JP2002541071A - Antibodies to phosphomannans, protective against candidiasis - Google Patents

Abstract

Abstract: Disclosed are antibodies that provide protection from candidiasis, particularly antibodies of the IgG class and antibodies that specifically bind to carbohydrate antigens on the cell wall of yeast from the genus Candida. Also disclosed are pharmaceutical compositions and therapeutics useful in the treatment of candidiasis, and diagnostics.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to an IgG class antibody that protects a host from candidiasis, and more particularly, to an antibody that specifically binds to a carbohydrate antigen on the cell wall of yeast derived from Candida. The present invention further relates to pharmaceutical compositions and therapies useful in the treatment of candidiasis, and diagnostic methods useful in diagnosing candidiasis and monitoring the course of treatment of candidiasis.

[0002] Acknowledgment to the Federal Government [0002] The disclosed invention is supported by the National Institute of Allergy and Infectious Diseases grant RO1AI2491.
2 and PO1AI37194. The United States Government has certain rights in this invention.

[0003]

[Prior art]

Candida yeast can cause a variety of clinical syndromes, commonly referred to as candidiasis, usually classified by the affected physiological site. The two most common syndromes include mucocutaneous candidiasis (eg, stomatitis or thrush, esophagitis and vaginitis) and invasive or deep organ candidiasis (eg, mycemia, endocarditis, and ophthalmia) Enditis). These syndromes include Dismukes, Ca
ndidiasis (Cecil's Textbook of Medici
Ne, 1827-1830, Bennett et al., 1996).

[0004] Patients suffering from mucocutaneous infections can be treated with any of several topical preparations, including nystatin, clotrimazole, econazole, ketoconazole, butoconazole, terconazole and miconazole (Id.). When treating more clinically severe Candida-related diseases (eg, candidemia or disseminated candidiasis), amphotericin B formulations (both deoxycholate-type and newer liposomal formulations) are administered intravenously. Administered only within. Fluconazole is administered orally. Treatment for Candida peritonitis is either intravenous amphotericin B or oral gluconazole (Id.).

[0005] Also, the medical literature has some C.I. Various classes of antibodies to the Albicans antigen (IgA, IgG and IgM) have received experimental attention and have been reported to be of diagnostic or therapeutic importance. For example, C.I. Torres-Rodriguez et al., 1997, Mycoses, 40: 4 for Albicans embryonic tubes.
39-44; Reboli, 1993, J.A. for dot immunobinding assays involving all Candida proteins. Clin. Microbiol. 31: 518
-23. A monoclonal antibody specific for the anti-C3b receptor, an integrin with antigenic and structural homology to Candida surface antigens, has been demonstrated to increase the survival of mice with disseminated candidiasis (Lee Et al., 19
97, Immunology, 92: 104-110). Similarly, antibodies to mannoprotein (MP) and aspartyl proteinase (Sap) have been shown to provide protection from vaginitis in rats (De Ber
Nardis et al., 1997, Infect. Immun. 65: 3399-34
05).

US Pat. Nos. 4,670,382 and 4,806,465 (both Bu)
ckley et al., 1989) include C.I. Although IgG monoclonal antibodies against a set of closely related cytoplasmic antigens of Albicans have been described, no therapeutic data showing efficacy against Candida infection is given. US Patent No. 5,288,6
No. 39 (Burnie et al., 1994) describes a monoclonal antibody against Candida stress or heat shock proteins, which is described in albicans at a dose of 3 in 24 hours in inoculated animals.
It was shown to have resulted in 3% survival. No. 5,641,760.
No. (Yu et al., 1997) include C.I. albicans infection is said to be useful in the treatment of C. albicans infection. A monoclonal antibody against the albicans fimbrial subunit has been disclosed. However, although this patent identified the antibody as a member of the IgG2 isotype, it did not show in vivo data indicating protection from Candida infection.

C. is known to contain adhesins. Some immunogenic phosphomannan preparations of C. albicans are available from C. albicans. Albicans has been used to prepare vaccines for treating disseminated candidiasis and to elicit antibodies against such disseminated candidiasis. For example, European Patent No. 344,320 (Kawamura et al., 1989) describes IgG and IgM class human monoclonal antibodies raised against mannan extracted from Candida. While antibodies of the IgG class are said to be preferred and aggregation activity is discussed, those of skill in the art understand that aggregation is distinct from protective effects, and Kawamura et al. Did not show.

[0008] Therapeutic efficacy describes immunization of mice with Candida phosphomannoproteins encapsulated in liposomes, and US Pat. No. 578,309 (Cutler et al., 19
96). Further, mannan adhesin extracted from cell walls ("L-adhidine" or "L-mannan" or "L-mann")
Mice were immunized with a liposome encapsulation of, and two IgM class monoclonal antibodies specific for yeast surface epitopes were described by Cutler et al. The two antibodies (B6.1 and B6) were strong aggregation factors,
On the other hand, only (B6.1) was shown to protect untreated mice from disseminated candidiasis. Each antibody is C.I. albicans recognized a distinct mannan cell wall determinant, and MAb B6.1 recognized a carbohydrate antigen. Han et al., 1997, Infect. Immun. 65: 4100-07. The B6.1 antibody also enhanced the feeding and killing of yeast cells by polymorphonuclear leukocytes (PMN) in the presence of serum complement (Caesar-TonWhat et al., 1997;
Infect. Immun. 65: 5354-57).

[0009] Thus, contrary to the disclosure of the B6.1 antibody, which is an antibody of the IgM class, the other references discussed above are clearly the IgG specific for the phosphomannan antigen on the Candida cell wall. The class of antibodies did not show protection from candidiasis. Despite the work by Cutler et al., It was surprising that protective IgG class antibodies specific for carbohydrate antigens were found by the inventors.

