HK1118200A - Treatment of fungal infections with polyene or beta glucan synthase inhibitor anti-fungals combined with anti hsp90 antibodies - Google Patents

Description

Treatment of fungal infections with beta glucan synthase inhibitors in combination with anti-HSP 90 antibodies

The application is a divisional application of Chinese patent application No01807383.2, which has the international application date of 3, 20 and 2001, has the international application number of PCT/GB01/01195 and is named as 'beta glucan synthase inhibitor and anti-HSP 90 antibody combined treatment of fungal infection'.

Technical Field

The present invention relates to novel compositions and formulations that are effective antifungal agents, and a novel antibody that can be incorporated into the compositions and formulations.

Background

Fungal infections are a major cause of patient mortality in intensive care units, and occur more frequently in immunocompromised and debilitating patients (Gold, j.w.m., 1984, am.j.med. (journal of american medicine)76: 458-463; klein, r.s. et al, 1984, n.engl.j.med. (british journal of national medicine)311：354-357；Burnie，J.P.，1997，Current Anaesthesia &Critical Care (modern anesthesia and first aid)8: 180-183). The existence and persistence of fungal infections can be attributed to the selective pressure of broad-spectrum antifungals, the frequent prolonged stay of patients in facilities such as intensive care units, problems in diagnosing infections, and the lack of efficacy of fungal agents used in therapy. Although strict hygiene controls may prevent fungal infections to some extent in hospitals or other settings, outbreaks of infections remain a serious problem and need to be addressed.

Systemic fungal infections such as invasive candidiasis and invasive aspergillosis may be caused by a variety of fungal pathogens, such as Candida albicans, Candida tropicalis, Candida krusei, and the less toxic species Candida parapsilosis and Torulopsis glabrata (the latter in some articles being Candida glabrata). Although candida albicans was once the most common fungal isolate obtained from intensive care units, recent studies have shown that candida tropicalis, candida glabrata, candida parapsilosis, and candida krusei now account for about half of these isolates (Pfaller, m.a. et al, 1998, j.clin.microbiol. (clinical microzyme)Biological magazine)36: 1886-1889; pavese, p. et al, 1999, pathobiology46: 579-583). The rise in non-Candida albicans species suggests that Candida developed resistance to traditional antifungal therapy (Walsh, T.J. et al, New Eng.J.Med. (New England journal of medicine)340：764-771)。

Detection and diagnosis of the fungal pathogen responsible for the infection is critical for subsequent treatment, since antifungal agents may be more effective against certain bacterial species. British patent 2240979 and EP 0406029, which are incorporated herein by reference in their entirety, disclose a fungal stress protein and its antibodies that can be used in a sensitive and highly specific assay for the detection of fungal pathogens.

Traditionally, the treatment of candida albicans, candida tropicalis and candida parapsilosis with the antifungal amphotericin B has been considered the "gold mark" for systemic antifungal therapy (Burnie, j.p., 1997, supra). Unfortunately, amphotericin B itself is highly toxic and its use is affected by its side effects such as chills, fever, myalgia or thrombophlebitis. Other antifungal agents include oral azole drugs (miconazole, ketoconazole, itraconazole, fluconazole) and 5-flucytosine. However, fungal species such as candida krusei and torulopsis glabrata, which are resistant to fluconazole, often occur in patients who have been administered fluconazole prophylactically. In addition, strains of Candida albicans that are resistant to fluconazole have been reported (Opportunistic Pathogens, 1997,1: 27-31). Thus, despite recent advances in therapeutic agents such as fluconazole, itraconazole and systemic liposome-based amphotericin B variants (Burnie, j.p., 1997, supra), the need for effective agents for treating fungal infections remains urgent.

Disclosure of Invention

The present invention addresses the above-identified need by providing a novel composition which is a significant improvement over prior art antifungal agents for the treatment of fungal infections in humans or animals, and also provides a novel antibody which can be incorporated into the composition. The compositions of the invention include antibodies that bind to one or more epitopes of a fungal stress protein in admixture with known antifungal agents. The inventors have very surprisingly found that the efficacy of the antifungal agent against fungal infections is significantly enhanced, thereby allowing the use of lower or better doses, which reduces unwanted side effects. In addition, the compositions of the present invention are effective in treating fungal infections that are resistant to the fungal agents used in the compositions themselves.

