present invention also provides human antibodies, humanized antibodies, and chimeric antibodies. The present invention further describes heavy and light chain variable regions of antibodies within the scope of the present invention.

Description

Related Cross-Reference This application application is based on December 21, 2001 U.S. Provisional Application No. 60 / 343,444, filed (Attorney Docket No. 07787.6004), also is intended to claim the benefit. This application relies on the entire disclosure of this provisional application, the disclosure of which is incorporated herein by reference. This application is related to U.S. Patent Application Serial No. 09 / 097,055 filed June 15, 1998, which is specifically incorporated herein by reference, and also is U.S. Patent Application No. 60 / 341,806, which is incorporated by reference to Methods for It is also related to the application filed earlier on December 21, 2001, entitled `` Blocking or Alleviating Staphylococcal Nasal Colonization by Intranasal Application of Monoclonal Antibodies '', and U.S. Patent Nos. 5,571,511 and 5,955,074. The entire disclosure of these applications is specifically incorporated herein by reference.

Description of the invention
FIELD OF THE INVENTION This invention is in the field of immunology and infectious diseases, and relates to protective antibodies specific for Gram positive bacteria, particularly bacteria with peptidoglycan exposed on the surface. The present invention includes monoclonal antibodies and fragments, regions and derivatives thereof.

Introductory humans have long struggled with infections caused by bacteria, especially gram-positive bacteria. The surface structure and cell walls of Gram-positive bacteria form a complex matrix, thereby realizing functions essential for the interaction between bacteria and the host. The cell wall consists of peptidoglycan macromolecules (repeated by units of N-acetylglucosamine and N-acetylmuramic acid) and accessory molecules including teichoic acid, lipoteichoic acid and carbohydrate attached to it (e.g. (9) and (See (24)). In addition, there are a number of surface proteins anchored to the bacterial cell wall (see, eg, (17)).

  The body uses a variety of means to protect against such bacteria. Mechanical barriers such as skin and mucous membranes are the body's first line of defense. If pathogens can pass through these barriers and begin to proliferate, white blood cells called polymorphonuclear leukocytes, or PMNs, are the next mechanism used by the body to respond to infection. Finally, acquired immune mechanisms intervene, where circulating antibodies and complements (soluble plasma proteins that can bind non-specifically to foreign targets) bind to invading pathogens and attract phagocytic cells. Then, phagocytes swallow and digest the pathogen. This latter mechanism is called phagocytosis, and antibodies and complements that bind to and promote phagocytosis are called opsonins. The enhancement of phagocytosis by opsonin is called opsonization. Opsonization may utilize a combination of antibody and complement (the “classical pathway”) or only complement (the “alternate pathway”). The opsonization and phagocytic system is important because incomplete phagocytosis and killing of staphylococci (and other Gram-positive bacteria) leads to host entry, infection, and sometimes death.

  Because this type of bacteria is prevalent on the skin and other surfaces, most mammals are exposed to gram-positive bacteria. Thus, polyclonal sera from mammals, including humans, appear to contain IgG that binds to many different cell wall and cell surface components of Gram positive bacteria. Such a collection of IgGs may help protect against gram positive bacteria. Because many polyclonal IgGs that bind to many epitopes on surface antigens or cell wall molecules (such as peptidoglycan, teichoic acid, lipoteichoic acid, protein, carbohydrate) become opsonized and phagocytose gram-positive bacteria. It can be promoted. Therefore, the combined function of antibodies in the polyclonal serum may be the functional activity of the serum. However, it is clear that such polyclonal IgGs are not necessarily protective as evidenced by the constant presence of infections of this kind. Clinicians administer vaccines based on this type of bacteria to increase the level of antibodies against Gram-positive bacteria. However, since many bacterial cell extracts used for immunization are not pure for one epitope or antigen, the activity of the resulting antibody may show activity against several different cell wall components. This is particularly problematic when the cell wall is targeted by the antibody and the purity of the cell wall preparation cannot be confirmed.

  In addition, polyclonal sera may be protective in some individuals but cannot be used to elucidate the functional role of antibodies against a single epitope. This is because polyclonal sera naturally contain many different antibodies and bind to many antigens and epitopes. Each antibody can contribute to the activity of a complex function. Thus, the ability of an antibody against a specific epitope on the cell wall to act as an opsonin factor for Gram-positive bacteria is not clearly defined.

  In addition, some antibodies to certain epitopes may promote phagocytosis, while others may have other functions, such as blocking bacterial attachment to cells. Therefore, the potential functions of specific antibodies, such as enhanced phagocytosis, prevention of bacterial adhesion, and neutralization of toxic activity, can be elucidated, thereby laying the foundation for treatment that provides the expected protection. Only have monoclonal antibodies against specific epitopes.

  Until recently, it has been difficult to determine the role of peptidoglycans or antibodies to peptidoglycans due to the impure peptidoglycan preparations. Teichoic acid and lipoteichoic acid are closely related to cell wall peptidoglycans. Furthermore, for certain bacteria such as Staphylococcus epidermidis, teichoic acid and lipoteichoic acid have the same glycerol phosphate backbone. Such teichoic acid moieties can easily be incorporated into peptidoglycan preparations prepared from cell wall extracts. Thus, the activity of serum against such preparations may be due to the activity of antibodies against contaminants rather than the activity of antibodies against peptidoglycans (see, eg, (36)). Recently, we have developed monoclonal antibodies against LTA with multiple functional activities, including opsonin activity against gram positive bacteria. Using such antibodies, it can be confirmed that the peptidoglycan preparation is free of LTA contaminants.

  Furthermore, since peptidoglycans are ubiquitous in the bacterial kingdom, there is unlikely to be highly specific opsonins or protective antibodies to peptidoglycans. In addition, questions remain about the role of protective antibodies. Peterson and coworkers have shown that normal human serum opsonizes cell wall extracts and peptidoglycans (20). However, it is clear that there were many different antibodies to many different epitopes in serum. At least three different antigenic sites have been identified on the peptidoglycan matrix. When Peterson and coworkers cut peptidoglycan with lysostaphin into small soluble fragments, the fragments were no longer opsonized in the presence of normal human serum. It can be explained that the small fragment failed to support the binding of a sufficient number of different antibodies and that the antibody against a single epitope on the peptidoglycan was not opsonin. Thus, although peptidoglycan can activate the alternative pathway that promotes S. aureus opsonization and phagocytosis only by complement, the role of antibodies and the classical pathway in opsonization and phagocytosis is still difficult to understand.

  The role of antibodies in such processes became even more uncertain when studies by the same group revealed that IgG-deficient sera were completely opsonized. This result is consistent with studies by other groups that showed normal levels of killing by PMN using sera after blocking neutrophil IgG Fc receptors with sera from which antibodies were removed. I did it. The fact that many cell wall epitopes are deep below the surface and may be covered with proteins and capsular polysaccharides in living and growing bacteria is even more complicated (18, 19).

  Therefore, it has not been known whether monoclonal antibodies against peptidoglycans that bind to a specific epitope can have functional activity without working in concert with another antibody having another antigen specificity or epitope specificity. It was also unknown whether antibodies with a single specificity could fulfill several functions important for host immunity and defense. Such monoclonal antibodies should be useful in the prevention or treatment of Gram positive infections, and the epitopes or antigens to which they bind should be useful as vaccines to elicit protective immunity in the host.

  The present invention relates to a therapeutic composition comprising a protective monoclonal antibody (MAb) against PepG that enhances phagocytosis, prevents colony formation and / or suppresses toxicity induced or promoted by peptidoglycan (PepG). . As mentioned above, phagocytosis is important for effective immunity against gram positive bacteria. The present invention provides a protective opsonin MAb against PepG that enhances phagocytosis and its killing effect on Gram-positive bacteria, and thus can prevent or treat systemic infection. Nasal colonization has been found to be the primary reservoir of staphylococci, and a strong correlation between staphylococcal nasal colonization and subsequent staphylococcal infection has been demonstrated. The present invention provides protective anti-PepG MAbs that block and / or reduce nasal colonization by gram positive bacteria such as staphylococci, thereby reducing the incidence and / or severity of infections associated therewith. Protective anti-PepG MAbs can reduce the toxic effects of cell wall components when administered intravenously, subcutaneously, intramuscularly, or by other routes of administration. Thus, this type of therapeutic composition prevents and treats infection by gram positive bacteria.

  Protective monoclonal antibodies of the present invention include both IgG and IgM PepG-specific anti-PepG MAbs and also include PepG-specific mouse, mouse / human chimera, humanized, or fully human MAbs. The protective monoclonal antibodies of the invention are directed to any multiple epitope on PepG. The antibodies of the present invention exhibit multiple binding properties and functional activities.

  These protective monoclonal antibodies are administered alone or in combination in the nostrils of normal or colonized human subjects or other mammals to prevent or reduce bacterial colonization in the nasal mucosa, thereby It can prevent systemic infection or reduce the spread of Gram-positive bacteria.

  The present invention provides a single protective property to enhance phagocytosis, prevent bacterial infections that can occur as a result of colonization in the nasal mucosa, and further reduce the toxic effects of PepG and other cell wall components or toxins. Also included are methods of using anti-PepG MAbs and methods of combining MAbs.

  Furthermore, PepG epitopes or PepG antigens and peptides that mimic such epitopes and antigens are useful as vaccines to elicit opsonin antibodies against gram positive bacteria.

Detailed Description of the Invention
Definitions As used herein, the term “antibody” includes full-length antibodies and portions thereof. A full-length antibody has one pair, or more usually two pairs of polypeptide chains, each pair comprising a light chain and a heavy chain. Each heavy or light chain is divided into two regions: a variable region (providing recognition and binding to the antigen) and a constant region (associated with localization and cell-cell interactions). Thus, full-length antibodies generally have two heavy chain constant regions (HC or CH), two heavy chain variable regions (HV or VH), two light chain constant regions (LC or CL), and two light chain variable Includes regions (LV or VL) (Figure 2). The one or more light chains may be lambda chains or kappa chains. Thus, in an embodiment of the invention, the antibody comprises at least one heavy chain variable region and one light chain variable region, such that the antibody binds an antigen.

  Another aspect of the invention includes variable regions comprising alternating complementarity determining regions or CDRs and framework regions or FRs. CDRs are sequences within the variable region and generally confer antigen specificity.

The invention also includes antibody portions that include variable region sequences sufficient to confer antigen binding. Antibody portions include, but are not limited to, Fab, Fab ′, F (ab ′) ₂ , Fv, SFv, scFv (single chain Fv), and include complete antibody such as papain cleavage and pepsin cleavage. Produced by proteolytic cleavage or by recombinant methods that manipulate complete heavy and light chain cDNAs to produce heavy and light chain fragments separately or as part of the same polypeptide Is included.

  The MAbs of the present invention comprise antibody sequences corresponding to human antibodies, non-human animal antibodies, and hybrids thereof. As used herein, the term “chimeric antibody” includes a variable region derived from an animal antibody, such as a rat or mouse antibody, that is a constant region domain derived from another molecule, eg, a human IgG, IgA, or IgM antibody. And the antibody fused.

  One type of chimeric antibody, a “humanized antibody”, has a variable region that has been altered (by mutagenesis or CDR grafting) to match (as much as possible) a known human variable region sequence. CDR grafting involves grafting CDRs from an antibody with the desired specificity onto the FRs of a human antibody, thereby exchanging most non-human sequences for human sequences. Thus, a humanized antibody closely matches the sequence of a known human antibody (its amino acid sequence). Humanization of mouse monoclonal antibodies reduces the degree of response to human anti-mouse antibodies, ie HAMA. The invention also includes fully human antibodies that avoid HAMA responses as much as possible.

  The invention also includes “modified antibodies”, which include, for example, a protein or peptide encoded by a gene encoding a truncated or modified antibody. Such proteins or peptides can function similarly to the antibodies of the present invention. Other modifications, such as the addition of other sequences, that can enhance effector function, including the ability to block or reduce nasal colonization by staphylococci are within the scope of the invention. Such modifications include, for example, addition of amino acids to the amino acid sequence of the antibody, deletion of amino acids in the amino acid sequence of the antibody, substitution of one or more amino acids in the antibody amino acid sequence with alternative amino acids, isotype switching, and Class switch is included.

  In certain embodiments, the Fc region of an antibody may be modified so that it does not bind bacterial proteins. The Fc region is usually the binding site for accessory cells of the immune system. When the antibody binds to and covers the bacteria, such accessory cells recognize the covered bacteria and respond to the infection. When bacterial proteins bind to the Fc region near the attachment site of the auxiliary cell, the normal function of the auxiliary cell is inhibited. For example, protein A, a bacterial protein found in the cell membrane of S. aureus, binds to the Fc region of IgG near the auxiliary cell binding site. Protein A, in doing so, inhibits the function of these accessory cells, thus preventing the bacteria from being cleared. To avoid interference with this antibacterial immune response, the Fc portion of the antibodies of the invention can be modified to prevent non-specific binding of protein A without losing binding to accessory cells (e.g. (See (10)).

  In light of these various forms, antibodies of the present invention include full-length antibodies, antibody portions, chimeric antibodies, humanized antibodies, fully human antibodies, and modified antibodies, and unless otherwise indicated, Call it “MAb”.

  As used herein, the term “epitope” refers to one or more regions of PepG where an antibody binds to PepG. The region to be bound may or may not be a continuous part of the molecule.

  The term “antigen” as used herein refers to a polypeptide sequence, a non-proteinaceous molecule, or any molecule that can be recognized by the immune system. The antigen may be a full size staphylococcal protein or molecule thereof, or a fragment thereof, the fragment being from a recombinant cDNA encoding a protein that is less than full length, or a full size molecule or protein, or It is generated from a fragment derived from that fragment. Such fragments can be generated by proteolysis. The antigen may be a polypeptide sequence that includes an epitope of a staphylococcal protein, and the epitope may not be contiguous with the linear polypeptide sequence of the protein. The DNA sequence encoding the antigen can be identified, isolated, cloned, transfected into prokaryotic or eukaryotic cells and expressed by procedures well known to those skilled in the art (25). The antigen may be 100% identical to a region of the amino acid sequence of the staphylococcal molecule or protein thereof, or may be at least 95% identical, at least 90% identical, or at least 85% identical. Antigens may be less than 100%, 95%, 90%, or 85% identical to the amino acid sequence of a staphylococcal molecule or its protein, provided that antibodies that still bind to the native staphylococcal molecule or its protein are used. Shall be able to lure out.

  The% identity of peptide antigens is described, for example, by Devereux et al. (Nucl. Acids Res. 12: 387, 1984) and uses the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG) However, it can be determined by comparing the target antigen or epitope sequence with a similar portion of the staphylococcal sequence (40). This GAP program uses the alignment method of Needleman and Wunsch (J. Mol. Biol. 48: 443, 1970), revised by Smith and Waterman (Adv. Appl. Math 2: 482, 1981), It can be applied to the determination of the percent identity of protein or nucleic acid sequences listed herein (41, 42). Preferred default parameters for the GAP program are described in the following: (1) edited by Schwartz and Dayhoff, “Atlas of Protein Sequence and Structure”, National Biomedical Research Foundation, pages 353-358, 1979. Unary comparison matrix for nucleotides (including 1 value for identical and 0 value for non-identical) and weighted comparison matrix of Gribskov and Burgess Nucl. Acids Res. 14: 6745, 1986; (2) per gap A penalty of 3.0 and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for the terminal gap (43, 44).