For antibody classes, class switching does not guarantee a similar level of efficacy. Thus, the reported efficacy of an IgM antibody, such as the B6.1 antibody, does not mean that different classes of antibodies are equally effective in a clinical setting, even when directed against the same antigen. For example, in disease of group B streptococci, IgM specific for cell wall carbohydrates was much more effective than IgG2a of the same specificity as IgM (Hill et al., 1992, Clin. Immunol. Immunon.
opathol. 62: 87-91). Similarly, with an isotype switch,
Protected antibodies can be converted to unprotected antibodies. For example, for Cryptococcus neoformans, isotype switching from IgG3 to IgG1 preserved the same epitope specificity while converting unprotected antibodies to protected antibodies in experimental cryptococcosis (Yuan Et al., 1995, J. Immuno.
l. 154: 1810-1816; and Yuan et al., 1998, Infect.
Immun. 66: 1057-1062).

As known to those skilled in the art, antibodies of the IgM class can fix complement and activate macrophages directly. IgG class antibodies can not only bind macrophages and neutrophils, but can also activate complement, so that immunity can be enhanced by antibody- and cell-mediated responses. IgG antibodies can also have greater binding affinity than IgM antibodies. The immune response mediated by IgM can have very different consequences on how the mammalian host infected by Candida recognizes and responds to this pathogen. This is because both the antibody response and the cellular response are known to be important in responding to Candida infection. Thus, the use of both an IgG antibody and an IgM antibody will help enhance the immune response of an infected mammalian host when administered together, or possibly sequentially. B6.
One antibody recognizes phosphomannan adhesin and is known to be protective, but until the invention described herein was recognized, it recognized a candida adhesin and No monoclonal IgG antibody has been described in the literature as being therapeutically effective in inhibiting or preventing the disease.

[0012]

[Means for Solving the Problems]

The present invention relates to candidiasis, in particular, disseminated candidiasis, mucocutaneous candidiasis (
For example, antibodies that protect the host against stomatitis or thrush, esophagitis and vaginitis or vaginal candidiasis) and invasive or deep organ candidiasis (eg, mycemia, endocarditis and endophthalmitis). More particularly, the invention relates to antibodies of the IgG class and IgG3 isotype. The present invention also relates to therapeutic methods useful in treating candidiasis, and diagnostic methods useful in diagnosing candidiasis and monitoring the course of treatment for candidiasis.

It is an object of the present invention to provide a purified or isolated IgG antibody that specifically binds to a carbohydrate antigen on the cell wall of a yeast derived from the genus Candida. In this case, the antibody protects the mammalian host from infection by yeast. More particularly, an object of the present invention is to provide an acid labile component of such a yeast cell wall phosphomannan complex (especially
It is an object of the present invention to provide an antibody that specifically binds to a β-1,2-linked oligomannosyl residue or a β-1,2-linked mannotriose residue.

The antibodies described above are protective against several types of candidiasis, including disseminated candidiasis and mucocutaneous candidiasis. Antibodies include IgG1, IgG2 and Ig.
It can be any IgG isotype, including G3 as well as its various sub-isotypes. Also included are human, chimeric or humanized antibodies.

[0015] It is a further object of the present invention to provide a pharmaceutical composition comprising said antibody, optionally combined with a pharmaceutically acceptable carrier and excipient. Possible yeast infections treatable by such a pharmaceutical composition include C.I. albi
cans, C.I. glabrata and C.I. tropicalis and their strains. Topical, systemic and aerosol formulations are specifically included, which contain the formulation in unit dosage form, and one or more other antifungal, antibody or other therapeutic agents. It is a composition in a composition. In a preferred formulation,
Both IgG and IgM antibodies are administered to the same yeast at about the same time or at different times.

Another object of the present invention is to utilize the above pharmaceutical composition in a method of treating disseminated candidiasis and mucocutaneous candidiasis. Also included are diagnostic kits comprising the above-described antibodies together with reagents for detecting the binding of the antibodies to carbohydrate antigens on the cell wall of yeast from the genus Candida, and also include hybridoma cells expressing such antibodies. Is done.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION

1. Definitions As used herein, the term "antibody," unless otherwise indicated, is widely used to refer to both antibody molecules and various antibody-derived molecules. Such antibody-derived molecules comprise at least one variable region (either the heavy or light chain variable region), and comprise Fab fragments, Fab 'fragments, F (ab') ₂ fragments, Fv fragments, Fabc fragments, single fragments. Chain Fv (acFv) antibodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions of antibody chains with other molecules, and the like.

The antibody of the present invention may be a hybridoma cell, vertebrate serum, a recombinant eukaryotic cell or a recombinant prokaryotic cell transfected with a nucleic acid encoding the antibody.
This can include plant cells), ascites, or from the milk of transgenic animals.

The term “antigen” refers to a molecule that an antibody specifically recognizes and binds to. The specific part of the antigen to which the antibody binds is called an "epitope".

The term “humanized antibody” refers to an antibody whose structure is substantially human. That is,
A humanized antibody has at least substantially all of its constant regions derived from human antibodies, even if all or part of its variable regions are derived from certain other species. "Human antibody" refers to an antibody encoded by nucleotides of human origin, and such nucleotides can be modified by those skilled in the art by known nucleotide manipulation techniques.

The antibodies described herein may also include amino acid sequence changes when compared to a naturally occurring antibody. That is, the antibodies of the invention need not necessarily consist of the exact amino acid sequence of the backbone that constitutes their native variable or constant regions, but may improve or supplement the binding properties of the antibody to its cognate antigen. It contains various substitutions that alter the binding of the antibody to the body or effector molecules such as the Fc receptor. In another format, a minimal number of substitutions are made to the scaffold region to ensure that the immunogenicity of the antibody in humans is reduced, and preferably minimized. In a preferred embodiment of the recombinant antibodies of the present invention, any non-human scaffold regions used can be varied with a minimum number of substitutions to the scaffold regions to avoid extensive introduction of non-human scaffold residues. it can.