The present invention provides a composition comprising an antibody or antigen-binding fragment thereof which specifically binds to one or more epitopes of a fungal stress protein and an antifungal agent comprising at least one antifungal agent selected from the group consisting of polyene antifungal agents and echinocandin (echinocandin).

The invention further provides a combined preparation comprising an antibody or antigen-binding fragment thereof specifically binding to one or more epitopes of a fungal stress protein, an antifungal agent comprising at least one selected from the group consisting of a polyene antifungal agent and an echinocandin antifungal agent, for simultaneous, separate or sequential use in the treatment of a fungal infection.

The antibody may be specific for a heat shock protein derived from a member of the genera candida or torulopsis. (Candida or Torulopsis are generally considered synonymous). In particular, the antibodies may be specific for heat shock proteins derived from hsp90 of candida albicans, as described in british patent 2240979 and EP patent 0406029.

The antibody or antigen-binding fragment thereof may be directed against a polypeptide comprising SEQ ID NO: 1 is specific for an antigenic determinant. Antibodies and their production and use are well known and elucidated, e.g. Harlow, E and Lane, d., Antibodies: a Laboratory Manual (antibodies: A Laboratory Manual), Cold spring harbor Laboratory Press, Cold spring harbor, New York, 1999.

Antibodies can be produced by standard methods well known in the art. Examples of antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, fragments produced by a Fab expression library, and antigen-binding fragments of antibodies.

Antibodies can be produced by a variety of hosts, such as goats, rabbits, rats, mice, humans, and the like. The host can be immunized by injection with a heat shock protein derived from Candida, such as hsp90 from Candida albicans, or any fragment or oligopeptide thereof having immunogenicity. Depending on the type of host, different adjuvants may be used to enhance its immune response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, polyether polyols, polyanions, polypeptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants for use in humans, BCG (Mycobacterium tuberculosis) and Corynebacterium parvum are particularly useful.

Monoclonal antibodies or any fragment or oligopeptide thereof against heat shock proteins derived from candida species, such as candida albicans hsp90, can be prepared from continuously culturable cell lines by any method for producing antibody molecules. These methods include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV (EB virus) hybridoma technology (Koehler et al, 1975, Nature (Nature),256: 495-497; kosbor et al, 1983, Immunl.today (immunization today)4: 72; cote et al, 1983, PNAS USA,80: 2026-; cole et al, 1985, Monoclonal Antibodies and Cancer Therapy (Monoclonal Antibodies and Cancer Therapy), Alan R.Liss Inc, New York, pp 77-96).

In addition, with the development of "chimeric antibody" technology, the appropriate antibody can be obtained by splicing the murine antibody gene with the human antibody geneMolecules with proto-specificity and biological activity (Morrison et al, 1984, PNAS USA)81: 6851-6855; neuberger et al, 1984, Nature (Nature),312: 604-608; takeda et al, 1985, Nature (Nature),314: 452-454). Alternatively, single chain antibodies specific for candida heat shock proteins can be produced using methods known in the art, using techniques already described for producing single chain antibodies. Antibodies with the relevant specificities, but with different idiotypic compositions, can also be produced by chain shuffling from randomly combined immunoglobulin libraries (Burton, d.r., 1991, PNAS USA,88，11120-11123)。

antibodies can also be produced by induction of lymphocyte cell lines in vivo, or by screening of recombinant immunoglobulin libraries, or by panel screening of high-specific binding reagents (Orlandi et al, 1989, PNAS USA,86: 3833-3837; winter G et al, 1991, nature,349：293-299)。

antigen-binding fragments, such as F (ab')₂Fragments may be obtained by digestion of the antibody molecule with pepsin, and Fab fragments by reducing F (ab')₂Disulfide bonds of the fragments. Alternatively, monoclonal Fab fragments of the desired specificity can be quickly and simply identified by constructing a Fab expression library (Huse et al, 1989, Science,256：1275-1281)。