  Alternatively, a short region of up to 10 or 20 units, or a relatively high homology region, eg, between antibody sequences or homologous parts thereof, for a defined region of a peptide or nucleotide sequence Percent identity can be determined by dividing the number of matching amino acids or nucleotides by the total length of the aligned sequences and multiplying by 100%. If there is an insertion, or gap, of 1, 2 or 3 amino acids in or adjacent to the MAb chain, eg, in the CDR, the insertion or gap is counted as a single amino acid mismatch.

  The antigen may be a bacterial surface antigen and / or a pathogenic antigen and / or an adherent antigen. A surface antigen is an antigen that is accessible to an antibody when the antigen is in the form of an intact whole bacterium, ie, the antigen is not inside the cytoplasm. Pathogenic antigens are antigens that are involved in the pathogenesis process and cause disease in the host. Pathogenic antigens include, for example, LTA, peptidoglycan, toxin, pilus, flagella, and adherent antigen. Adhesive antigens mediate the ability of staphylococcal bacteria to attach to the nostril surface. Antigens may be non-protein components of staphylococci such as carbohydrates and lipids. For example, peptidoglycan and lipoteichoic acid are two non-protein antigens found in the cell wall of staphylococci. An antigen may comprise or include a fragment of a non-protein molecule as long as it elicits an immune response.

  Antigens used herein include molecules that can elicit an antibody response against PepG. The antigen can be a PepG molecule, or a fragment thereof, which can be obtained enzymatically or otherwise from the entire molecule or a fragment thereof. The antigen may be a PepG fragment containing an epitope of PepG, and the epitope may not be contiguous with the macromolecular structure of the molecule. The antigen may have 100% identity to the region of PepG, or 95%, 90%, or 85%. The identity of the antigen with the PepG molecule may be lower, provided that it can elicit antibodies that bind to PepG. An antigen may be an unrelated molecule that can elicit antibodies that bind to PepG with some structural similarity. In certain embodiments of the invention, the antigen elicits an antibody that binds to PepG on the bacterial surface. In certain embodiments, the antigen is a peptide that can elicit antibodies that bind to PepG and be encoded by cDNA. The procedures are generally described in "Molecular Cloning: A Laboratory Manual", 2nd edition, which is incorporated herein by reference for any purpose (25).

  Specific antigens of the invention include the hybridomas 11-232.3, 11-248.2, 11-569.3, 11-232.3 IE9, 99-110FC12 IE4 (MAb-11 232.3, MAb-11-248.2, MAb described herein. -11-569.3, MAb-11-232.3 IE9, and MAb-99-110FC12 IE4), A130, or antigens that bind to any of the monoclonal antibodies produced by M130.

  The antibody is at least 2-fold, at least 3-fold, at least 5-fold, and at least 10-fold greater than the background signal by a protein ELISA or other assay, i.e., at least twice the signal obtained for non-specific binding If an antibody obtains a signal that is at least 3-fold, at least 5-fold, or at least 10-fold greater, it is said to specifically bind an antigen, epitope, or protein. An antibody is said to specifically bind bacteria if, for example, a methanol-immobilized bacterial ELISA or a live bacterial ELISA results in a signal that is at least 1.5, 2, or 3 times greater than the background signal from the antibody.

  By “enhanced phagocytosis” is meant herein an increase in phagocytosis over background levels as analyzed by the method of this application or other equivalent assay. The level considered valuable will vary considerably depending on the specific infection situation, including the type of bacteria and the severity of the infection. For example, the enhanced phagocytic activity can be as much as or more than 75%, 80%, 85%, 90%, 95%, or 100% of the background phagocytosis. The enhanced phagocytosis may be as much as, or more than, 50%, 55%, 60%, 65%, or 70% of the background phagocytosis. As used herein, opsonin activity may be assessed by an assay that measures neutrophil-mediated opsonophagocytic bactericidal activity.

  The MAbs of the present invention are useful for the prevention and other treatments of systemic and local staphylococcal infections. In this regard, whether exposed by intentional staphylococcal instillation, whether exposed systemically, prior to, simultaneously with, or after exposure to staphylococci, the number of colonies in the nostril of the mammal If it can be reduced, the MAb of the present invention is said to “reduce” nasal colonization of staphylococci. For example, in the nasal colonization animal model described below, MAb or MAb aggregates have a reduced degree of colonization or the number of bacterial colonies that can grow from a sample of nasal tissue after administration of MAb or MAb aggregates. Are considered to reduce colony formation. MAb or MAb aggregate is used to reduce colony count to at least 25%, at least 50%, at least 60%, at least 75%, at least 80%, or at least 90% in the nasal colony formation assay described herein. , Reduce colony formation. A 100% reduction may be called eradication.

  If it can prevent nasal colonization of human or non-human mammals when administered to the nostrils before or simultaneously with exposure to staphylococci, whether by intentional instillation or by other methods, MAb is said to “block” staphylococcal colonization. In the nasal colony formation assay described herein, staphylococcal colonies from a nasal tissue sample taken from a mammal treated with a MAb of the present invention, such as 12 hours or more or 24 hours or more compared to a control mammal MAb prevents colony formation if it cannot grow over a long period of time. MAbs also block colonization in the nasal colony formation assay described herein when the number of colonized animals is reduced compared to control animals. For example, after administration of substances and Gram-positive bacteria, if the number of animals with colonization decreases to at least 25%, at least 50%, and at least 75% of control animals, or from samples taken from treated individuals If the colonies do not grow for longer periods, such as 12 hours or more or 24 hours or more, MAbs are considered to prevent colony formation.

  Clinically, nasal swabs are cultured in an appropriate bacterial medium to determine the presence or absence of nasal colonization in human patients. These cultures are scored for the presence or absence of staphylococcal colonies. In this type of qualitative assay system, it may be difficult to distinguish between prevention and reduction of staphylococcal colonization. Thus, for the purpose of qualitative assays such as nasal swabs, MAbs can form colonies if they prevent subsequent colonization for longer periods, such as 12 hours or more or more than 24 hours, in human patients who do not show signs before colonization. "Block" A MAb “reduces” colony formation if the number of positive cultures taken from human patients who are already staphylococcal positive prior to administration of the MAb of the present invention is discernably reduced.

  Accordingly, intranasal administration of MAbs of the present invention can be performed to prevent and / or reduce nasal colonization of staphylococci. Upon administration (instillation) of an `` effective amount '' of MAb, the mammal is: 1) no nasal colonization by staphylococci for at least 12 hours after administration, 2) the number of gram positive or staphylococcal colonies in the nostrils, Identifiable, medically meaningful or statistically significant reduction, or 3) Identifiable, medical significance of the frequency of cultured gram-positive or staphylococcal bacteria taken from the nostril 4) Either a distinct, medically meaningful or statistically significant decrease in 4 or gram positive or staphylococcal infection incidence.

  “Drip infusion” includes any delivery system capable of delivering an effective amount of MAb to the nostril of a mammal.

  The object of the present invention is to reduce the incidence of staphylococcal infections, including nosocomial infections. Effective doses may include staphylococcal infections involving body parts other than the nostrils, such as systemic infections, trauma or surgical site infections, which are identifiable, medically meaningful, or statistically Those well-proven to be significantly reduced are included. Such proofs include, for example, animal studies or preterm infants, those undergoing hospitalization or outpatient surgery, burn victims, indwelling catheters, stents, patients undergoing joint replacement, elderly patients, genetics And clinical trials of patients at risk of Gram-positive bacterial infection, including but not limited to patients whose immune system is suppressed by chemicals or by viruses.

  As used herein, `` treatment '' of a patient includes administration of a composition of the invention that results in `` therapeutically beneficial outcome '', whereby 1) of existing Gram positive bacterial infection or colonization, Identifiable, medically meaningful or statistically significant reduction, remission, or reduction, or 2) identifiable, medically meaningful for subsequent bacterial attack, infection, or colonization Or a statistically significant inhibitory action or prevention, or 3) an identifiable, medically meaningful or statistically significant reduction in the likelihood of nosocomial infection. Thus, treatment is an identifiable, medically meaningful or statistically significant reduction in the number of Gram-positive bacteria in patients with colonization or infection, as well as a reduction in the likelihood of subsequent colonization or infection. including. As used herein, “colony formation” refers to the presence of gram positive bacteria in a patient, particularly without symptoms in the nostrils, and “infection” refers to an infection with symptoms in any body part.

As used herein, “medically meaningful” refers to improving patient status, improving patient prognosis, reducing patient morbidity or mortality, or within a patient population. Include any treatment that reduces disease incidence or mortality due to bacterial infections addressed here. Whether a result is “statistically significant” is specifically determined or confirmed depending on the exact statistical test used. Those skilled in the art will readily recognize whether any statistical test used is statistically significant, as determined by the parameters of the test itself. Examples of such well-known statistical tests include X ² test (chi-square test), Student t test, F test, M test, Fisher exact test, binomial exact test, Poisson exact test, repeated measurement Includes but is not limited to directional or two-way analysis of variance and correlation coefficient calculation (Pearson and Spearman).

  The MAbs of the present invention include “protective MAbs”. Protective MAbs show 1) strong binding to PepG, 2) enhance opsonization and killing of gram-positive bacteria (opsonophagocytic killing), and 3) weaken bacterial colonization. Such MAbs also inhibit toxicity induced or promoted by PepG. In another embodiment, such protective MAbs comprise a therapeutic composition for treating Gram positive bacterial infection.

  A vaccine is thought to confer a protective immune response if it stimulates the production of protective opsonin antibodies against gram positive bacteria. The production of protective opsonin antibodies can be measured by comparing the presence of such antibodies in the serum of subjects who have received the vaccine, compared to controls which have not been administered the vaccine. The presence of protective opsonin antibodies in serum can be measured by the activity assays described herein, or other equivalent assays. When using the opsonophagocytic bactericidal assay, immunity is considered enhanced if the test serum kills at least 50%, at least 75%, at least 100% more bacteria than the control serum.

Embodiments of the Invention One aspect of the present invention relates to protective anti-PepG MAbs that bind to whole bacteria. Bacteria include all gram positive bacteria, especially staphylococci and streptococci. Since many of the PepG epitopes may not be utilized on the surface of Gram positive bacteria, the present invention provides protective MAbs that bind not only to whole bacteria but also to isolated PepG. Such protective MAbs can neutralize the toxic effects of such molecules by binding PepG.

  Another aspect of the invention relates to protective MAbs that function as opsonins that bind in a manner that allows interaction with phagocytic cells, thereby promoting phagocytosis. Such protective MAbs may be those that prevent or reduce Gram positive bacterial infection. Such protective anti-PepG MAbs may be used alone or in combination with MAbs of different specificities, such as LTA-specific MAbs, to treat diseases caused by Gram-positive bacteria and / or other organisms. Can be treated. Another aspect of the invention is a protective anti-PepG MAb that can prevent or reduce bacterial nasal colonization.

  Certain embodiments of the present invention include monoclonal antibodies MAb-11-232.3, MAb-11-248.2, MAb-11-569.3, MAb-11-232.3IE9, MAb-99-110FC12IE4, A130 described herein. Or a protective MAb comprising the antigen binding domain of M130.

  The present invention also includes a protective chimeric anti-PepG MAb in which a variable region derived from a mouse monoclonal antibody is fused to a human constant region, which is produced during mammalian cell culture.

  For example, a chimeric heavy chain can comprise a heavy chain variable region antigen binding region of a protective mouse anti-PepG MAb of the invention linked to at least a portion of a human heavy chain constant region. This chimeric heavy chain may be combined with a chimeric light chain comprising a protective murine anti-PepG MAb light chain variable region antigen binding region linked to at least a portion of a human light chain constant region.

  In certain embodiments of the invention, the protective chimeric antibody is a human / mouse chimeric A130 antibody as described herein. In another embodiment, the protective chimeric antibody is a monoclonal antibody MAb-11-232.3, MAb-11 248.2, MAb-11-569.3, MAb-11-232.3IE9, MAb-99-110FC12IE4, described herein, It contains the antigen binding domain of either A130 or M130.

  Epitopes and antigens bound by protective anti-PepG monoclonal antibodies are also embodiments of the invention. Another embodiment of the present invention includes epitopes and antigens that elicit opsonin antibodies that bind to the Gram positive bacterium PepG in vertebrates. Such epitopes and antigens elicit protective opsonin antibodies when introduced into humans, mice, rats, rabbits, dogs, cats, cows, sheep, pigs, goats, or chickens. Peptides that mimic such epitopes and antigens and can elicit opsonin antibodies against PepG of Gram positive bacteria are also included in the present invention. Such epitopes, antigens, peptides, and fragments of PepG can also be used as vaccines to protect or reduce infection caused by Gram-positive bacteria.

  The present invention also discloses therapeutic compositions comprising the protective, anti-PepG MAbs of the present invention that are chimeric, humanized, or fully human antibodies, and fragments, regions, and derivatives thereof. Such compositions may include a pharmaceutically acceptable carrier. The therapeutic composition of the present invention can alternatively comprise an isolated antigen, epitope, or portion thereof, together with a pharmaceutically acceptable carrier.

  In certain embodiments, therapeutic compositions of the present invention include, but are not limited to, monoclonal antibodies MAb-11-232.3, MAb-11-248.2, MAb-11-569.3, Protective antibodies comprising the antigen binding domain of either MAb-11-232.3IE9, MAb-99-110FC12IE4, A130, or M130 are included.

  Pharmaceutically acceptable carriers include, but are not limited to, sterile liquids such as water and oils, including petroleum oils, animal oils, vegetable oils, peanut oil, soybean oil, mineral oil, sesame oil, and the like. . Saline, aqueous dextrose, and glycerol solutions can also be used as liquid carriers. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences”, 18th Edition (8), which is incorporated herein by reference for any purpose.

  Furthermore, the present invention may be practiced with various delivery excipients and / or carriers. Such excipients can extend the half-life of MAbs during storage and upon administration including but not limited to mucosal application, for example, inhalation or instillation into the nostril. . Such carriers include natural polymers, semi-synthetic polymers, synthetic polymers, liposomes, and semi-solid dosage forms (8, 16, 22, 26, 29, 30, 37). Natural polymers include, for example, proteins and polysaccharides. The semi-synthetic polymer is a modified natural polymer such as chitosan which is a deacetylated form of chitin which is a natural polysaccharide. Synthetic polymers include, for example, polyphosphate esters, polyethylene glycol, poly (lactic acid), polystyrene sulfonate, and poly (lactide coglycolide). Semi-solid dosage forms include, for example, dendrimers, creams, ointments, gels, and lotions. Such carriers can be used to microencapsulate the MAb or covalently bind to the MAb.