The term “conventional molecular biology method” refers to polynucleotide manipulation techniques well known to those skilled in molecular biology. Examples of such known techniques are described in Molecu.
lar Cloning: A Laboratory Manual (2nd edition,
See Sambrook et al., Cold Spring Harbor, N.W. Y. (19
89)) and Current Protocols In Molecular.
Biology (Ausebel et al., 4 volumes in total, John Wiley & Son
s, N.C. Y. ) Can be found. Examples of conventional molecular biology techniques include in vitro ligation, restriction endonuclease digestion, polymerase chain reaction (PCR),
Includes, but is not limited to, cell transformation, hybridization, electrophoresis, DNA sequencing, cell culture, and the like.

As used herein, the term “isolated” or “substantially pure”
Refers to an antibody that is substantially free of other antibodies, proteins, lipids, carbohydrates or other substances that are naturally associated, or for example, fragments thereof. One of skill in the art would use conventional methods for antibody or protein purification using Mab C3.1.
Antibodies can be isolated or substantially purified.

The term “protective” or “therapeutically effective” means that the antibody is effective at inhibiting the adhesion of the yeast cell to its target tissue or cell in the host, or in the bloodstream or infection. It is generally meant to be effective in reducing or preventing an increase in fungal cell levels at an organ site or other site of infection. More specifically, the phrase "protective" or the term "therapeutically effective" refers to the ability of an antibody or pharmaceutical composition according to the present invention to cause a Candida pathogen to cause phagocytosis and killing of macrophages, monocytes or neutrophils. It means that it can be opsonized to promote or that macrophages that can amplify cellular and immune responses can be activated. Preferably, the method of treatment of the present invention comprises at least about 20%, more preferably 40%, even more preferably 60%, and most preferably 90% or more of the Candida organism in the infected mammalian host during the course of treatment. Effective for killing.

The terms “variable region” and “constant region” as used herein in connection with antibody and immunoglobulin molecules refer to the conventional terms given to this term by those of ordinary skill in immunology. Meaningful. Both the antibody heavy and light chains are "variable regions."
And a “constant region”. The split point between the variable and constant regions can be determined by one skilled in the art by reference to standard books describing the structure of the antibody. For example, Kabat et al., “Sequence of Proteins of Immunological Attention: No. 5
Edition, US Department of Health and Human Services, Government Printing Office (1991).

[0026] 2. Candida-related symptoms Among the more than 150 recognized species of Candida, albicans is the most commonly identified pathogen in humans. Other clinically important species include C
. guilliermondi, C.I. krusei, C.I. parapsilos
is, C.I. pseudotropicalis and C.p. tropicalis. Mucocutaneous infections include thrush or oropharyngeal candidiasis, stomatitis, esophagitis, gastrointestinal candidiasis, intertrigo, acneitis, vulva vaginitis, balanitis, candida cystitis, and chronic mucocutaneous candidiasis. included. Numerous diagnostic classes exist for severe or deep candida infections, including such candidiasis, disseminated candidiasis, systemic candidiasis, invasive candidiasis, visceral candidiasis, and hepatopancreas. Terms that indicate the involvement of certain organs, such as candidiasis and ocular candidiasis, are included. See, for example, Dismukes, 1996. Severe or deep candida infections are frequently found in immunocompromised or immunocompromised patients, such as acquired immunodeficiency syndrome (AIDS) patients.

[0027] 3. Generation of Hybridomas Producing Monoclonal Antibodies Against Candida Antigen To generate antibodies, various species of host animals are immunized by injecting L-man antigen, or appropriately prepared Candida extracts or whole cells. can do. Suitable animals for this purpose include, but are not limited to, rabbits, mice and rats. Various adjuvants can be used to increase the immunological response, depending on the host species. Such adjuvants include Freund (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and Potentially useful human adjuvants such as, but not limited to, BCG (Calmet-Guerin) and Corynebacterium parvum.

[0028] Monoclonal antibodies to the Candida antigen can be prepared using any technique provided for the production of antibody molecules by continuous cell lines in culture. Such techniques are described in Kohler and Milstein (Nature,
1975, 256: 495-497), the human B cell hybridoma technology (Kosbor et al., 1983, Immuno).
logic Today 4:72; Cote et al., 1983, Proc. Natl
. Acad. Sci. 80: 2026-2030), and EBV-Hybridoma technology (Cole et al., 1985, Monoclonal Antibodies).
and Cancer Therapy, Alan R.A. Liss, Inc.
, Pp. 77-96), but are not limited thereto. Furthermore, a technique developed to create a “chimeric antibody” by splicing a gene derived from a mouse antibody molecule having an appropriate antigen specificity with a gene derived from a human antibody molecule having an appropriate biological activity (Morrison Et al., 1984, Proc.
. Acad. Sci. 81: 6851-6855; Neuberger et al., 19
84, Nature, 312: 604-608; Takeda et al., 1985, N.
attribute, 314: 452-454). Alternatively, the techniques described for making single-chain antibodies (US Pat. No. 4,946,778)
It can be adapted to generate single-chain antibodies specific for Candida.

[0029] 4. Isolation of B cells specific for Candida antigens B cells specific for antigens can be obtained from convenient samples, such as peripheral blood lymphocytes, obtained from human patients infected with Candida by techniques known and available in the art. Can be isolated by For example, using the fusion protein of the invention, B cells expressing immunoglobulins that specifically bind to phosphomannan antigen can be purified by affinity chromatography, fluorescence activated cell sorting (FACS), and Zn-chelated Sepharose or It can be detected and isolated by other widely used techniques such as Protein A Sepharose (Harlow et al., Antib.
odies: A Laboratory Manual, Cold Spring
g Harbor Laboratory, 1988).

As another example, lymph nodes obtained from a candidiasis patient can be minced and passed through a wire mesh using a rubber policeman. Pure B cells can be isolated using CD19-coated immunomagnetic beads. B cells specific for the antigen can be isolated by affinity chromatography or fluorescence-activated cell sorting using the appropriate fusion protein. Thereafter, B cells specific for the Candida antigen can be immortalized using known techniques, such as EBV immortalization. Using any effective lymphotropic virus or other transforming factor capable of transforming B cells to grow in continuous culture and continue to produce monoclonal antibodies specific for Candida-related antigens Can be.

[0031] 5. Isolation of antigens specific for immunoglobulin heavy and light chain sequences In addition to providing antibodies specific for Candida phosphomannan (and β-1,2-mannnotriose), the present invention provides , A polynucleotide encoding an antibody specific for Candida. The polynucleotide may have a wide range of sequences due to the degeneracy of the genetic code. One of skill in the art can readily vary a given polynucleotide sequence encoding a Candida-specific antibody according to the present invention to a different polynucleotide encoding the same antibody. For example, the polynucleotide encoding the antibody can be varied in view of factors affecting expression, such as codon frequency and RNA secondary structure.

[0032] 6. Production of Recombinant Human Antibodies The antibodies of the invention can be produced by various methods useful for producing polypeptides, such as, for example, in vitro synthesis, recombinant DNA production, and the like. Preferably, humanized antibodies are produced by recombinant DNA technology. The antigen-specific antibodies of the present invention may be produced using recombinant immunoglobulin expression techniques.
Recombinant production of immunoglobulin molecules, including humanized antibodies, is disclosed in US Pat. No. 4,816,816.
No. 4,316 (Boss et al.) And U.S. Pat. No. 4,816,567 (Cabilly).
Et al.), GB 2,188,638 (Winter et al.) And GB 2,209,757 (Winter et al.). In addition, technology relating to recombinant expression of humanized immunoglobulin is described by Goeddel et al., Gene.
Expression Technology Methods ENZYMO
LOGY Vol. 185 Academic Press (1991), and Borreback, ANTIBODY ENGINEERING, W.M. H. F
See also Leeman (1992). Further information regarding the production, design and expression of recombinants may also be found in Mayforth, DESIGNING A.
NTIBODIES, Academic Press, San Diego (1
993).

As an example, a recombinant antibody of the invention may be produced by the following process.

A) By conventional molecular biology methods, the minimum portion of CDRs and variable region framework required to maintain donor antibody binding specificity is derived from human immunoglobulin and the remainder of the antibody is Construction of an expression vector comprising a nucleotide sequence encoding the antibody heavy chain as derived from other human immunoglobulins yields a vector for expression of the humanized antibody heavy chain.

B) By conventional molecular biology methods, the minimum amount of CDRs and variable region framework required to maintain donor antibody binding specificity is derived from human immunoglobulin and the remainder of the antibody is Construction of an expression vector containing a nucleotide sequence encoding the antibody light chain as derived from other human immunoglobulins yields a vector for expression of the humanized antibody light chain.

C) The expression vector is delivered to a host cell by conventional molecular biology methods to produce a transfected host cell.

D) Culturing the transfected cells by conventional cell culture techniques to produce a recombinant antibody.

The host cells are co-transfected with the two expression vectors of the present invention (a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide). Good. The two vectors may contain different selectable markers, but are preferably identical, except for the heavy and light chain coding sequences. This procedure provides for equal expression of the heavy and light chain polypeptides. Alternatively, a single vector encoding both heavy and light chain polypeptides may be used. The heavy and light chain coding sequences may include cDNA or genomic DNA, or both.

The host cells used to express the recombinant antibodies of the invention may be bacterial cells, such as E. coli, or the antibody binding fragments may be expressed on available phage display systems ( Winter et al. (1994) Ann.
Immunol. 12: 433-455 and Little et al. B
iotechnol. 41 (2-3): 187-195)). Preferably, mammalian cells such as eukaryotic cells or, most preferably, Chinese hamster ovary cells may be used. The choice of the expression vector depends on the choice of the host cell,
One skilled in the art may choose to have the desired expression and regulatory properties in the chosen host cell.

General methods for the construction of vectors of the invention, transfection of cells to produce host cells of the invention, and culture of cells to produce antibodies of the invention are all conventional molecular methods. It is a biological method. Similarly, once produced, the recombinant antibodies of the present invention can be purified by standard procedures in the art, including cross-flow filtration, ammonium sulfate precipitation, affinity column chromatography, gel electrophoresis, and the like.

[0041] 7. Preparation of Diagnostic, Therapeutic and Prophylactic Compositions The antibodies of the invention may be used in conjugation with or attached to other antibodies (or portions thereof), such as human or humanized monoclonal antibodies. These other antibodies
The antibodies of the present invention may be reactive with other markers (epitope) characteristic of the disease to which they bind, or may have different properties selected, for example, to recruit molecules or cells of the human immune system to the diseased cells. . The antibodies (or portions thereof) of the present invention can be administered together with such antibodies (or portions thereof) as a separately administered composition or with two agents linked by conventional chemical or molecular biological methods. It may be administered as a single composition. Further, the diagnostic and therapeutic value of the antibodies of the present invention is that the humanized antibody is labeled with a label that produces a detectable signal (either in vitro or in vivo), or with a label that has therapeutic properties. Can be increased. Some labels, such as radionuclides, produce a detectable signal and may have therapeutic efficacy. Examples of radionuclide labels include ¹²⁵ I and ¹³¹ I. Examples of other detectable labels include fluorescent chromophores such as fluorescein for fluorescent microscopy, phycobiliprotein or tetraethylrhodamine, enzymes that produce fluorescent or colored products for detection by fluorescence, absorption, visible color or aggregation. This may include producing fertile products for verification by electron microscopy, or ferritin, peroxidase or gold beads for direct or indirect electron microscopy visualization. Labels having therapeutic properties include drugs for the treatment of candidiasis as described below.