  In one embodiment, the MAbs of the present invention comprise carrier particles that can be formulated as powders, sprays, aerosols, creams, gels, etc. for application to the nostril or infection site, or are covalently bound to the outside of the carrier particles. Or it is non-covalent. In one embodiment, the carrier particle core is coated with a soluble film MAb that may contain a mucoadhesive substance. The carrier particle core may be inert or soluble.

  The present invention further includes a delivery system that can deliver an effective amount of MAb to the nostril or other site of infection in a mammal. Representative non-limiting methods include drops, sprays, powders, aerosols, mists, catheters, tubes, syringes; microparticles, pellets, cream applicators and the like. Within the scope of the invention are suitable delivery devices or applicators for the composition, such as catheters, tubes, sprays, syringes, nebulizers, or other applicators for creams, microparticles, pellets, powders, liquids, gels, etc. Also included are kits that include a composition that is combined and contains one or more MAbs of the present invention.

  Finally, the present invention provides a method for treating a patient infected with or suspected of having a gram positive bacterium. This method provides a patient with one or more protective anti-PepG MAbs (including monoclonal antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, fragments, regions, and derivatives thereof) and a pharmaceutically acceptable Administering a therapeutically effective amount of the therapeutic composition comprising a carrier. The patient can be any human or non-human animal in need of prophylaxis or other treatment. Representative patients include humans, ruminants, fur beasts, herds including mice, rats, rabbits, dogs, cats, pigs, sheep, goats, horses, primates, beef cattle, dairy cows, buffalo, camels S. aureus, staphylococci, or non-human animals such as herd animals, laboratory, zoo and farm animals, kennel and barn animals, domestic pets, and domestic animals Included are mammalian subjects who are infected with or are carriers of Gram positive bacteria.

  A therapeutically effective amount is an amount reasonably considered to provide a measurable reduction, support, prevention or prevention effect in the treatment of infection. The treatment described above or below may be a primary treatment or a capture treatment for additional treatments such as Gram positive bacterial infections, infections caused by different pathogens, or antibiotic therapy for unrelated diseases. Combination therapies with other antibodies are specifically included within the scope of the present invention.

  Another embodiment of the invention is a method of preventing or reducing such infection, comprising a protective anti-PepG MAb (including monoclonal, chimeric, humanized, or fragments, including fragments, regions, and derivatives thereof) A method comprising the administration of an effective amount of a therapeutic composition comprising a fully human antibody) and a pharmaceutically acceptable carrier.

  An effective amount may be an amount reasonably considered to prevent or reduce infection to some extent by gram positive bacteria. The treatment described above or below may be used as a primary treatment or as a capture treatment for additional treatments such as staphylococcal infection, infection caused by different Gram-positive bacterial pathogens, or antibiotic therapy for unrelated diseases. Also good. Indeed, combination therapies with other antibodies are specifically included within the scope of the present invention.

  In another embodiment, a peptide that mimics any PepG epitope is useful for preventing Gram positive bacteria from binding to epithelial cells, thereby inhibiting colony formation. For example, a therapeutic composition containing one or more such peptides can be administered intranasally to prevent or suppress colonization and thus prevent or reduce further infection.

  Yet another embodiment of the present invention is a vaccine comprising one or more PepG antigenic epitopes, or one or more peptides that mimic PepG epitopes, in a pharmaceutically acceptable carrier. When introduced into a host, the vaccine elicits antibodies that are broadly protective and opsonic against gram-positive bacteria. A vaccine may comprise an epitope, a peptide that mimics an epitope, a mixture of an epitope and a peptide that mimics an epitope, its antigen, another antigen, or an epitope, a peptide that mimics an epitope, and any combination of antigens. Standard techniques for immunization and subsequent analysis of antibody responses are described in `` Antibodies: A Laboratory Manual '' (Harlow & Lane, 1988), Cold Spring Harbor Laboratory Press; `` Conjugate Vaccines '', (JM Cruse RE Lewis, Jr., 1989), Karger, Basel; US Pat. Nos. 5,955,079 and 6,432,679, each of which is incorporated herein by reference.

  The protective anti-PepG MAbs, vaccines and therapeutic compositions of the present invention are useful for infants and elderly patients, immunocompromised patients, patients undergoing invasive treatment, patients undergoing chemotherapy, radiation It is particularly beneficial for individuals who are known or suspected of being at risk of infection by Gram-positive bacteria, such as patients undergoing therapy and health care workers. This includes infants whose immune system is immature, patients who are receiving biografts such as valves, patients who have indwelling catheters, patients who are refraining from surgical procedures involving damage or damage to the skin or mucosal tissue, It includes health workers and patients who are expected to damage the immune system for some form of treatment, such as chemotherapy or radiation therapy. Among the non-human patients, those at risk include zoo animals, herds, and animals managed in cruel places.

  The MAb of the present invention comprises antibiotics such as mupirocin and bacitracin; antistaphylococcal agents such as lysostaphin, lysozyme, mutanolysin, cellozyl muramidase; antibacterial peptides such as nisin; and lantiobio It may be administered with other anti-staphylococcal antibiotics, including lantibiotics or other lantion-containing molecules such as nisin and subtilin.

In view of the disclosure provided, for the administration of MAbs of the present invention, the particular formulation and delivery method chosen is within the knowledge and experience of one of ordinary skill in the art. In particular, the amount of MAb required, how to combine with an appropriate carrier, dosing schedule, and dosage will vary widely based on standard knowledge in the field without departing from the claimed invention. It may be. In one embodiment, the MAb of the present invention may be administered by intravenous infusion or divided into several doses, the number of times being in the range of 1 to 4 or more times per day, 0.1-20 mg may be given. In one embodiment, the amount of MAb administered is effective for an inoculum consisting of 10 ⁸ S. aureus, the amount of bacteria known to ensure 100% colonization in animal models. Is a known dose, 0.1-3 mg at a time, 2-4 times a day. This regimen is effective for patients who are hospitalized for surgical procedures, patients under various conditions predisposing to staphylococcal infection, those in recovery, and infants with an immature immune system. Or should be effective prior to patient discharge.

  Protective anti-PepG antibodies, vaccines, and therapeutic compositions of the present invention include intravenous, intraperitoneal, intracorporeal injection, intra-articular, intraventricular, intrathecal, intramuscular, subcutaneous, intranasal, transdermal, skin It can be administered internally, intravaginally, orally, or by other effective administration methods. The composition may be administered locally, such as by intramuscular or subcutaneous injection at a specific site of infection. Administration can include direct administration of the therapeutic composition to the patient by application, immersion, dipping, wiping. This procedure may be contaminated by indwelling catheters, heart valves, cerebrospinal fluid shunts, artificial joints, other implants on the body, gram positive bacteria, or may introduce such contamination to patients The present invention can also be applied to an object to be placed in a patient body such as another object, device, or instrument.

  Detailed aspects of the invention will now be presented in the form of the following “Materials and Methods” and specific examples. Of course, these are included for illustrative purposes only and do not limit the invention.

Materials and methods
Bacteria
Type 5 S. aureus has been deposited with the ATCC under accession number 49521.

  Type 8 S. aureus has been deposited with the ATCC under accession number 12605.

  The S. epidermidis strain Hay has been deposited with the ATCC under accession number 55133 on 19 December 1990.

  S. hemolyticus has been deposited with the ATCC under accession number 43252.

Hybridoma Hybridoma 96-105CE11 IF6 (M110) was deposited with the ATCC under accession number HB-12368 on June 13, 1997.

  Hybridoma 99-110 FC12 IE4 was deposited with the ATCC on September 21, 2000 with the Patent Deposit PTA-2492.

  Hybridoma 11-232.3 IE9 (M130) was deposited with the ATCC at PTA-3659 on August 21, 2001.

Isotyping assay Isotypes were determined using a mouse immunoglobulin isotype kit obtained from Zymed Laboratories (Cat. No. 90-6550).

Binding Assay In the binding assay of the present invention, immunoglobulin is incubated with a staphylococcal whole cell preparation or a bacterial cell wall component preparation such as LTA or PepG. The binding assay may be an agglutination assay, a coagulation assay, a colorimetric assay, a fluorescent binding assay, or other suitable binding assay known to those skilled in the art. Particularly suitable assays are enzyme-linked immunosorbent assays (ELISA) or radioimmunoassays (RIA). Binding may be detected directly, but can also be detected indirectly using competitive or non-competitive binding methods known to those skilled in the art.

  While staphylococcal whole cell preparations, LTA preparations, PepG preparations, or combinations of these preparations include plates, wells, beads, microbeads, paddles, propellers, or rods using standard techniques It can be fixed to an appropriate solid support without being limited thereto. The solid support may be composed of, for example, glass or plastic. In certain embodiments of the invention, the solid support is a microtiter plate.

  In general, a binding assay requires the following steps. First, the fixed preparation is incubated with an immunoglobulin source. In one embodiment of the assay, the immunoglobulin source is a biological sample such as, for example, tissue culture supernatant or ascites, plasma, serum, whole blood, body tissue. In another embodiment, the immunoglobulin may be further isolated or purified from its source by means known to those skilled in the art, and the purified or isolated immunoglobulin is used in subsequent assays. The amount of binding is determined by comparing the binding observed in the test sample to the binding of the negative control. A negative control is defined as any sample that does not contain antigen-specific immunoglobulin. In a binding assay, the result is a positive binding reaction when the amount of binding observed in the test sample exceeds the amount of binding in the negative control. Positive binding may be determined from a single positive / negative binding reaction or from an average of a series of binding reactions. A series of binding reactions can include a sample containing a measured amount of immunoglobulin that specifically binds to the immobilized antigen, thereby creating a calibration curve. This calibration curve can be used to quantify the amount of antigen-specific immunoglobulin in an unknown sample.

  In an alternative embodiment of the assay, the antibody is immobilized on a solid support and an unknown immunoglobulin sample is characterized by its ability to bind to a bacterial preparation. Other embodiments of the assay discussed above are utilized as appropriate.

  The specific binding assay used in the examples is described below.

Live Bacteria ELISA (LBE) : An LBE assay was performed to measure the ability of antibodies to bind to live bacteria. Various types of bacteria may be used in the assay, including S. aureus type 5, 5-USU, type 8, Staphylococcus epidermidis strain Hay, and hemolytic streptococci. Bacteria from overnight plate cultures were transferred to 35 ml Tryptic Soy Broth (TSB) and grown at 37 ° C. for 1.5-2.0 hours with gentle shaking. The bacteria were then pelleted by centrifugation at 1800-2000 × g for 15 minutes at room temperature. The supernatant was removed and the bacteria were resuspended in 35-45 ml phosphate buffered saline (PBS / BSA) containing 0.1% bovine serum albumin. Bacteria were pelleted again by centrifugation, the supernatant was discarded, and the bacteria were resuspended in PBS / BSA to achieve a permeability (% T) of 65% to 70% at 650 nm. From that suspension, the bacteria are further diluted 15-fold in 0.9% sterile sodium chloride (Sigma catalog number S8776, or equivalent) and 100 μl of this suspension is added to a series of wells in a flat bottom sterile 96 well plate. It was.

  Each antibody to be tested was PBS / BSA (PBS / BSA / Tween / Prot A-HRP) containing 0.05% Tween-20 and horseradish peroxidase-conjugated protein A (protein A-HRP, Zymed Laboratories) at a 1: 10000 dilution. ) And diluted to the desired concentration. Protein A-HRP is allowed to bind to the antibody for 30-60 minutes at room temperature prior to use, thereby producing an antibody-protein A-HRP complex that is antibody to protein A found on the surface of S. aureus The possibility of nonspecific binding could be minimized. In general, several dilutions of test antibody were used in each assay. From each antibody dilution, 50 μl of antibody-protein A-HRP complex is added to a series of wells, and the bacterial and antibody-protein A-HRP complex mixture is gently rotated on a rotary shaker ( Incubated at 37 ° C. for 30-60 minutes (at 50-75 rpm).

  After incubation, the bacteria were pelleted in the plate by centrifugation at 1800-2000 xg. The well supernatant was carefully removed and 200 μl of PBS / BSA containing 0.05% Tween-20 (PBS / BSA / Tween) was added to all wells to dilute unbound reagent. The bacteria were pelleted again by centrifugation and the supernatant was removed. 100 microliters of TMB substrate (BioFx, Inc. catalog number TMBW-0100-01) was added to each well and the reaction was allowed to proceed for 15 minutes at room temperature. 100 μl of TMB stop reagent (450 nm Stop Reagent, BioFx, Inc. catalog number STPR-0100-01) was added to stop the reaction. The absorbance of each well was determined using a microplate reader equipped with a 450 nm filter.

  In this assay, the intensity of color development was directly proportional to antibody binding to bacteria. Control wells contained bacteria and protein A-HRP without antibody.

Immunoassay with methanol-fixed bacteria : Suspend heat-killed bacteria in 0.9% sterile sodium chloride (Sigma catalog number S8776, or equivalent) at 70-75% permeability (% T) at 650 nm. Made cloudy. Ten milliliters of the bacterial suspension was diluted 15-fold in 0.9% sterile sodium chloride and then pelleted by centrifugation at 1800 xg for 15 minutes at 10-15 ° C. The supernatant was discarded and the pellet was resuspended in 1500 ml methanol (MeOH). 100 microliters of bacteria-MeOH suspension was dispensed into each well of a Nunc Maxisorp strip well (Nunc catalog number 469949). MeOH was evaporated and bacteria were fixed in plastic wells. Bacteria coated strip wells were placed in plastic bags and stored in the dark at room temperature and prepared and used within 2 months.

To evaluate the antibodies, bacteria-coated plates were washed four times with phosphate buffered saline (PBS-T) containing 0.05% Tween20 as follows. About 250 μl of PBS-T was added to each well. The plate buffer was removed by flipping over the sink, and the remaining buffer was removed by inverting the plate and tapping on absorbent paper. The antibody was diluted in PBS-T and then added to the wells. Supernatant, ascites, or purified antibody was tested at the dilution shown in the examples. Control wells received only PBS-T. After the antibody was added, the wells were incubated for 30-60 minutes in a draft-free environment at room temperature. The wells were again washed 4 times with PBS-T. 95 microliters of detection antibody was then added to each well. The detection antibodies are: rabbit anti-mouse IgG ₃ , rabbit anti-mouse IgM, or goat anti-antigen, which binds to horseradish peroxidase (HRP) and is diluted 6000 times in PBS-T. One of human IgG (gamma specific) (Zymed catalog numbers 61-0420, 61-6820, 62-8420, respectively).

  After further incubation at room temperature for 30-60 minutes, the wells were washed 4 times with PBS-T and 100 μl of TMB substrate solution (BioFx # TMBW-0100-01) was placed in each well. The plate was incubated for 15 minutes in the dark at room temperature and the reaction was stopped by adding 100 μl of TMB stop solution (BioFx # STPR-0100-01). The absorbance of each well was measured at 450 nm using a Molecular Devices Vmax plate reader.