The present invention also provides various methods for treating and / or detecting Candida cells. These methods involve the administration of labeled or unlabeled Candida-specific antibodies to the patient. One method of detecting Candida cells in humans is described in (
Administering a labeled Candida-specific antibody (labeled with a detectable label) to a human, followed by detecting bound labeled antibody by the presence of the label. Alternatively, a Candida-specific antibody may be linked or conjugated to a therapeutic molecule such as ricin or other toxin.

The recombinant antibodies of the present invention may also be used for the selection and / or isolation of human monoclonal antibodies, and peptides that may be useful in diagnostic and therapeutic applications similar to antibodies. Or may be used for the design and synthesis of non-peptide compounds (mimics) (eg, Saragovi et al., 1991 Science 2).
53: 792-795).

When a Candida-specific antibody of the invention is used in vivo, the antibody typically comprises
It is administered in a composition comprising a pharmacological carrier. The pharmacological carrier can be any compatible, non-toxic substance suitable for delivering the monoclonal antibody to the patient. Sterile water, alcohol, fats, waxes and inert solids may be included in the carrier.
Also, pharmacologically acceptable buffers or dispersants may be incorporated into the pharmacological composition.

The antibody composition of the present invention can be administered to a patient in various ways. Preferably, the compositions are administered parenterally, ie, subcutaneously, intramuscularly or intravenously. Aerosol formulations are also specifically contemplated. Injectable forms of administration are also sometimes preferred due to systemic infection, and systemic maximal effects on respiratory and deep tissue infections. If long-term administration by injection is required, an intermediate port, an indwelling catheter or an automatic pump mechanism may be used. Accordingly, the present invention provides a solution or acceptable carrier of a human antibody,
There is provided a composition for oral administration comprising a solution thereof, preferably dissolved in an aqueous carrier. A variety of aqueous carriers can be used, eg, water, buffered water, 0.4%
Saline, 0.3% glycine and the like can be used. These solutions are sterile,
Generally, there is no problem of fine particles. These compositions may be sterilized using conventional, known sterilization techniques.

The composition may be pharmacologically required to approximate physiological conditions such as pH control and buffering agents, eg, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, toxicity regulators. It may contain chemically acceptable auxiliary substances. The concentration of antibody in these formulations may vary widely, for example, from less than about 0.5% by weight or at least about 1% to 15 or 20% by weight, depending on the particular mode of administration selected. It may be chosen based primarily on capacity, viscosity, etc.

Preferred doses of the antibodies of the present invention for systemic administration range from about 0.1 to about 5 mg / kg of body weight. A more preferred dose is about 0.5 to about 2.0 mg
/ Kg, most preferably in the range of about 1.0 to about 1.5 mg / kg. A human or other mammalian subject is treated with an appropriate schedule and multiple doses of the antibody drug. Such a schedule results in essentially saturating antibody levels or significant opsonization levels in the blood or infected tissue of patients undergoing treatment according to the methods of the present invention, and is retained. This is the schedule. For example, a single dose of a chimeric antibody is described by Clark et al., Effect of a chimer.
ic antibody to tumour necrosis factor
-(Alpha) on cytokine and physiological
responses in patients with severes
epsis-Randomized, clinical trial.
Crit. Care. Med. 26: 1650-59 (1998).

[0048] Topical or mucocutaneous infections are treated by topical administration of a therapeutic antibody composition of the present invention. For example, for oral delivery, the pharmacological composition may be administered in the form of a cream or wash which can be applied by a swab or the like or by rinsing at intervals. These compositions may also be formulated in buccal suppositories, for example, for prolonged release from the mouth area. In other embodiments, tablets or oral inserts or gums may be used as delivery vehicles. For vaginal delivery, the composition may be applied in a cream formulation, pessary or insert, as is known to those skilled in the art.

A pharmacologically effective amount is the amount of the designed pharmacological composition required to produce a positive effect. A positive effect includes reducing the amount of microorganisms in a subject, killing or inactivating microorganisms, or completely or almost completely eliminating the infection of microorganisms from the body. Preferably, the patient has an infection as measured by any of the test parameters, which infection is reduced such that it is at least 100-fold, more preferably 1,000-fold, and even more preferably undetectable after treatment.

Yet another embodiment of the present invention is directed to a composition of the present invention that can be used in combination with other agents (eg, antifungal agents) to maximize the effect of the composition in an additive or synergistic manner. Have been. Agents that may be effective in combination with the compositions of the present invention include other agents and treatments known or expected to have a positive effect on Candida microorganisms. Such agents include, but are not limited to, flucytosine, mycoconazole, fluconazole, itraconazole, ketoconazole, griseofulvin, amphotericin B, and derivatives, variants and combinations of these agents. Other agents are described, for example, in US Pat. No. 5,679,648 to McCafrey et al. (1977).

The actual methods for preparing parenterally administrable compositions and modulators required for administration to a subject are known, or will be apparent, to those skilled in this art; REMINGTON'S PHARMACEUTICAL SCIENC
E, 15th Ed. , Mack Publishing Company, E
aston, Pa. (1980), which is incorporated herein by reference.

[0052] 8. Diagnostic Kits for Detection of Diseased Tissue and Candida Cells A kit can be provided that includes an antibody according to the present invention that can bind to diseased tissue or Candida. These kits can be used in combination with conventional histological staining techniques to more quickly and more accurately detect which disease is present and the extent of infection or stage of disease. This is useful for the purpose of diagnosing, detecting and / or determining which treatment or therapy may be suitable for treating the disease of a particular subject.

Preferred kits include, for example, antibodies prepared for contact with a tissue or biological fluid sample. The sample is then incubated with the antibody, as is known in the art as conventional methods. After incubation with the antibodies of the kit, the cells and / or tissues are tested for the presence or absence of binding. Standard assays to be used in such kits include latex agglutination, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA).
) Or other suitable antibody detection systems, including, but not limited to.

In view of the above general discussion, the specific examples proposed below are illustrative only and are not intended to limit the intent of the present invention. Other general configurations will be apparent to those skilled in the art.