Immunoassay using protein A : To assess the binding of MAb to S. aureus, the immunoassay was modified for the methanol-immobilized bacteria described above. Staphylococcus aureus expresses protein A on its surface, and protein A binds strongly to the constant region of the gamma globulin heavy chain, resulting in false positive results for nonspecific binding of antibody to protein A. May occur. Immunoassay wells were coated with bacteria as described above, but to overcome this difficulty, MAb was conjugated to HRP and a solution of recombinant protein A (before adding antibody to the bacteria-coated wells ( Zymed Laboratories catalog number 10-1123) and diluted 8000 times in PBS-T. The binding reaction was allowed to proceed for 30 minutes at room temperature. The wells were washed 4 times with PBS-T and 100 μl of each protein A-HRP-MAb mixture solution was added to the wells. The presence of protein A-HRP obtained by pretreatment prevented MAb from binding to protein A on S. aureus. Furthermore, the binding of protein A-HRP to the heavy chain constant region does not interfere with the antibody binding site on the MAb, thereby allowing the MAb to be evaluated for S. aureus or other bacteria.

  The protein A-HRP-MAb solution was allowed to bind for 30-60 minutes at room temperature in the coated wells. The wells were then washed with PBS-T and TMB substrate solution was added as described above to complete the assay.

Immunoassay with LTA and PepG : Binding of MAb to LTA was measured by immunoassay using wells coated with S. aureus LTA (Sigma catalog number 2515). 100 microliters of a 1 μg / ml LTA solution in PBS was dispensed into a series of Nunc Maxisorp strip wells and incubated overnight at room temperature. Unbound material was removed from the wells by washing 4 times with PBS-T. The antibody diluted in PBS-T was then added to the wells and the assay continued as described in the immunoassay with methanol-immobilized bacteria above.

For immunoassay for PepG, Nunc Maxisorp strip well plates were also prepared by the procedure described in 5 μg / ml PepG solution (S. Foster, Example 2) in 0.1 M carbonate buffer (pH 9.2-9.6). 100 μl) and coated overnight at room temperature. Unbound antigen was removed from the plate by washing 4 times with PBS-T. A series of wells were replicated by adding sample supernatant, ascites, or antibody diluted in PBS-T to the wells. The plate was covered with a plate sealer and incubated for 30-60 minutes in a room temperature, no draft environment. Plates were washed again with PBS-T and 95 μl of γ-specific rabbit anti-mouse IgG (Zymed Laboratories) conjugated to horseradish peroxidase (HRP) was added to all wells. The plate was re-covered and incubated for 30-60 minutes in a windless environment at room temperature. The plate was washed with PBS-T and 100 μl of TMB substrate solution was added to each well. After incubation at room temperature in the dark for 15 minutes, 100 μl of TMB stop solution was added to all wells, and the absorbance of each well was measured using a Molecular Devices V _max plate reader equipped with a 450 nm filter.

Antibodies that bind to activity assay antigens do not necessarily enhance opsonization or enhance protection from infection. Therefore, an opsonization assay was used to determine the functional activity of the antibody.

  Opsonization assays include colorimetric assays, chemiluminescent assays, fluorescent or isotope label incorporation assays, cellular bactericidal assays, or measuring the potential opsonization of a substance, thereby identifying reactive immunoglobulins. Other suitable assays known to those skilled in the art may be used. In an opsonization assay, infectious pathogenic and eukaryotic cells are incubated together with the opsonized substance to be tested or an opsonized substance plus a substance that is thought to enhance opsonization.

  In certain embodiments, the opsonization assay is a cellular bactericidal assay. In this in vitro assay, infectious pathogens such as bacteria, phagocytic cells, and opsonic substances such as immunoglobulins are incubated together. Eukaryotic cells with phagocytic or binding ability may be used for cellular bactericidal assays. In certain embodiments, the phagocytic cell is a macrophage, monocyte, neutrophil, or a combination of these cells. Complement proteins may be included to promote opsonization by both the classical and alternative pathways.

  The amount or number of infectious pathogens remaining after incubation determines the antibody's ability to opsonize. The fewer infectious pathogens that remain after incubation, the greater the opsonic activity of the tested antibody. In cellular bactericidal assays, opsonin activity is measured by comparing the number of viable bacteria in two similar assays, one of which contains the antibody being tested. Alternatively, opsonin activity is determined by measuring the number of living organisms before and after incubation with the sample antibody. A decrease in bacterial count after incubation in the presence of antibody indicates positive opsonization activity. In a cellular bactericidal assay, the incubation mixture is cultured under appropriate bacterial growth conditions to determine that opsonization is positive. If the number of surviving bacteria is reduced by comparing the sample before and after the incubation, or the sample containing the immunoglobulin and the sample not containing it, the reaction is positive.

Neutrophil-mediated opsonophagocytic bactericidal assay : This assay was performed using neutrophils isolated from adult venous blood by sedimentation using PMN separation medium (Robbins Scientific catalog number 1068-00-0). 40 microliters of antibody, serum, or other immunoglobulin source was added at various dilutions to replicate the wells of the round bottom microtiter plate. Then 40 microliters of neutrophils (about 10 ⁶ cells per well) were added to each well, followed immediately by about 3 × 10 ⁴ logarithmically growing bacteria (E. epidermidis strain Hay, ATCC 55133, or 5 10 μl Tryptic Soy Broth (Difco catalog number 906374, or equivalent) with type S. aureus, ATCC 49521) was added. Finally, 10 μl of human serum free of immunoglobulin was added as a source of active complement. (Immunoglobulin was removed from human serum complement by pre-incubating the serum with protein G agarose and protein L agarose prior to use in the assay. In this way, removal of the immunoglobulin removes the anti-antibody in the complement. (The concentration of staphylococcal antibodies is minimized, thereby reducing bacterial killing caused by antibodies originally present in the complement solution.)
Plates were incubated at 37 ° C with constant vigorous shaking. At zero time, ie when sample antibody was first added, and after 2 hours of incubation, 10 μl aliquots were taken from each well. To determine the number of viable bacteria in each aliquot collected from each sample well, each aliquot is diluted 20-fold with 0.1% aqueous BSA (to dissolve the PMN) and mixed vigorously by rapid pipetting. And incubated overnight at 37 ° C. on blood agar plates (Remel, catalog number 01-202, or equivalent). Opsonin activity was measured by comparing the number of bacterial colonies observed from the sample collected at 2 hours with the number of bacterial colonies observed from the sample collected at 0 hour. Colonies were counted using an IPI mini-count colony counter.

  This cellular bactericidal assay has been shown to correlate with in vivo efficacy, as described in Examples 11 and 12 of US Pat. No. 5,571,511.

Nasal Colony Formation Assay : The mouse nasal colonization model of Staphylococcus aureus was based on the work of Kiser et al. (11). Briefly, streptomycin resistant type 5 Staphylococcus aureus is grown on high salinity Columbia agar (Difco) to promote capsule formation. Bacteria are washed with sterile saline (0.9% NaCl aqueous solution) to remove media components and saline (0.9% NaCl aqueous solution) containing various concentrations and combinations of anti-staphylococcal MAbs or irrelevant control MAbs. ) At about 10 ⁸ bacterial / animal dose. After 1 hour preincubation, the bacteria are pelleted and resuspended in either saline or antibody-containing saline, with a final volume of 10 μl per animal dose. Mice raised for 24 hours in water containing streptomycin are sedated by anesthesia. The staphylococci are instilled into the nostril of the mouse by pipetting without touching the nose.

  After 4-7 days while the animals are kept in streptomycin-containing water, the animals are sacrificed and the nose is surgically removed and dissected. The nasal tissue is vortexed vigorously in 0.5% Tween-20-added saline (0.9% NaCl aqueous solution) to release adherent bacteria, and this saline solution is added to Columbia blood agar containing streptomycin (Remel ) And tryptic soy agar (Difco) to determine if there is colony formation.

  The invention described so far will be better understood by reference to the examples. The following examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any way.

Production of hybridomas and monoclonal antibodies against immunization of mice with S. aureus PepG 5-6 week old female BALB obtained from Harlan Sprague Dawley (Indianapolis, IN) to produce monoclonal antibodies against S. aureus PepG Immunization was performed using / c mice. The immunogen for primary immunization was S. aureus PepG (a gift from Mr. Roman Dziarski; PepG can also be prepared as described in Example 2). 5 μl PepG (7 mg / ml suspension) was mixed with 345 μl PBS and 350 μl RIBI adjuvant (RIBI Immunochemicals, Hamilton, NH). The resulting suspension contained 50 μg / ml PepG. Each mouse was immunized by subcutaneous (sc) administration of 0.1 ml (5 μg per mouse).

  A booster immunization was given approximately 4 weeks after the first immunization. PBS (873 μl) was mixed with 7.1 μl PepG (7 mg / ml suspension) and 120 μl Alhydrogel (Accurate Chemical and Scientific, Co., Westbury, NY). 0.1 ml (5 μg PepG per mouse) was administered subcutaneously to each mouse.

After an additional 8 weeks, mice were immunized with a 50 μg / ml PBS solution (0.2 ml / mouse) containing 50% Alum adjuvant (Pierce cat # 77161). Sera from immunized mice were tested by ELISA as described above. As shown in Table 1, mouse 8813 serum bound most strongly to PepG. Three days before hybridoma generation, the mice received 10 μg PepG in PBS intraperitoneally as the last boost before fusion.

Hybridoma generation Hybridomas were prepared by the general method of Shulman, Wilde and Kohler, and Bartal, AH and Hirshaut (2, 28). Spleen cells of mouse 8813 and SP2 / 0 mouse myeloma cells (ATCC number CRL1581) were mixed at a ratio of 10 spleen cells per SP2 / 0 cell and centrifuged (400 × g, 10 minutes at room temperature) And washed in serum free DMEM (Hyclone catalog number SH30081.01, or equivalent). The cell mixture is removed by removing the supernatant and adding 1 ml of a 50% w / v solution of polyethylene glycol (PEG, molecular weight 1500, Boehringer Mannheim catalog number 783641) over a period of 60-90 seconds in a sterile 50 ml centrifuge cone. Completed the fusion. The serum-free medium was then slowly added sequentially in 1, 2, 4, 8, 16, and 19 ml volumes. The hybridoma cell suspension was gently resuspended in medium and the cells pelleted by centrifugation (500 × g, 10 minutes at room temperature). The supernatant was removed and the cells were RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 0.05 mM hypoxanthine, and 16 μM thymidine (HT medium; Life Technologies catalog number 11067-030, or equivalent) Resuspended. 100 μl of suspended hybridoma cells were seeded in 96-well tissue culture plates. In eight wells (row 1 of plate A), 100 μl of a solution containing about 2.5 × 10 ⁴ SP2 / 0 cells was placed. The SP2 / 0 cells were used as a control for killing effect by selective medium added 24 hours later.

  24 hours after preparing the hybridoma, RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 0.1 mM hypoxanthine, 0.8 μM aminopterin, and 32 μM thymidine (HAT medium, Life Technologies catalog number 11067). -030, or equivalent) 100 μl was added to each well.

  96 hours after preparing the hybridoma, SP2 / 0 cells in plate A row 1 were killed, indicating that the HAT selection medium successfully killed unfused SP2 / 0 cells. Twelve days after hybridoma preparation, supernatants from all wells were tested by ELISA for the presence of antibodies bound to PepG.

Based on the results of this preliminary assay, cells from 28 of the original 760 wells were transferred to 24-well culture dishes. Four days later, the supernatants of these cultures were retested by ELISA for the presence of antibodies bound to PepG using isotype specific test reagents. Briefly, the culture supernatant was added to a 96-well plate coated with PepG to allow binding. A series of wells were incubated with HRP-conjugated rabbit anti-mouse IgA, HRP-conjugated rabbit anti-mouse IgG, and HRP-conjugated rabbit anti-mouse IgM to simultaneously detect antibody binding to PepG and antibody isotype. The wells were then washed and developed by standard methods. As shown in Table 2, one of the cultures that produced IgG antibodies (99-110CF10) was further passaged. The other 15 cultures that produced IgM antibodies were also further passaged.

Cultures 99-110CF10 and 99-110FC12 were subcloned by limiting dilution as follows. Hybridomas were counted using a hemocytometer and adjusted to a concentration of 225 cells / ml. The 1 ml cell solution was then added to 36 ml RPMI 1640 medium, 7.5 ml heat-inactivated fetal calf serum, 0.5 ml 10 mg / ml kanamycin solution (GIBCO BRL catalog number 15160-054), and Hybridoma Serum Free medium (Life Technologies catalog number). 12045-084) mixed with 5 ml. The resulting suspension contained 4.5 cells / ml. 200 μl of this suspension was then added to each well of two 96-well culture dishes. As shown in Tables 2 and 3, culture 99-110CF10 did not produce antibodies that bound to PepG. Subsequent subclones of culture 99-110CF10 likewise did not elicit PepG specific antibodies.

The clones from culture 99-110FC12, shown in Table 4, continued to produce IgM antibodies bound to PepG. 32 clones from 99-110FC12 were tested by ELISA on PepG coated plates. Of these, 31 species were strongly positive with absorbances of 3.159 or higher. Four clones designated 99-110FC12 IE4, ID3, IIH5, and IIC6 were grown and stored frozen. Clone IE4 was selected for further analysis.

Clone 99-110FC12 IE4 was grown in an Integra Biosystems culture system designed to produce large amounts of immunoglobulin in the culture supernatant. The supernatants of IE4 clones were tested by ELISA on wells coated with methanol-immobilized Staphylococcus epidermidis strains Hay, PepG, and LTA. As shown in Table 5, this antibody bound strongly to S. aureus PepG but not to methanol-fixed bacteria or S. aureus LTA.

Preparation of hybridoma and monoclonal antibodies against Bacillus subtilis PepG
Purified peptidoglycan B. subtilis HR (a gift from Howard Roger, University of Kent, UK) The vegetative cell wall has been used under stringent conditions using materials that do not contain lipopolysaccharide. (6, 38, which are incorporated herein for any purpose). Proteins were removed from the peptidoglycan by pronase treatment, and teichoic acid and other attached polymers were removed by HF (48% v / v) treatment at 4 ° C. for 24 hours. Insoluble peptidoglycan was pelleted by centrifugation (13,000 g, 5 min at 4 ° C.) and resuspended in distilled water to 2 mg / ml PepG. This step was repeated once. The peptidoglycan was then pelleted and resuspended in 50 mM pH 7.5 Tris HCl to 2 mg / ml PepG, and this step was repeated once. Finally, the peptidoglycan was pelleted three more times and resuspended in distilled water to 2 mg / ml PepG. Peptidoglycan was resuspended in distilled water at about 10 mg / ml and stored at -20 ° C.