Examples Example 1: Materials and Methods Microorganisms and culture conditions for preparing a cell line producing the C3.1 antibody are as follows. C. albicans CA-1 has already been described (Han et al.,
1995 Infect. Immun. 63: 2714-9, Han et al., 199.
8 Infect. Immun. 66: 5771-5776, and Kanbe et al., 1993 Infect. Immun. 61: 2578-2584), starting with a frozen glycerol stock and glucose-yeast extracted peptone (GYEP).
) Grow as hydrophilic stable mother cells at 37 ° C in culture. Wash the hydrophilic yeast cells and add Dulbecco's phosphate buffered saline (DPBS) to the desired yeast cell concentration.
, Sigma Chemical Co., St. Lo
uis, MO) and used to infect mice.

[0056] Charles River Labs
oratories, Kingdom, N.W. Y. The BALB / c female mice obtained were 6-8 weeks old and were used throughout the study.

MAb C3.1 was one of several MAbs, including protected MAb B6.1, isolated via hybridoma technology from L-man-vaccinated mice (Han et al., 1995). Was. MAb C3.1 is available from LigoCyte Pharmaceuticals, Ind.
c. , Bozeman, MT) in serum free solvent and ammonium sulfate precipitated. Already described (Han et al., 1997 J. Infe
ct. Dis. 175: 1169-1175, Han et al. 1998) was used as a positive control antibody. MAb B6.
1 is LigoCyte Pharmaceuticals (LigoCyte Pharm)
accessories, Inc. , Bozeman, MT) by ammonium sulfate precipitation in a serum-free medium. Antibody titers were determined by total C.I. albic
It was determined by aggregation with either ans yeast cells or mannan-coated latex beads (see Han et al., 1995 and Han et al., 1998).

The MAbC3.1 isotype determination was performed as follows. MAb C3.1
Isotypes were detected by a capture enzyme immunosorbent assay (ELISA) and confirmed by the immune double diffusion (Ouchterory) technique. All anti-mouse immunoglobulins were purchased from Sigma.

Mannan Extraction and Acid Hydrolysis C. cultivated as described above. albicans yeast cells were treated with β-mercaptoethanol to produce a mannan extract, which was previously (Kanbe et al., 1992).
93 Infect. Immun. 61: 2578-2584) on a concanavalin A affinity column. The extract is made up to 10 mM H
It is hydrolyzed by boiling in Cl, neutralized and described (Han et al., 1997).
Infect. Immun. 4100-4107) on a P-2 (Bio-Rad, Richmond, CA) column. Each fraction eluted from the P-2 column was tested for its ability to react with MAb C3.1 in a similar manner as described previously for identification of the epitope specificity of MAb B6.1 (Id.).

Fluorescence microscopy The distribution of the C3.1 epitope on yeast cells was determined by indirect immunofluorescence (IFA). MAb C3.1 was reacted with yeast cells, washed, and reacted with fluorescein-labeled anti-mouse IgG or anti-mouse IgM. The cells were observed with a fluorescence microscope and a phase contrast microscope.

Example 2: MAb C3.1 conveys protection against sporadic Candida Protection against Candida infection by passive transport. MAb C3.1 was used immediately or stored at -20 ° C, treated at 56 ° C for 30 minutes before use, or C.I. a
lbicans yeast cells (Han et al., 1995; Han et al., 199).
8, Han et al., 1998 J. Mol. Infect. Dis. (In press))
. The preventive effect of MAb C3.1 was tested against experimental diffuse candidiasis and Candida vaginal infection as follows.

1) Versus sporadic candidiasis 0 hr. Treat with MAb C3.1 intraperitoneally (ip). 4 hours intravenously (iv), C.I. sensitize with Albicans (5 × 10 ⁵ yeast cells). 48 hours Continue to measure kidney colony forming unit (CFU) or determine survival time.

2) Vaginal infection with Candida 0 hours Estradiol is injected subcutaneously (sc) at 0.5 mg / mouse. 72 hours i. p. Or i. vg. With MAb C3.1 (0.5 mg / mouse) (given 100 μl of Mab C3.1 for i.vg.). 76 hours i. vg. And C. sensitize with Albicans (5 × 10 ⁵ yeast cells). Measure 120-hour vaginal CFU (Han et al., 1998 Infect Im
mun 66: 5771-5776).

The statistical significance of the difference in survival time was determined by Kaplan-Meier test (Syst
at 7.0, SPSS Inc. (Chicago, IL). For other analyses, a Student's t-test was used. The P-value is
A value of less than 0.05 was considered statistically significant.

FIG. 1 suggests that MAb C3.1 has a protective effect against Candida. In panel (A), BALB / c mice received unheated MAb C3.1 (0
-1), MAb (H-C3.1) heated at 56 ° C for 30 minutes, C.I. alb
icans absorption MAb C3.1 (A-C3.1) or DPBS (buffered diluent)
To i. p. Given in.

In panel B of FIG. 1, mice received MAb C3.1, MAb B6.1 or DPBS as a control. In both panels, animals were given i. v
. Sensitized with 5 × 10 ⁵ viable yeast cells in
Assessed by measuring U (A) and by survival curve (B).

Mice given unheated or heated MAb C3.1 (FIG. 1) had 86% and 88% less CFU than mice given DPBS, respectively (P <0.00).
1). Mice receiving the absorbed serum had almost the same number of CFUs as control mice receiving DPBS. Bars indicate standard error. Mice receiving MAb C3.1 had a similar survival time to animals receiving MAb B6.1. These mean survival times were significantly longer than those of animals receiving DPBS (P <
0.05). The conclusion is that MAb C3.1 enhances the resistance of mice to diffuse Candida.

Example 3 MAb C3.1 has a protective effect on Candida vaginal infection i.v. by yeast cells (5 × 10 ⁵ ) vg. Prior to sensitization, MAb C3.I was injected intraperitoneally (ip) (FIG. 2A) or vagina (ivg.) (FIG. 2B) of simulated estrus mice.
1 was given. Vaginal CFU was prepared using the unheated MAb C3.1 (C
3.1), heat treatment at 56 ° C. for 30 minutes (H-C3.1), C.I. albican
Compared to CFU from animals receiving s-absorbed C3.1 (A-C3.1) or DPBS (diluent).

In FIG. 2A, mice receiving unheated (C3.1) or heated (H-C3.1) D
There were 60% and 49% less CFU, respectively, compared to PBS-control mice. Mice receiving absorbed C3.1 (A-C3.1) or DPBS had similar CFUs.

In FIG. 2B, mice receiving unheated or heated MAb C3.1 were treated with DPB
Approximately 86% less CFU compared to S control mice. This CF
The reduction in U is similar to that observed with administration of MAb B6.1. C
. Mice receiving albicans-absorbed C3.1 resulted in CFU similar to DPBS control mice.

In both panels, a significant difference was observed between unheated MAb C3.1 or heated MAb.
Seen between mice given any of C3.1 and DPBS controls (
P <0.05). Bars indicate standard error.

Example 4: C.I. IgG class antibodies that are protective against C. tropicalis L-man immunogen was prepared according to the technique described in Example 1 using T. tropicalis species. BALB / c female mice were purchased from Charles River Laboratories, Ki
ngston, N.M. Y. ), From 6 to 8 weeks of age and licensed. Han
Et al., 1995, hybridomas were produced from L-man-vaccinated mice via hybridoma technology and selected. Antibody titers were determined according to Han et al., 199.
5 and Han et al., 1998, all C.I. Measured by aggregation with either tropicalis yeast cells or mannan-coated latex beads. Selected antibodies to be isotyped are detected in a capture enzyme-linked immunosorbent assay (ELISA) and the isotype is determined by immunodouble diffusion (Ouch).
terry) technology. Protection against Candida infection by passive transport was measured by the technique described in Example 3.

Example 5: Additional IgG Antibodies Against Candida albicans Albicans yeast cells were used to prepare an L-man immunogen according to the technique described in Example 1. BALB / c female mice were from Charles River Laboratories,
Kingston, N .; Y. ), From 6 to 8 weeks of age and licensed. H
per et al., 1995, hybridomas were produced from L-man-vaccinated mice via hybridoma technology and selected. Antibody titers were determined by Han et al., 1
995 and Han et al., 1998, all C.I. Albicans was measured by aggregation with either yeast cells or mannan-coated latex beads.
The selected antibodies to be isotyped are captured in a capture enzyme linked immunosorbent assay (EL
ISA) and the isotype is determined by immunodouble diffusion (Ouchterory)
Confirmed by technology. Protection against Candida infection by passive transport was measured by the technique described in Example 3.

Example 6 Treatment of Candida Infection in Human Patients For the treatment of disseminated disease, patients showing signs of disseminated disease were given i. v.
Or i. p. Or i. m. Should provide the antibody alone or in combination with other antimicrobial agents. High-risk patients should be identified for the prevention of Candida (eg, undergoing abdominal surgery, open heart surgery, kidney transplantation, bone marrow transplantation, or indwelling catheters, corticosteroids, broad spectrum antibiotics) Patients), the antibody pharmacological composition described above is administered i.p. prior to treatment. v.
Or i. m. Should be administered at For the treatment of vaginal Candida infection, the antibody pharmacological composition described above comprises i. v. Or i. p. Or i. m. Like, in the vagina,
Administered alone or in combination with other antimicrobial agents.

Example 7 Use of a Test Kit to Detect Candida Infection The antibodies described above are used in a capture antigen format to capture Candida antigens in serum or vaginal secretions from infected patients. Such a kit is further provided with an agent for detecting the binding of the antibody to such an antigen.

It should be understood that the foregoing discussion and examples exist only in detail description of certain preferred embodiments. Thus, it should be apparent to one skilled in the art that various modifications and equivalents may be made without departing from the spirit and spirit of the invention. All articles, patents and patent specifications identified above are incorporated by reference in their entirety.

References 1. Shibata, N., K. Iuta, T. Imai, Y. Satoh, R. Satoh, A. Suzuki, C.
Kojima, H. Kobayashi, K. Hisamichi and S. Suzuki, 1995.Existence of b
ranched side chains in the cell wall mannan of pathogenic yeast, Candida
albicans. Structure-antigenicity relationship between the cell wall ma
nnans of Candida albicans and Candida parapsilosis. J. Biol. Chem. 270: 111
3-1122. 2. Han, Y. and JE Cutler, 1995. Antibody response that protects
against disseminated candidiasis.Infect.Immun. 63: 2714-2719. 3.Han, Y. and JE Cutler, 1997.Assessment of a mouse model of n.
eutropenia and the effect of an anti-candidiasis monoclonal antibody in
These animals. J. Infect. Dis. 175: 1169-1175. 4. Han, Y., T. Kanbe, R. Cherniak and JE Cutler, 1997. Biochemic
al characterization of Candida albicans epitopes that can elicit protect
ive and nonprotective antibodies. Infect.Immun. 65: 4100-4107. 5. Han, Y., RP Morrison and JE Cutler, 1998. A vaccine and mon.
oclonal antibodies that enhance mouse resistance to Candida albicans vag
inal infection. Infect.Immun. 66: 5771-5776. 6. Han, Y., MA Ulrich and JE Cutler, 1998. Candida albicans ma.
nnan extract-protein conjugates induce a protective immune response agai
nst experimental candidiasis. J. Infect. Dis.In Press. 7. Hill, HR, DK Kelsey, LA Gonzales and HV Raff, 1992. Mon
oclonal antibodies in the therapy of experimental neonatal group B strep
62: 87-91. 8.Kanbe, T., Y. Han, B. Redgrave, HH Riesselman and JE Cutler, tococcal disease.
1993.Evidence that mannans of Candida albicans are responsible for ad
herence of yeast forms to spleen and lymph node tissue.Infect.Immun. 6
1: 2578-2584. 9. Shibata, N., K. Hisamichi, T. Kikuchi, H. Kobayashi, Y. Okawa an
d S. Suzuki, 1992. Sequential nuclear magnetic resonance assignment of
β-1,2-linked mannooligosacharides isolated from the phosophomannan of t
he pathogenic yeast Candida albicans NIH B-792 strain.Biochemistry 31:
5680-5686. 10. Yuan, R., A. Casadevall, G. Spira, and MD Scharff, 1995. Iso
type switching from IgG3 to IgG1 converts a nonprotective murine antibod
y to Cryptococcus neoformans into a protective antibody.J. Immunol. 154
: 1810-1816. 11. Yuan, RR, G. Spira, J. Oh, M. Paizi, A. Casadevall and MD
Scharff, 1998. Isotope switching increases efficacy of antibody protect
ion against Cryptococcus neoformans infection in mice.Infect.Immun. 66
: 1057-1062.