  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed no evidence of contaminating proteins when analyzing PepG preparations as previously described (39) . The purity of peptidoglycan was further confirmed by obtaining only the predicted amino acid from the amino acid analysis of hydrolyzed PepG and by analyzing the enzyme digested material by reverse phase chromatography. It was carried out as previously described (1). This level of purity has never been ensured in the production of anti-peptidoglycan antibodies.

Preparation of Muropeptide Conjugates for Immunization Peptidoglycan conjugates were made according to the manufacturer's protocol for Imject SuperCarrier EDC system for peptides (Pierce Cat # 77152).

  Muropeptides were generated by Cellosyl digestion. Cellosyl is a muramidase that cleaves the bond between N-acetylmuramic acid and N-acetylglucosamine in the glycan backbone of PepG. Completely cellosyl digestion of PepG yields a small soluble muropeptide. Collect 1 ml of purified peptidoglycan at 11.5 mg / ml by centrifugation (13,000 g, 5 min, 4 ° C.) and resuspend in 1 ml binding buffer supplemented with Imject SuperCarrier EDC system for peptides (Pierce, catalog number 77152). It became cloudy. 25 μl of cellosyl (2 mg / ml, Hoechst) was added to the PepG suspension and incubated for 7 hours at 37 ° C. with rotary mixing. The sample was then boiled for 10 minutes and insoluble material was removed by centrifugation as described above. 300 μl of cellosyl digested PepG was added to 200 μl of binding buffer.

  Preparation of SuperCarrier (Pierce, catalog number 77152), conjugation, and purification of the conjugate were performed according to the manufacturer's protocol. PepG conjugate was stored at -20 ° C.

Immunization of mice Four 8-12 week old BALB / C mice obtained from Sheffield University Field Laboratories were immunized to produce monoclonal antibodies against Bacillus subtilis PepG. The immunogen for primary immunization was Bacillus subtilis PepG prepared as a muropeptide conjugate (above). For each mouse, 50 μL of PBS containing 50 μg of conjugate was mixed with 50 μl of Freund's complete adjuvant and this mixture was injected subcutaneously.

  Approximately 14 days, 29 days, 113 days, and 232 days after the primary immunization, each mouse was mixed with 50 μl PBS containing 50 μg conjugate with 50 μl Freund's incomplete adjuvant. The placed one was injected intraperitoneally. Hybridomas were made 4 days after the last boost with PepG conjugate.

Hybridoma generation Hybridomas were prepared by the general method of Shulman, Wilde, and Kohler, and Bartal and Hirshaut (2, 28). Spleen cells from immunized mice 1 and 2 are pooled and mixed with SP2 / 0 mouse myeloma cells at a ratio of 5 spleen cells per SP2 / 0 cell and centrifuged (770 × g, 30 At 5 ° C.) and washed in serum-free RPMI. 1 ml of PEG 1500 (50% in 75 mM HEPES, Boehringer catalog number 783641) was added over 1 minute followed by 1 ml RPMI over 1 minute followed by 9 ml RPMI over 2 minutes. The cells were then pelleted by centrifugation at 430 xg for 15 minutes at 30 ° C. Cells at approximately 1 × 10 ⁶ cells / ml in RPMI-1640 / HAT containing 20% FCS (1 ml of 50 × HAT concentrate (Invitrogen catalog number 21060-017) in 50 ml of RPMI-1640) Resuspended. 100 ml of cell suspension was added to each well of 10 96-well plates and grown at 37 ° C. Unfused SP2 / 0 cells were used as a selection control but died 5 days after seeding.

  Thirteen days after hybridoma preparation, supernatants from all wells were assayed by ELISA for the presence of antibodies that bind PepG. Of the 829 wells tested, 13 were positive.

  Based on ELISA results, positive cells were grown in 24-well culture plates and retested by ELISA for stable antibody secretion. Six series were found to secrete antibodies against PepG. Lineage BB4 was found to produce antibodies with the highest affinity for the Bacillus subtilis cell wall. At approximately 75% confluence, BB4 was cloned by limiting dilution and subclones were retested for anti-PepG antibody secretion. Two clones, BB4 / A4 and BB4 / A5, were found to secrete antibodies against Bacillus subtilis PepG. Isotype determination using Isotype Strips (Roche Diagnostics, catalog number 1493027) indicated that both antibodies were IgG.

Substrate affinity of PepG antibody
Cellosyl digestion of PepG
Bacillus subtilis, Staphylococcus aureus, Streptococcus mutans, Bacillus megaterium, Enterococcus faecalis, Staphylococcus epidermidis and epidermidis PepG from monocytogenes (Listeria monocytogenes) was purified as described in Example 2. 1 ml of each PepG (10 mg / ml) was placed in 25 mM pH5.6 sodium phosphate buffer and digested with 250 μg / ml cellosyl (Hoechst AG) at 37 ° C. for 15 hours. Samples were boiled for 3 minutes to stop the reaction and insoluble material was removed by centrifugation (14,000 × g, 8 minutes at room temperature). Soluble cellosyl digested PepG was stored at -20 ° C.

  Staphylococcus PepG has a unique pentaglycine bridge that can be cleaved by lysostaphin, a glycine-glycine endopeptidase. Lysostaphin (25 μg / ml, Sigma catalog number L0761) was added to the cellosyl digest of S. aureus and S. epidermidis PepG to cleave this peptide bridge. S. aureus was digested without lysostaphin.

ELISA to determine the affinity of antibodies for PepG and cellosyl digested PepG
Three hybridoma lines (QED Biosciences) identified as 11-232.3, 11-248.2, and 11569.3 were generated by immunizing mice with UV-inactivated complete S. aureus, and these lines Was shown to bind to PepG. 11-232.3 (when purified, this MAb is referred to as 702PG), 11-248.2, and 11-569.3, BB4 / A4 and BB4 / A5 produced MAbs with different affinities for PepG from various bacteria The affinity for cellosyl digested PepG from various bacteria was compared by ELISA as follows. A 96-well immunoassay plate (NUNC Immunoplate Maxisorp) was added to 0.05 M pH 9.6 carbonate / bicarbonate buffer (0.015 M in 100 μg / ml poly-L-lysine (Sigma Chemicals, catalog number P6407). Na ₂ CO _3, NaHCO of 0.035 M _3, pH 9.6, in the following reference is referred to as carbonate buffer) was coated over 1 hour time at room temperature. The carbonate buffer was removed and the wells were washed once with carbonate buffer. The wells were then coated overnight at 4 ° C. with 100 μl of purified PepG substrate or cellosyl digested PepG substrate in carbonate buffer at 5 μg / ml. The substrate solution was removed and the wells were washed twice with PBS-T. The wells were then blocked for 2 hours at 37 ° C. with 150 μl of PBS-T (Sigma catalog number A7030, blocking buffer) containing 0.2% w / v bovine gelatin. Blocking buffer was removed and the wells were washed 4 times with PBS-T.

50 μl of one of the MAbs listed above (appropriately diluted in addition to blocking buffer) was added to each well and the binding reaction was incubated at 37 ° C. for 2 hours. The monoclonal antibody was removed and the wells were washed 3 times in PBS-T. 50 μl of HRP-conjugated goat anti-mouse IgG (Biorad) diluted 20,000-fold in blocking buffer was added to each well and the binding reaction was incubated at 37 ° C. for 1 hour. The secondary antibody was removed and the wells were washed 3 times with PBS-T. 50 μl of TMB enzyme substrate (Biorad catalog number 172-1068) was added to each well and allowed to develop for 15 minutes at room temperature. The reaction was stopped by adding 50 μl of 2M H ₂ SO ₄ to each well, and the absorbance at 450 nm was read with a VICTOR plate reader (Wallac). The results of this ELISA are shown in Table 6.

Table 6 demonstrates that the described PepG antibodies show different ranges of specificities and affinities for various PepG substrates, so that each is not identical. Specifically, 702PG, MAb-11-232.3, and MAb-11-248.2 have high affinity for S. aureus PepG and low affinity for Bacillus subtilis PepG, whereas BB4 / A4 and BB4 / A5 ( Antibodies produced by MAb-BB4 / A4 and MAb-BB4 / A5) show the opposite specificity. MAb-BB4 / A4 and MAb-BB4 / A5 also showed high affinity for Staphylococcus epidermidis but not for other bacteria.

  Bacillus subtilis 168 HR was awarded by Howard Roger, University of Kent, UK.

  Staphylococcus aureus 8325/4 was a gift from Richard Novick of the Skiball Institute, New York.

  Streptococcus mutans LTII was a gift from Roy Russell of the University of Newcastle.

  Bacillus megaterium KM was awarded by Keith Johnstone of Cambridge University, UK.

  Staphylococcus epidermidis 138 was a gift from Paul Williams of the University of Nottingham.

  Listeria monocytogenes EGD was awarded by W. Goebel from the University of Wurzburg.

  When subjected to cellosyl digestion that cleaves glycan chains, any PepG substrate is less likely to be bound by antibodies 702PG, MAb-11-232.3, MAb-11-248.2, MAb-BB4 / A4, and MAb-BB4 / A5. Thus, these antibodies may interact with epitopes that require complete glycan chains. On the other hand, the affinity of MAb-11-569.3 is unaffected by cellosyl digestion, suggesting that it can interact with epitopes unrelated to the cleaved bond. The results of cellosyl / lysostaphin further ensure the results of a single digestion.

  Finally, S. aureus PepG has a higher level of O-acetylation at its glucosamine residue than PepG from Bacillus subtilis, and this O-acetylation is due to antibodies 702PG, MAb-11-232.3, and MAb- It is important for binding by 11-248.2, suggesting that binding by MAbBB4 / A4 and MAb-BB4 / A5 can have a negative impact.

Binding of monoclonal antibodies to LTA, PepG, and staphylococci
MAb-11-232.3, MAb-11-248.2, MAb-11-569.3, and MAb-99-110FC12 IE4 are S. aureus PepG, S. aureus LTA, methanol-immobilized S. aureus, and methanol-immobilized Whether it binds to Staphylococcus epidermidis was tested. In addition to these MAbs, a human / mouse chimeric anti-LTA antibody designated A110 was included in this assay as a positive control for LTA and Staphylococcus epidermidis binding. A description of the generation and chimerization of A110 can be found in US patent application Ser. No. 09 / 097,055 filed Jun. 15, 1998.

As shown in Table 7, MAb-11-232.3, 11-248.2 ascites, and 99-110FC12IE4 supernatant all bound strongly to S. aureus PepG. As expected, A110, an anti-LTA antibody, does not bind to PepG.

When tested for LTA, only A110 showed strong binding, as shown in Table 8. No binding was obtained with MAb-11-232.3, MAb-11-569.3, and 99-110FC12IE supernatants. Some cross-reactivity was obtained with 100-fold diluted 11-248.2 ascites, which may be due to the presence of high concentrations of nonspecific immunoglobulins in the ascites.

  These data indicated that MAb-11-232.3 and MAb-11-248.2 produced against UV-killed S. aureus were specific for PepG on the bacterial surface. MAb-11-569.3, produced against S. aureus, also killed with UV, may be specific for PepG because it binds much less to PepG and does not bind to LTA, It was shown that it is also specific for another surface antigen and may cross-react with PepG. As expected, MAb-99110FC12IE4 produced against purified S. aureus PepG does not bind to LTA and binds PepG fairly strongly.

  Each MAb was also tested on plates coated with methanol-immobilized S. epidermidis strain Hay and methanol-immobilized S. aureus as shown in Tables 9 and 10, respectively.

All antibodies except MAb-99-110FC12IE4 bound to S. epidermidis strain Hay. Interestingly, MAb-11-569.3 bound more strongly to Staphylococcus epidermidis strain Hay than MAb-11-232.3, but MAb-11-569.3 was as strong as MAb-11-232.3 to PepG and Staphylococcus aureus. Did not combine. This result is due to the fact that the antigen on the surface of S. aureus from which MAb-11569.3 was produced is conserved in S. aureus and S. epidermidis, whether PepG or PepG. MAb-11569.3 suggests that both bacteria are strongly bound. IgM antibodies (derived from hybridomas 11-248.2 and 99-110FC12IE4) were not tested against S. aureus. This is because the immunoassay Protein A method used for S. aureus coated plates does not work with IgM antibodies that do not bind to the protein.

  As mentioned above, peptidoglycan is a cell wall component found in gram positive bacteria. These assays show that MAb-11-232.3, MAb-11-248.2, and MAb-99-110FC12IE4 bind PepG strongly and not to LTA, another cell wall component common to Gram-positive bacteria. Yes. The binding of PepG by MAb-11-569.3 in the ELISA (Table 7) is not as strong as that of type 5 S. aureus in the methanol-immobilized ELISA (Table 10). The difference observed in binding by MAb may be because there is a specific epitope bound by the MAb and that specific epitope is presented in protein and complete bacterial ELISAs. Alternatively, MAb-11-569.3 may bind to a different antigen but cross-react with PepG. MAb-11-232.3, 11-248.2 ascites, and MAb-11-569.3 also bind to S. epidermidis strain Hay in an ELISA assay. Furthermore, MAb-11-232.3 and MAb-11-569.3 also bind to S. aureus in ELISA assays (binding to S. aureus in the 11-248.2 ELISA could not be measured). The absence of binding to the Staphylococcus epidermidis strain Hay by the 99-110FC12IE4 supernatant in ELISA indicates that this antibody binds to an epitope found on S. aureus PepG but is not expressed in S. epidermidis strain Hay, Or suggest that it is not available for binding.

Antibodies that bind to the opsonic phagocytic active antigen of a monoclonal antibody do not necessarily enhance opsonization or enhance protection from infection. Therefore, the functional activity of anti-PepG MAbs against S. aureus and S. epidermidis strain Hay was determined using a neutrophil-mediated bactericidal assay. Neutrophils (PMN) were isolated from adult venous blood using neutrophil (PMN) separation medium (Robbins Scientific catalog number 1068-00-0). To the round bottom wells of the microtiter plate, 40 μl of PMN was added (approximately 2 × 10 ⁶ cells per well) with approximately 3 × 10 ⁴ logarithmically growing bacteria. Human serum treated with protein G and protein L to remove antibodies that bind to S. aureus and S. epidermidis strain Hay was used as the source of active complement. 40 μl of antibody was added to the wells at various dilutions and the plate was incubated at 37 ° C. with constant vigorous shaking. A 10 μl sample was taken from each well after zero and 2 hours of incubation. Each was diluted, vortexed vigorously to disperse the bacteria, placed on a blood agar plate and cultured overnight at 37 ° C. to quantify the number of viable bacteria.

The results show the observed reduction in the number of bacterial colonies as a percentage of the control sample. In the opsonophagocytic bactericidal assay, as shown in Table 11, the 99-110FC12IE4 supernatant was active against type 5 S. aureus but not against S. epidermidis strain Hay.

Mice were immunized with PepG to produce hybridoma 99-110FC12IE4, and mice were immunized with UV inactivated complete S. aureus to produce hybridomas 11-232.3, 11-248.2, and 11-569.3. Produced. The activity of each anti-PepG MAb from the hybridoma line was tested in an opsonophagocytic bactericidal assay. In addition, A110 binding LTA was also included in the assay. MAb produced by 11-232.3 and 11-569.3 are κ light chain antibodies of murine IgG _3, were used after purification. Is a human / mouse chimeric antibody with human IgG ₁ and κ light chain A110 were also used after purification. MAb-99-110FC12IE4 and MAb-11-248.2 are mouse IgM, kappa light chain antibodies and were used as cell culture supernatant (99-110FC12IE4) or ascites (11-248.2). Opsonin studies were performed to determine whether MAbs enhance phagocytosis and killing effects on both groups of staphylococci.

これまでのアッセイでは、A110が由来するマウス・モノクローナル抗体M110によって黄色ブドウ球菌がより強力にオプソニン化されることが示されている(表12Bおよび米国特許出願第09/097,055号)。本発明者らは、A110とM110との間の活性における差が、キメラ抗体と非キメラ抗体間の活性の差というよりも、アッセイにおける用量の影響によるものであると考える。表13に示されるように、A110は、表皮ブドウ球菌に対する活性を失っていない。

表皮ブドウ球菌株Hayを標的生物体として使用したとき、MAb-11-232.3、11-248.2腹水、およびMAb-11-569.3についての結果は、表13に示すように、5型黄色ブドウ球菌で得られた結果と同様であった。300μg/mlでは、MAb-11-232.3およびMAb-11-569.3でそれぞれ、66%および83%が死滅した。ハイブリドーマ11-248.2由来の未希釈の腹水では、95%が死滅した。しかし、99-110FC12 IE4由来の上清では死滅が起こらず、このことは、表9においてメタノール-固定化表皮ブドウ球菌への結合が非常に弱かったことと一致する。最後に、A110では、試験したすべての用量(11.1μg/ml〜300μg/ml)で強力な死滅(>98%)が起こり、抗体なしでPMNと補体を混合して得られた背景死滅は、22%であった。

As shown in Table 12A, each of the anti-PepG antibodies showed enhanced killing action of S. aureus. When PMN was mixed with complement without antibodies, S. aureus killing was less than 20%. However, adding MAb-11-232.3 or MAb-11-569.3 at 100 μg / ml killed 76% and 82%, respectively. Using undiluted ascites from 11-248.2 (mouse IgM) killed 89% and undiluted supernatant from 99-110FC12IE4 (also mouse IgM) killed 75%. Surprisingly, although A110 binds strongly to S. aureus LTA (Table 8) and methanol-fixed S. aureus (Table 10), S. aureus opsonization shown in this assay is very weak .

Previous assays have shown that S. aureus is more strongly opsonized by the mouse monoclonal antibody M110 from which A110 is derived (Table 12B and US Patent Application No. 09 / 097,055). We believe that the difference in activity between A110 and M110 is due to dose effects in the assay rather than the difference in activity between chimeric and non-chimeric antibodies. As shown in Table 13, A110 does not lose activity against S. epidermidis.

When S. epidermidis strain Hay is used as the target organism, results for MAb-11-232.3, 11-248.2 ascites, and MAb-11-569.3 are obtained with type 5 S. aureus, as shown in Table 13. The results were similar. At 300 μg / ml, MAb-11-232.3 and MAb-11-569.3 killed 66% and 83%, respectively. In undiluted ascites from hybridoma 11-248.2, 95% died. However, the supernatant from 99-110FC12 IE4 did not die, which is consistent with the very weak binding to methanol-immobilized Staphylococcus epidermidis in Table 9. Finally, with A110, strong killing (> 98%) occurred at all doses tested (11.1 μg / ml to 300 μg / ml), and background killing obtained by mixing PMN and complement without antibodies is 22%.

  These data indicate that MAb-11-232.3, 11-248.2 ascites, and MAb-11-569.3 can enhance phagocytosis and killing effects on type 5 Staphylococcus aureus and S. epidermidis strain Hay . This data also shows that A110 is not as effective against S. aureus as MAb-11-232.3, 11-248.2 ascites, and MAb-11-569.3, but is more active against S. epidermidis strain Hay. . 99-110FC12IE4 supernatant is active against S. aureus but not against S. epidermidis strain Hay. Although there are some notable exceptions to A110 that bind strongly to methanol-immobilized S. aureus but are weakly opsonizable to live S. aureus, these data indicate binding to methanol-immobilized bacteria. And demonstrate the correlation between the ability to enhance opsonization of such bacteria.

Nasal Colonization Assay Using a mouse staphylococcal nasal colonization model, it was demonstrated that intranasal instillation of MAb-11-232.3 significantly reduced colonization in the nasal cavity.

To ensure that the inhibitory effect of nasal colonization caused by the test MAb was specific for anti-staphylococcal antibodies, the ability of an irrelevant control chimeric IgG to block nasal colonization of staphylococci was examined. The control was Medi 493 (Medimmune, Inc.), a chimeric IgG ₁ MAb against RSV. In the same experiment, the ability of MAb-11-232.3 to block colony formation was also tested.

Streptomycin-resistant type 5 Staphylococcus aureus (SA5, 1-3 × 10 ⁸ bacteria / mouse), physiological saline (0.9% NaCl aqueous solution), MAb-11-232.3-containing physiological saline (13 × 10 ⁸ bacterial mice) Pre-incubated for 1 hour in 2-3 mg purified IgG per dose), or saline containing Medi 493 (2-3 mg purified IgG / 1-3 × 10 ⁸ bacterial mouse dose). After pre-incubation, the bacteria are pelleted and saline (10 μl / mouse dose), saline containing MAb-11-232.3 (10 μl / mouse dose), or saline containing Medi 493 (10 μl / mouse dose) Resuspended. Eight or nine mice were instilled with SA5 in saline, SA5 in MAb-11-232.3, or SA5 in Medi 493, respectively. Seven days later, the mice were sacrificed, the nasal tissue was dissected and seeded on Columbia blood agar containing tryptomycin and trypsinized soy agar to determine if there was colony formation. Table 14 shows that MAb-11-232.3 reduced staphylococcal nasal colonization in mice, whereas Medi 493, an anti-RSV MAb, had no effect.

  Specifically, Table 14 shows that both the number of colonized mice and the number of colonies are antibody-specifically reduced by MAb-11-232.3 specific for anti-S. Aureus surface antigens. In the saline and irrelevant chimeric IgG control groups, all mice had colonization with S. aureus, but in the MAb-11-232.3 group, only 3 out of 8 mice had colonies. It was. This reduction in the number of colonized mice demonstrates that MAb-11-232.3 administered is protective because 5 out of 8 mice escape bacterial colonization . In the MAb-11-232.3 group, the number of colonies recovered per mouse was also dramatically reduced compared to the other two groups. In the saline control group, the average number of colonies of nine mice with colony formation was 70, and in the control group with irrelevant antibody, the average number of colonies of mice with colony formation was 187. In contrast, in the treated group, only 3 out of 8 animals showed evidence of colonization, and the level of colonization was greatly reduced, averaging 8 colonies per mouse nasal cavity . Such a decrease in recovered colonies is also beneficial for prevention in vivo. Therefore, administration of anti-PepG MAb provides protection from nasal colonization by S. aureus. These data show that this effect is specific to the anti-staphylococcal surface antigen MAb and does not fall into the general result that the antibody binds to surface protein A on staphylococci through its Fc portion. It also proves that. Additional MAbs against S. aureus peptidoglycans, MAb-11-248.2 and MAb-11-569.3, will also show a similar inhibitory effect on colonization by S. aureus as described above. Research is underway to affirm the effectiveness of MAb-11-248.2 and MAb-11-569.3 in the in vivo mouse model described above.

Hybridoma 11-232.3 Subcloning of hybridoma 11-232.3 to generate IE9
QED cell culture 11-232.3 was cloned by limiting dilution. Briefly, cells were diluted to a concentration of 225 viable cells per ml. 1 ml of this suspension was added to 36 ml RPMI 1640. The cell suspension was further diluted by adding 7.5 ml FBS, 0.5 ml 10 mg / ml kanamycin solution (Gibco BRL catalog number 15160-054) and 5 ml Hybridoma SFM medium (Gibco BRL catalog number 12045-084). The final volume of the suspension was 50 ml and the cell concentration was 4.5 cells / ml. 200 μl of cell suspension was added to each well of two 96-well tissue culture dishes. In C0 ₂ 5% humidified atmosphere in air and the culture was incubated at 37 ℃ 10 days. The presence of the clone was confirmed by microscopic observation of the foci of the cells that entered the individual wells. Approximately 40% of all wells contained 11-232.3 proliferating clones. All supernatants bound peptidoglycan when tested by ELISA. Four cultures 11232.3-IG9, -IE9, -IH7, and -IB6 were grown and stored frozen. The binding of these four clones to peptidoglycan and LTA is shown in Table 15. The MAb produced by the hybridoma 11-232.3IE9 was subsequently designated M130.

As shown in Table 15, monoclonal antibody M130 produced by hybridoma 11-232.3IE9 bound S. aureus in the LBE assay. Surprisingly, M130 exhibits opsonic activity against S. epidermidis strain Hay (Table 7), but did not bind S. epidermidis strain Hay in this assay. The opsonin assay uses antibodies up to a concentration of 300 μg / ml, but the LBE assay uses up to a concentration of 3 μg / ml, so the difference in concentration used is significant, and therefore the M130 activity in the two assays There may be a difference.

Cloning and sequencing of the M130 variable region
Total RNA was isolated from 2 × 10 ⁶ frozen IE9 (232-3) hybridoma cells using the Midi RNA isolation kit (Qiagen) and following the manufacturer's protocol. RNA was dissolved in 10 mM Tris and 0.1 mM EDTA (pH 8.4) containing 0.25 μg / gl Prime Rnase inhibitor (0.03 U / μg, Sigma).

FIG. 1 shows a strategy for cloning variable region genes. Table 17 shows the oligonucleotide primer sequences (SEQ ID NOs: 5-12) used in the procedure. Superscript II-MMLV reverse transcriptase (Life Technologies), mouse kappa chain specific primer (JSBX-18, SEQ ID NO: 8, Sigma-Genosys), and mouse heavy chain specific primer (JSBX-25A, SEQ ID NO: 9, Sigma- Total RNA (2 μg) was converted to cDNA using Genosys) according to the manufacturer's procedure (see Table 12 for primer sequences). The first strand cDNA synthesis product was purified using a Centricon-30 concentrator (Amicon). Of the 40 μl of the recovered cDNA, 5 μl was used as template DNA for PCR. PCR amplification reaction (50 μl) is template DNA, 30 pmoles of appropriate primers (JSBX-11 A, -12A and -18 for light chain, SEQ ID NOs: 6-8; JSBX-5 and -25A for heavy chain) , SEQ ID NO: 5 and SEQ ID NO: 9), 2.5 units of ExTaq polymerase (PanVera), 1 × ExTaq reaction buffer, 200 μM of each dNTP, 2 mM MgCl ₂ . Initially, the template was denatured by incubation at 96 ° C. for 3 minutes. The product was amplified by repeating a temperature cycle consisting of 96 ° C for 1 minute, 60 ° C for 30 seconds and 72 ° C for 30 seconds 30 times. PCR products from successful reactions were purified using the Nucleospin PCR purification system (Clontech) according to the manufacturer's procedure.

  Then, following the manufacturer's procedure, using a 3: 1 molar ratio of the inserted sequence to the vector used, the PCR product (about 400 base pairs) was transformed into the bacterial vector pGEM T (Promega T ). One half (5 μl) of the ligation reaction was used to transform Ultracompetent XL1 Blue cells (Stratagene) according to the manufacturer's procedure. Diagnostic restriction enzyme digests with DraIII and BsiWI (for heavy chain clones) or DraIII and EcoRV (for light chain clones) (New England Biolabs) were used to identify bacterial clones containing plasmids with DNA inserts. The plasmid containing the appropriate size (approximately 400 bp) insert was then sequenced. The PGEM vector containing the M130 heavy chain variable region is called pJSB18-6, and the pGEM vector containing the M130 light chain variable region is called pJSB4-4. The final common DNA sequences for the light and heavy chain variable regions are shown in FIG. 2 (SEQ ID NO: 2 and SEQ ID NO: 4, respectively).

Cloning of mouse / human chimeric antibody A130 The heavy and light chain variable regions of M130 were then subcloned into a mammalian expression plasmid vector to generate a recombinant mouse / human chimeric antibody molecule under the control of a CMV transcription promoter. . Variable region of the M130 is fused directly to the human IgG ₁ constant region. On the other hand, the light chain of M130 has a mouse κ intron domain 3 ′ of the variable region coding sequence. After splicing, this variable region becomes fused to an exon of the human kappa constant region. A selectable marker for both vectors in mammalian cells is neomycin (G418).

The variable region gene fragments were reamplified by PCR using primers that had the fragments adapted for cloning into expression vectors. (Figure 1, Table 17). The heavy chain forward primer (JSBX-44, SEQ ID NO: 11) contains a 5 'tail encoding the C-terminus of the heavy chain leader and a BsiWI restriction site for cloning, and a heavy chain reverse primer (JSBX- 45, SEQ ID NO: 12) has a 3 ′ EcoRI restriction site for cloning. Thus, two amino acids glutamine (E) and phenylalanine (F) is added between the heavy chain variable region and a human IgG ₁ constant region.

  The light chain forward primer (JSBX-11A, SEQ ID NO: 6) is appended with a 5 ′ tail encoding the two C-terminal amino acids of the light chain leader and an AgeI restriction site for cloning purposes. The light chain reverse primer (JSBX-27, SEQ ID NO: 10) is added with a 3 ′ DNA sequence for the splice junction of the junction region-κ exon, followed by a BstBI restriction site for cloning. ing. PCR was performed as described above, using pJSB18-6 as the heavy chain template and pJSB4-4 as the light chain template. PCR parameters were incubated at 96 ° C. for 3 minutes, followed by 30 temperature cycles of 58 ° C. for 30 seconds, 70 ° C. for 30 seconds, and 96 ° C. for 1 minute.

  The heavy chain PCR product is digested with BsiWI and EcoRI (New England Biolabs), purified using a Nucleospin PCR purification column (Clontech) as per manufacturer's instructions, and manufactured using the Takara ligation kit (Panvera) Ligated with BsiWI / EcoRI / PflMI digested and gel purified pJRS383 as per the procedure of the person. The ligation mixture was then transformed into XL1 Blue cells (Stratagene), clones were selected, and those that were correctly inserted were screened to yield the mammalian vector pJSB22 (FIG. 3).

  The light chain PCR product (approximately 350 base pairs) was digested with AgeI and BStBI (New England Biolabs) and purified using a Nucleospin PCR purification column (Clontech) as described by the manufacturer. This fragment was then ligated to pJRS384 that had been digested with GelI / BstBI / XcmI and gel purified using the Takara ligation kit (Panvera) according to the manufacturer's procedure. The ligation mixture is then transformed into XL1 Blue cells (Stratagene), clones selected and screened for correctly inserted to obtain the mammalian expression plasmid pJSB6.1 (see FIG. 4). It was.