[Brief description of the drawings]

1A and 1B show that active transport of MAb C3.1 protects BALB / c mice from disseminated candidiasis.

2A and 2B show that MAb C3.1 has a prophylactic effect on Candida vaginal infection in pseudoestrus mice.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/10 A61P 31/10 C12N 5/10 G01N 33/53 V G01N 33/53 33/569 A 33 / 569 C12N 5/00 B (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL) , SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, B G, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN , IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Cutler , Jim, E. Ash Drive, Batesman, Montana, United States of America, 1426 (72) Inventor Han, Yong Moon United States of America, Montana, Batesman, Treasure Avenue 306 (72) Inventor, Rieselman, Marsha United States of America, Batesman, Hitching Post Road 30 F-term (reference) 4B065 AA91X AB05 BA08 CA25 CA44 CA46 4C076 AA24 BB21 BB31 CC07 CC34 DD80 FF54 FF68 4C084 AA02 AA03 AA14 AA17 BA01 BA08 BA23 BA44 CA59 MA02 MA05 MA13 MA55 MA63 NA05 NA10 NA11 NA14 AB31AB CBA4C 4H045 AA11 AA30 CA15 DA76 EA23 EA52 FA72

Claims (30)

[Claims] 1. An isolated IgG antibody that specifically binds to a carbohydrate antigen on the cell wall of a yeast derived from the genus Candida, wherein said antibody is protective against infection of a mammalian host by the yeast. 2. The antibody according to claim 1, wherein the antibody specifically binds to an acid labile component of a phosphomannan complex in a yeast cell wall. 3. The antibody according to claim 2, wherein the acid-labile component that specifically binds to the antibody is a β-1,2-linked oligomannosyl residue. 4. The antibody according to claim 2, wherein the acid-labile component to which the antibody specifically binds is a β-1,2-mannnotriose residue. 5. The method according to claim 5, wherein the yeast is C. elegans. albicans, C.I. glabrata and C.I. The antibody according to any one of claims 1 to 4, which is selected from the group consisting of C. tropicalis and strains thereof. 6. The method according to claim 6, wherein the yeast is C. The antibody of claim 5, which is an albicans. 7. The antibody according to claim 1, wherein the antibody is protective against candidiasis of the type selected from the group consisting of disseminated candidiasis and mucocutaneous candidiasis. 8. The antibody according to claim 1, wherein the antibody is an isotype selected from the group consisting of IgG1, IgG2, and IgG3. 9. The antibody according to claim 8, wherein the antibody is of the IgG3 isotype. 10. The antibody according to claim 9, wherein the antibody is antibody C3.1. 11. The isolated antibody according to claim 5, wherein the antibody is selected from the group consisting of a human antibody, a chimeric antibody and a humanized antibody. 12. A pharmaceutical composition comprising the isolated antibody of claim 5, formulated with a pharmaceutically acceptable carrier and excipient. 13. The pharmaceutical composition according to claim 12, wherein the composition is formulated for topical administration. 14. The pharmaceutical composition according to claim 12, wherein the composition is formulated for systemic administration. 15. The composition of claim 1, wherein the composition is formulated as an aerosol.
3. The pharmaceutical composition according to 2. 16. The pharmaceutical composition according to claim 12, wherein the composition is formulated in a therapeutically effective unit dosage form. 17. The method of claim 12, further comprising one or more other therapeutic agents.
A pharmaceutical composition as described. 18. The pharmaceutical composition according to claim 17, wherein said one or more other therapeutic agents is an antifungal agent. 19. The pharmaceutical composition according to claim 17, wherein said one or more other therapeutic agents is an antibody. 20. A method for treating or preventing candidiasis in a mammalian subject, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 12. Method. 21. The method of claim 20, wherein the candidiasis is disseminated candidiasis. 22. The method of claim 20, wherein the candidiasis is mucocutaneous candidiasis. 23. A method of treating candidiasis in a subject, comprising the step of administering to the subject a therapeutically effective amount of the antibody of claim 5. 24. A method for inhibiting or preventing the development of candidiasis in a mammalian subject, wherein the subject in need thereof specifically binds to a carbohydrate antigen on the cell wall of yeast derived from the genus Candida. Administering a protective amount of an IgG antibody. 25. The yeast, wherein the yeast is C. albicans, C.I. glabrata and C.I. 25. The method of claim 24, wherein the method is selected from the group consisting of C. tropicalis and strains thereof. 26. The method of claim 24, further comprising administering a protective amount of an anti-Candida IgM antibody. 27. The method of claim 26, wherein the IgM antibody and the IgG antibody are administered at about the same time. 28. The method of claim 26, wherein the IgM antibody and the IgG antibody are administered at different times. 29. A diagnostic kit comprising the antibody according to claim 1 together with a reagent for detecting the binding of the antibody to a carbohydrate antigen on the cell wall of yeast derived from the genus Candida. A hybridoma cell expressing the antibody according to any one of claims 1 to 4.