  The two individual plasmids were then combined to create a single expression construct containing both light and heavy chain expression cassettes. Plasmid pJSB6.1 was digested with BamH and NheI (New England Biolabs) and purified using a Nucleospin PCR purification column (Clontech) as described by the manufacturer. Plasmid pJSB22 was digested with BglII and NheI (New England Biolabs), separated on an agarose gel, and a fragment containing the heavy chain expression domain was isolated using a Nucleospin Gel Fragment DNA purification column (Clontech). These fragments were ligated together and transformed into XL1 Blue cells (Stratagene) using the Takara ligation kit (Panvera) according to the manufacturer's procedure. Subsequently, the obtained di-cistronic expression vector pLG1-MEGA was transfected into mammalian cells for producing the A130 chimeric antibody after confirming the sequence of the variable region (FIG. 5).

Transient generation of recombinant mouse / human chimeric A130 antibody
Using Superfect (Oiagen), COS-7 (ATCC number CRL 1651) cells were transfected with plasmid pLG1-MEGA in 6-well tissue culture dishes as described by the manufacturer. Two days later, supernatants were assayed for production of chimeric antibody and the ability of the expressed antibody to bind to S. aureus peptidoglycan antigen as follows.

An 8-well strip (Maxisorp F8, Nunc, Inc.) was coated with goat anti-human Fc (Pierce) diluted 5x in PBS. The plate was then covered with a pressure sensitive film and incubated overnight at 4 ° C. The plates were then washed once with 1x Wash solution (KPL catalog number 50-63-01). 100 μl of each culture supernatant dilution was then added to duplicate wells and incubated for 60 minutes at room temperature on a plate rotating plate. The plate was washed 7 times with Wash solution. Diluted goat anti-human κHRP (Zymed) conjugate sample / conjugate diluent (0.02 M of pH7.4 Tris, NaC1 _{2, 2%} gelatin in 0.25M, Tween-20 0.1%) to 8 million times in addition to did. 100 μl was added to the sample, then placed on a plate rotating plate and incubated for 60 minutes at room temperature. Samples were washed 7 times as above and then incubated with 100 μL / well TMB substrate (BioFx, catalog number TMBW-0100-01) at room temperature for less than 1 minute. Stop the reaction with 100 μL / well of TMB stop reagent (BioFx, catalog number STPR0100-01) and use an automated microtiter plate ELISA reader (Spetramax Plus, Molecular Devices, Inc.) for absorbance values at 450 nm. It was determined. FIG. 6 shows that mouse / human chimeric A130 antibody is bound by goat anti-human IgGκ antibody, and transfection of cells with pLG1-MEGA plasmid produces a molecule containing both human IgG and κ domains It shows that cells are obtained.

  The supernatant was then assayed for the ability of the expressed antibody to bind to peptidoglycan. Using a 5 μg / ml solution of S. aureus peptidoglycan (prepared by the method described in Example 2) in pH 9.5 carbonate coating buffer (0.1 M sodium carbonate), an 8-well strip (Maxisorp F8, Nunc, Inc.) was coated overnight at 4 ° C. The plate was washed once with PBS. 100 μl of each culture supernatant dilution was added to duplicate wells and incubated on a plate rotating plate for 60 minutes at room temperature. The plate was washed 7 times with Wash solution. 100 μl of goat anti-human IgG H + L-HRP (Zymed) diluted 4,000 times in addition to sample / conjugate diluent was added to the sample and the plate was placed on a plate rotating plate and incubated for 60 minutes at room temperature. Samples were washed 7 times with Wash buffer and then incubated for 10-15 minutes at room temperature with 100 μL / well TMB substrate (BioFx) on a plate rotating plate. The reaction was stopped with 100 μL / well of TMB stop reagent (BioFx) and the absorbance value at 450 nm was determined using an automated microtiter plate ELISA reader (Spectramax Plus, Molecular Devices).

  As a positive control, the original mouse monoclonal antibody M130 was used, and assayed using a goat anti-mouse FcHRP conjugate diluted 2000 times. FIG. 7 shows that transfection of the pLG1 MEGA plasmid into the cells yields cells that produce molecules that bind to S. aureus peptidoglycan. These results suggest that the mouse / human chimeric antibody A130 retains the peptidoglycan binding ability of the mouse monoclonal antibody M130 from which it is derived.

Specific binding of monoclonal antibody to PepG A sandwich assay was performed to confirm that the monoclonal antibody was specific to PepG and did not bind to contaminants in the PepG preparation used in the ELISA assay shown in Table 7. . Briefly, a multiwell plate is coated with a “capture” antibody specific for PepG, LTA, or an unknown antigen, then PepG is added to the well, bound by the antibody, and then the “detection” antibody And the affinity for PepG captured by the capture antibody was measured.

  Specifically, monoclonal antibodies M110, M130, MAb-11-230.3, MAb-11-232.3, MAb-11-391.4, MAb-11-557.3, MAb-11-564.4, MAb-11-580.5, and MAb-11 -586.3 was diluted to 3 μg / ml in PBS. Hybridomas 11-230.3, 11-232.3, 11-391.4, 11-557.3, 11-564.4, 11-580.5, and 11-586.3 were obtained from mice immunized with UV-killed S. aureus. As mentioned above, MAb-11-230.3, MAb-11-232.3, MAb-11-557.3, MAb-11-564.4, and MAb-11-586.3 bind to PepG, MAb11-391.4 binds to LTA, MAb-11-580.5 has been previously known to bind to an unknown epitope on S. aureus. These MAbs were used to coat four columns of Nunc Maxisorp strip wells (Nunc catalog number 469949), which are referred to as “capture” antibodies. Coating with each capture antibody was performed by adding 100 μl of a 3 μg / ml solution to the appropriate wells and incubating overnight (18-26 hours) at room temperature. The unbound material was then removed from the wells by washing 4 times with PBS-T. For each capture antibody, S. aureus peptidoglycan (prepared by S. Foster as described in Example 2) diluted to 10 μg / ml in PBS-T was added to 2 wells in 4 rows. PBS-T without peptidoglycan was added to another two rows of wells.

  Wells were incubated at room temperature for 30-60 minutes and washed 4 times with PBS-T. Next, 100 μl of A130 at the concentration shown in Table 17 (2-fold dilution from 5 μg / ml to 0.078 μg / ml) was placed in the well. The anti-PepG monoclonal antibody A130 described above is a “detection” antibody and was diluted in addition to PBS-T (Axell Cat BYA20341, Lot H2415) containing 0.1% human serum and no IgG, IgA, and IgM. This detection antibody was expected to react with any PepG bound to the capture MAb used for coating. At the same time, PepG should not be captured in wells coated with LTA-specific MAbs M110 and 391.4, or with MAb-11-580.5, an MAb of unknown specificity. Expected.

  After further incubation for 30-60 minutes, the wells were washed with PBS-T and each well was diluted 6000 times with HRP-conjugated γ-specific goat anti-human IgG (Zymed Cat 62-8420) in PBS-T. 95 μl was added. After 30-60 minutes, the wells were washed with PBS-T and 100 μl of TMB substrate solution (BioFX Cat TMBW-0100-01) was added to each well. The reaction was allowed to proceed for 15 minutes in the dark at room temperature, and 100 μl of TMB stop reagent (BioFX Cat STPR-0100-01) was added to each well to stop the reaction. The absorbance of each well was measured using a Molecular Devices Vmax microplate reader equipped with a 450 nm filter.

Table 18 shows the results of a capture assay using a series of capture MAbs followed by PepG and then using A130 as the detection MAb. After the wells were coated with MAb-11-230.2, MAb-11-232.3, MAb-11-557.3, MAb-11-564.4, or MAb-11-586.3 and incubated with PepG, detection antibody A130 was Bind to captured PepG. When capturing using an anti-LTA antibody such as that produced by hybridoma 391.4 or M110, A130 does not bind, probably because the capture antibody does not bind PepG. Furthermore, MAb-11-580.5, whose specificity is unknown, does not capture PepG as much, as evidenced by insufficient binding by A130. As a positive control, when Mep having the same variable region as A130 is used to capture PepG, A130 binds strongly to the plate. These results show that when PepG is captured by a series of different antibodies that are thought to bind to PepG, A130 binds to the captured material, so A130 binds to PepG and binds to contaminants in the PepG preparation. Suggests not to.

  To confirm that the anti-PepG antibody does not bind to possible LTA contaminants in the PepG preparation, use A110 binding to LTA as the capture antibody, bind LTA to the capture antibody, and then add the detection antibody A similar sandwich assay was performed in which binding to captured LTA was measured.

  Nunc Maxisorp strip wells (Nunc catalog number 469949) were coated with 100 μl of 3 μg / ml A110 in PBS and incubated overnight (18-26 hours) at room temperature. After overnight incubation, unbound material was removed from the wells by washing 4 times with PBS-T. Replicate wells then received 100 μl of LTA solution (Sigma catalog number 2515, PBS-T diluted to 1 μg / ml) or PBS-T alone. The wells were then incubated for 30-60 minutes at room temperature and washed 4 times with PBS-T.

  Monoclonal antibody titrated to 2-fold dilution from 5 μg / ml to 0.078 μg / ml in PBS-T (Axell Cat BYA20341, Lot H2415) with 0.1% human serum and no IgG, IgA, and IgM Selected from M110, M130, MAb-11-230.3, MAb-11-232.3, MAb-11-391.4, MAb-11-557.3, MAb-11-564.4, MAb-11-580.5, and MAb-11-586.3 100 μl of antibody was placed in the well.

  Each Mab was titrated in 4 columns with LTA in 2 rows and PBS-T alone in the other 2 rows. These mouse MAbs were used as detection antibodies similar to the A130 detection antibody used in Table 18.

  After further incubation for 30-60 minutes, the wells were washed again with PBS-T and each well contained HRP-conjugated γ-specific anti-mouse IgG (Jackson Immunoresearch Cat 115-035-) diluted 10,000-fold in PBS-T. 164) 95 μl was added. After 30-60 minutes, the wells were washed with PBS-T, and 100 μl of TMB substrate solution (BioFX Cat TMBW-0100-01) was added to each well. The reaction was allowed to proceed for 15 minutes in the dark at room temperature, and 100 μl of TMB stop reagent (BioFX Cat STPR-0100-01) was added to each well to stop the reaction. The absorbance of each well was measured using a Molecular Devices Vmax microplate reader equipped with a 450 nm filter.

Table 19 shows the results of a sandwich assay using A110 as a capture antibody followed by LTA and then using a series of detection MAbs. This assay confirms that antibodies that are thought to bind to PepG do not actually bind to possible LTA contaminants in the PepG preparation. A110 was used as a capture antibody to capture LTA on the plate. MAb-11-230.2, MAb-11-232.3, MAb-11-557.3, MAb-11-564.4, MAb-11-586.3, and M130 all bind to PepG but bind to LTA captured on the plate do not do. As expected, anti-LTA antibodies, including those produced by hybridoma 391.4 and M110, bind strongly to captured LTA. Finally, MAb-11-580.5 of unknown specificity does not bind to captured LTA. These results further confirm that anti-PepG monoclonal antibody containing M130 does not bind to LTA.

  Finally, to further confirm that M110 does not capture contaminants in the PepG preparation recognized by M130, the following sandwich assay was performed. M110 was bound to the plate as a capture antibody, and then either LTA or PepG was bound to the plate followed by either A110 or A130 as the detection antibody.

  Nunc Maxisorp strip wells (Nunc catalog number 469949) were coated with 100 μl of 3 μg / ml M110 and incubated overnight (18-26 hours) at room temperature. Unbound material was removed from the wells by washing 4 times with PBS-T. Next, 100 μl / well of LTA solution (Sigma catalog number 2515, diluted in PBS-T and diluted to 1 μg / ml) was placed in 4 columns of wells and 100 μl / well of PepG solution (10 μg / well in PBS-T). ml) was placed in four vertical wells, and only PBS-T was placed in four vertical wells. The wells were incubated for 30-60 minutes at room temperature and then washed 4 times with PBS-T. 5 μg / ml in PBS-T (Axell Cat BYA20341, Lot H2415) with 0.1% human serum and no IgG, IgA, and IgM in each two rows of LTA, PepG, and PBS-t 100 μl of A130 titrated to a 2-fold dilution from 1 to 0.078 μg / ml. A110, similarly diluted and titrated, was placed in two rows of LTA binding, PepG binding, and PBS-t, respectively. These chimeric antibodies were used as “detection” antibodies similar to the A130 detection antibody used in Table 18.

  After incubating for 30-60 minutes, the wells were washed with PBS-T, and each well received 95 μl of HRP-conjugated γ-specific goat anti-human IgG (Zymed Cat 62-8420) diluted 6000 times in PBS-T. added. After 30-60 minutes, the wells were washed with PBS-T, and 100 μl of TMB substrate solution (BioFX Cat TMBW-0100-01) was added to each well. The reaction was allowed to proceed for 15 minutes in the dark at room temperature, and 100 μl of TMB stop reagent (BioFX Cat STPR-0100-01) was added to each well to stop the reaction. The absorbance of each well was measured using a Molecular Devices Vmax microplate reader equipped with a 450 nm filter.

Table 20 shows the results of a sandwich assay using M110 as the capture antibody followed by PepG or LTA and then A110 or A130 as the detection antibody. These results again demonstrate that M110 does not capture an antigen that can be bound by A130. Furthermore, capturing PepG preparations with anti-LTA M110 antibody does not capture enough LTA to show binding above the background level of A110, so this result indicates that the PepG preparation used in these assays is Is not included at such a high level.

Human antibodies that bind PepG Rather than humanizing mouse antibodies to minimize the HAMA response during treatment as described above, those skilled in the art will isolate protective anti-PepG antibodies that are fully human. can do. Some of the alternative strategies that can be used by those skilled in the art to generate fully human recombinant antibodies are well known. One is the production of antibodies using phage display technology (50, 54). Specifically, human RNA is used to create a cDNA library of antibody heavy and light chain fragments expressed on the surface of bacteriophage. Such a library can be used to search for the antigen of interest (ie, PepG) and then isolate the phage that bind because the antibody is expressed on the surface. The sequence of the DNA encoding the variable region is determined, and cloning is performed to express the antibody.

  Another method of generating human antibodies utilizes “humanized” mice. Such transgenic mice produce human antibodies when inoculated with an antigen (48, 50, 51, 52, 54) because their antibody genes have replaced part of the human antibody gene complex. The resulting antibody producing cells can then be incorporated into standard hybridoma technology to establish cell lines that produce specific monoclonal antibodies.

  Recombinant human antibodies can also be generated by isolating antibody producing B cells from human volunteers with strong anti-PepG responses. Using fluorescence activated cell sorting (FACS) and fluorescently labeled PepG, cells that produce anti-PepG antibodies can be separated from other cells. RNA can then be extracted and the variable region of the reactive antibody can be sequenced (49, 53). The functional variable region DNA sequence may be synthesized or cloned into a mammalian expression vector for large-scale production of recombinant human antibodies.

Conclusion Monoclonal antibodies were raised in mice against S. aureus PepG, an abundant cell surface molecule on Gram-positive bacteria. One hybridoma clone, 99-110FC12IE4, binds strongly to PepG in an ELISA assay, but produces IgM antibodies that do not bind to LTA, another surface molecule common to S. epidermidis strain Hay, or Gram-positive bacteria ( Example 1, Table 5).

  Monoclonal antibodies were also raised against Bacillus subtilis PepG. Hybridomas BB4 / A4 and BB4 / A5 produce IgG antibodies that bind to Bacillus subtilis PepG (Example 2). PepG from several different bacteria for the affinity of monoclonal antibodies produced by BB4 / A4, BB4 / A5, 11-232.3, 11-248.2, 11-569.3, and antibody 702PG purified from hybridoma 11-232.3 Binding to was tested in an ELISA assay. MAb-BB4 / A4 and MAb-BB4 / A5 produced from the same mouse bind strongly to PepG from Bacillus subtilis and Staphylococcus epidermidis, while MAb-11-232.3 and MAb-11-248.2 are PepG Staphylococcus aureus (Example 3, Table 6). These results indicate that a monoclonal antibody against PepG from one gram-positive bacterium can bind to PepG from another gram-positive bacterium, which indicates binding to a conserved epitope on PepG. Maybe it shows.

  An ELISA assay tested whether A110, MAb-11-232.3, MAb-11-248.2, MAb-99-110FC12IE4, and MAb-11-569.3 bind to S. aureus PepG. MAb-11-232.3, MAb-11-248.2, and MAb-99-110FC12IE4 produced against either UV-killed S. aureus or S. aureus PepG are highly resistant to S. aureus PepG. Combined. MAb-11-569.3, which was also produced against UV-killed S. aureus, has weak binding to PepG, and this antibody may bind to non-PepG epitopes on the surface of S. aureus Was suggested. A110, an anti-LTA antibody, did not bind to S. aureus PepG (Example 4, Table 7). In a similar assay measuring the binding of antibody to S. aureus LTA, only A110 showed significant binding (Example 4, Table 8). These results demonstrate that antibodies raised against UV killed S. aureus may indeed be specific for PepG on the surface of S. aureus.

  Each antibody was then tested to bind to methanol-immobilized Staphylococcus epidermidis strain Hay. A110 produced against S. epidermidis LTA bound most strongly to methanol-immobilized Staphylococcus epidermidis. MAb-11-569.3 and MAb-11-248.2 also bound strongly to methanol-immobilized Staphylococcus epidermidis despite being produced against UV killed S. aureus. This suggests that these antibodies may bind to a conserved epitope on the surface of two cell lines. MAb-11-232.3, which was also produced against UV killed Staphylococcus aureus, did not bind very strongly to methanol-immobilized Staphylococcus epidermidis. Similarly, MAb99-110FC12IE4 produced against S. aureus PepG did not bind to methanol-immobilized S. epidermidis (Example 4, Table 9).

  Finally, it was tested whether A110, MAb-11-232.3, and MAb-11-569.3 bind to methanol-immobilized S. aureus. Not surprisingly, MAb-11-232.3 and MAb-11-569.3 bound strongly because they were produced against UV-killed S. aureus. Although produced against heat-killed Staphylococcus epidermidis, A110 also binds to methanol-immobilized S. aureus, indicating that this antibody can bind to a conserved epitope between the two bacteria. (Example 4, Table 10).

  The opsonin activity against Staphylococcus aureus and Staphylococcus epidermidis was tested in the supernatant of hybridoma 99-110FC12IE4 in the presence of complement and PMN from human serum from which antibodies against S. aureus and S. epidermidis were removed. MAb-99-110FC12IE4 was opsonic against S. aureus but not opsonic against S. epidermidis (Example 5, Table 11). As discussed in previous paragraphs, MAb-99-110FC12IE4 bound to Staphylococcus aureus PepG, but not to complete S. epidermidis, suggesting a correlation between binding ability and opsonin activity.

  MAb-11-232.2, MAb-11-569.3, MAb-11-248.2 produced against UV-killed S. aureus, MAb-99-110FC12IE4 produced against purified S. aureus PepG, respectively, and A110 Were tested in a similar opsonophagocytic assay against type 5 S. aureus. Antibodies MAb-11-232.2, MAb-11-569.3, and MAb-11-248.2 produced against UV-killed Staphylococcus aureus, and MAb-110FC12IE4 produced against S. aureus PepG were used in this assay. At least 75% of S. aureus was killed. A110, an anti-LTA antibody raised against LTA from Staphylococcus epidermidis, showed only 23% killing (Example 5, Table 12). Surprisingly, A110 bound strongly to both S. aureus LTA and methanol-immobilized complete S. aureus, but was not highly opsonic to S. aureus. M110, a non-chimeric form of A110, was slightly more opsonic to S. aureus in the above assay (Example 5, Table 12B). However, this variability appears to be due to inter-assay differences and dose effects rather than differences in activity between chimeric and non-chimeric antibodies since A110 does not lose activity against S. epidermidis. (Example 5, Table 13). On the other hand, antibodies raised against S. aureus PepG and against UV killed S. aureus showed strong opsonin activity against the bacteria as expected.

  In a similar assay for S. epidermidis strain Hay, MAb-11-232.2, MAb-11-569.3, and MAb-11-248.2 showed at least 66% killing, while MAb-99-110FC12IE4 was almost or It did not show any death. A110 killed at least 98% of S. epidermidis strain Hay, in contrast to the previous assay (Example 5, Table 13). These results and those discussed above show a strong correlation between the ability to bind to methanol-immobilized bacteria and the opsonin activity against that bacteria. However, A110 is an exception to note because it can bind to methanol-immobilized S. aureus but is not opsonic to live S. aureus. In addition, these results demonstrate that monoclonal antibodies raised against UV-killed Staphylococcus aureus can exhibit opsonin activity against S. epidermidis, while the MAb produced against one is Suggests the existence of a conserved determinant between the two bacteria that makes them opsonic. These results are weak for opsonin activity since MAb-11.232.3 was still slightly opsonic to this bacterium, despite weak binding to Staphylococcus epidermidis in methanol-immobilized bacterial ELISA It also shows that a bond may be sufficient.

  The ability of MAb-11-232.3 to block colonization in the nasal cavity was tested in mice. In the control group, 9 out of 9 mice were colonized by Staphylococcus aureus, whereas after preincubating type 5 S. aureus with MAb-11-232.3, only 3 out of 8 mice There was colonization of Staphylococcus aureus. Furthermore, among the mice with colony formation, the number of bacterial colonies of mice that received S. aureus preincubated with MAb-11232.3 was 1/10 that of control mice (Example 6, Table 14). . These results indicate that MAb-11-232.3, which binds to and is opsonic to S. aureus, can block bacterial colonization in the nasal cavity and reduce the number of bacteria in colonized mice. It suggests.

  Hybridoma 11-232.3 was subcloned and antibody M130 produced by subclone 11-232.3IE9 was further analyzed. A live bacterial ELISA assay was used to test whether M130 binds to several different bacteria. M130 bound to three different strains of live S. aureus but not hemolytic streptococci or S. epidermidis (Example 7, Table 16). These results are consistent with a methanol-immobilized bacterial ELISA in which MAb-11-232.3 bound strongly to S. aureus but only weakly to S. epidermidis. Therefore, although MAb-11.232.3 and M130 are specific for Staphylococcus aureus, MAb-11-232.3 is slightly opsonic to Staphylococcus epidermidis, although its binding strength is inferior. As such, it has a broad reactivity against various bacterial strains.

  The variable region of M130 was cloned to produce a human / mouse chimeric antibody having an M130 variable region and a human constant region (Examples 8 and 9). Such a chimeric antibody designated A130 retained the ability to bind to S. aureus PepG (Example 10, FIG. 7). Such human / mouse chimeric antibodies are expected to have a reduced HAMA response in humans and may be therapeutically advantageous.

  Finally, a sandwich assay was performed to confirm that the anti-PepG antibodies of the present invention are indeed specific for PepG and do not bind to contaminants in the PepG preparation. In the first assay, a series of antibodies was used to capture PepG on the plate, and then A130 was used as a detection antibody to detect the captured PepG (Example 11, Table 18). As expected, antibodies known to bind to PepG were able to capture PepG, as detected by binding of the A130 antibody. Antibodies that bind to LTA did not capture the antigen that could be detected by A130, suggesting that A130 does not bind to LTA. In the second assay, anti-LTA A110 antibody was used to capture LTA on the plate, which was then detected with the same series of antibodies used for capture in the first assay (Example 11, Table 1). 19). As expected, anti-LTA antibody was able to detect LTA captured by A110. On the other hand, the anti-PepG antibody could not detect the antigen captured by A110, suggesting that this type of antibody does not bind to LTA. Finally, M110 was used to capture either LTA or PepG on the plate, and then the captured antibody was detected with A110 or A130 (Example 11, Table 20). As expected, M110 failed to capture antigens that were detectable by A130 from PepG or LTA preparations. However, M110 was able to capture LTA that was detectable by A110. Importantly, M110 was unable to capture LTA from PepG preparations enough to be measurablely detected by A110, suggesting that PepG preparations contain very little LTA. It was. This level of purity of the PepG preparation has not been demonstrated to date.

  So far, the polyclonal sera used contain many different antibodies that bind to many different epitopes on the bacterial surface, and the sum of the combined binding and activity may be the activity of the whole serum. As such, it was unclear whether monoclonal antibodies could enhance the phagocytosis of Gram-positive bacteria. Thus, the inventors have demonstrated that a monoclonal antibody that binds to a single epitope on the surface of a bacterium can be opsonized against that bacterium. We have also demonstrated that monoclonal antibodies against PepG can have such activity, and that this type of antibody can be opsonized against several different types of Gram-positive bacteria.

  The antibodies of the present invention can prevent or reduce nasal colonization. Thus, the antibodies of the present invention can be useful protective molecules in combating antibiotic-resistant gram positive bacterial infections.

上記のとおり本発明を完全に説明したが、当業者ならば、本発明の思想および範囲から逸脱することなく、過度の実験を要することもなく、本発明を均等および条件の範囲内で実施できることがわかるであろう。また特定の実施形態および実施例に照らして本発明を説明してきたが、本発明者らは、それらはさらに変更可能なものであると考える。本出願は、上述の一般原理に従う本発明の変形、使用または改変にまで及ぶことを意図している。 References

Although the present invention has been described completely as described above, those skilled in the art can implement the present invention within the scope of equality and conditions without departing from the spirit and scope of the present invention and without undue experimentation. You will understand. Also, although the present invention has been described in the context of particular embodiments and examples, the inventors believe that they can be further modified. This application is intended to cover any variations, uses, or modifications of the invention according to the general principles set forth above.

  This specification includes a list of references, which are specifically incorporated herein by reference.

FIG. 2 shows the cDNA cloning strategy for the heavy chain variable region and light chain variable region of M130. It is a figure which shows the polypeptide sequence and nucleic acid sequence of (A) M130 antibody light chain variable region (sequence number 1 and sequence number 2) and (B) M130 antibody heavy chain variable region (sequence number 3 and sequence number 4). (A) shows the polypeptide and nucleic acid sequences of the M130 antibody light chain variable region (SEQ ID NO: 1 and SEQ ID NO: 2) and (B) M130 antibody heavy chain variable region (SEQ ID NO: 3 and SEQ ID NO: 4) ( (Continued in Figure 2-1.) It is a figure which shows pJSB22 heavy chain expression plasmid. It is a figure which shows the pJSB6 light chain expression plasmid. FIG. 2 shows a pLG1 bicistronic expression plasmid. It is a figure which shows the coupling | bonding of the anti-human IgG to the mouse / human chimeric antibody A130. It is a figure which shows the binding activity with respect to S. aureus peptidoglycan of the mouse / human chimeric antibody A130.

[Sequence Listing]

Claims (22)

  A medicament comprising a therapeutically effective amount of at least one MAb that binds to peptidoglycan (PepG) of gram positive bacteria, wherein the MAb produces a therapeutically beneficial result upon administration to a patient.   The medicament according to claim 1, wherein the administration reduces the number of Gram-positive bacteria in the patient.   The medicament according to claim 1, wherein the at least one MAb binds the Gram positive bacterium PepG at a level at least twice as large as the background in ELISA.   2. The medicament of claim 1, wherein the at least one MAb enhances opsonophagocytosis on Gram positive bacteria by at least 50%.   The medicament according to claim 1, further comprising a pharmaceutically acceptable carrier.   The medicament according to claim 1, further comprising at least one anti-staphylococcal drug.   2. The medicament of claim 1, further comprising at least one MAb that binds to lipoteichoic acid (LTA) of gram positive bacteria.   The medicament according to claim 1, wherein the at least one MAb prevents colonization by Gram positive bacteria upon instillation into the patient's nostril.   At least one MAb is Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Bacillus subtilis, Bacillus megaterium, Enterococcus faecalis The medicament according to claim 1, which specifically binds to PepG of a Gram-positive bacterium selected from Enterococcus faecalis) and Listeria monocytogenes.   The at least one MAb is selected from MAb-11-232.3, MAb-11-248.2, MAb-11-569.3, MAb-11-232.3IE9, MAb99-110FC12IE4, A130, and M130. Medicine.   The medicament of claim 1, wherein the at least one MAb comprises a variable region comprising a polypeptide having an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 3.   The medicament according to claim 1, wherein the at least one MAb comprises a variable region comprising a polypeptide having at least 80% identity with an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 3.   The medicament according to claim 1, wherein the at least one MAb is selected from a chimeric MAb, a humanized MAb, and a human MAb.   2. The medicament of claim 1, wherein at least one MAb comprises a modified Fc portion, wherein the modification reduces non-specific binding of the Mab through the Fc portion. The medicament according to claim 1, wherein the at least one MAb is selected from Fab, Fab ', F (ab') ₂ , Fv, SFv, and scFv.   A method for treating a patient, comprising administering to the patient a therapeutically effective amount of the medicament according to any one of claims 1 to 15.   The patient is selected from hospitalized infants, premature babies, burn victims, elderly patients, immunocompromised patients, immunosuppressed patients, patients undergoing invasive procedures, and health care workers The method according to claim 16.   The protective monoclonal antibody is administered by a route selected from intravenous, intraperitoneal, intracorporeal injection, intra-articular, intraventricular, intrathecal, intramuscular, subcutaneous, intranasal, intravaginal, and oral. The method according to 1.   A hybridoma cell line deposited with ATCC under accession number PTA-2492.   A hybridoma cell line deposited with ATCC under accession number PTA-3659.   A vaccine comprising at least one purified PepG, peptide, fragment thereof and epitope in a pharmaceutically acceptable carrier.   23. A method for treating a patient, comprising administering a therapeutically effective amount of the vaccine of claim 21.

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