various immunoassays can be used to screen for antibodies identified with the desired specificity. A variety of such methods are well known in the art and competitive binding or immunoradiometric assays can be performed using either polyclonal or monoclonal antibodies of established specificity. Such immunoassays specifically involve the measurement of complex formation of heat shock proteins derived from candida species, such as hsp90 of candida albicans or any fragment or oligopeptide thereof, with specific antibodies thereto. Can adopt a double-point single gramImmunological analysis of the clones, in which two unrelated epitopes specific for Candida heat shock proteins were used, but a competitive binding assay (Maddox et al, 1983, J.Exp.Med. (J.Imagel.),158：1211-1216)。

the antibody may comprise SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

The antifungal polyene agent may contain, for example, amphotericin B, a derivative of amphotericin B, or nystatin. Derivatives of amphotericin B may be used including preparations such as liposomal amphotericin B (samples provided by NexStar pharmaceutical company, cambridge, england), amphotericin B liposome complex (Abelcet), amphotericin B colloidal dispersion (Amphocil) and amphotericin B intralipid (Burnie JP, 1997, supra). Amphotericin B can be used in combination with another antifungal agent, 5-fluorocytosine (Burnie JP, 1997, supra).

The echinocandin antifungal agent may be, for example, LY303366(Eli Lilly corporation, Indianapolis, usa). Echinocandins are cyclic lipopeptides that inhibit the synthesis of beta-1, 3 glucan in fungi (Redding, JA et al, 1998, antimicrob. Agents Chemo. ther. (antimicrobial chemotherapeutics)42(3)：1187-1194)。

The present invention also provides a composition as described herein for use in the treatment of fungal infections. The fungal infection that can be treated with the composition or combined preparation may be caused by Candida, Cryptococcus, Histoplasma, Aspergillus, Torulopsis, Mucor, Trichosporon, Coccidioides or Paracoccidioides. Coccidioidomycosis is also referred to in the art as coccidioidomycosis, while paracoccidioidomycosis is a synonym for coccidioidomycosis. Fungal infections may be resistant to the antifungal agents themselves, i.e., fungal infections themselves cannot be treated with specific agents, since these specific antifungal agents are traditionally ineffective for themselves.

The invention also provides a composition or combined preparation as described herein for use in a method of treatment of a fungal infection of the human or animal body.

The invention also provides a method of manufacture of a medicament for the treatment of fungal infections in humans or animals, characterised by the use of a composition or combined preparation as described herein. Methods for the production of pharmaceutical agents are well known. For example, the medicament may additionally contain a pharmaceutically acceptable carrier, diluent or excipient (Remington's Pharmaceutical Sciences and USPharmacopoeia, 1984, Mack Publishing Company, Easton, Pa., USA).

The invention also provides the use of a composition or combined preparation as described herein in a method of manufacture of a medicament for the treatment of a fungal infection. Fungal infections may be resistant to the infecting agent itself.

The invention also provides a method for the treatment of fungal infections of the human or animal body comprising administering to a patient in need thereof a composition or combined preparation according to the present application. The exact dose (i.e., pharmaceutically acceptable dose) of the composition or combined preparation administered to a patient can be readily determined by one skilled in the art, e.g., by applying simple dose-dependent experiments. The composition or combined preparation can be administered orally.

The present invention further provides a kit comprising an antibody or antigen-binding fragment thereof which specifically binds to one or more epitopes of a fungal stress protein, a fungicide comprising at least one member selected from the group consisting of a polyene antifungal agent and an echinocandin antifungal agent. The kit can be used for treating fungal infection.

The invention also provides a polypeptide comprising SEQ ID NO: 1 or an antigen-binding fragment thereof.

The antibodies of the invention may have diagnostic value. Thus, for diagnostic purposes, the antibodies can be used to detect the presence of a stress protein in a host to determine whether the host has a specific fungal infection, such as an infection due to Candida, Cryptococcus, Histoplasma, Aspergillus, Torulopsis, Mucor, Trichosporon, Coccidioides or Paracoccidioides. For example, for diagnosing fungal abscesses, particularly hepatic candidiasis, and/or monitoring the course of treatment for such infections. Such diagnostic methods form a further object of the invention and may use standard techniques such as immunological methods like enzyme-linked immunosorbent, radioimmunoassay, latex agglutination or immunoblotting.

The antibodies of the invention may be labelled with a detectable label or conjugated using conventional means with an effector molecule such as a drug, for example an antifungal agent such as amphotericin B or flucytosine or a toxin such as ricin, or an enzyme. The invention extends to such labelled antibodies or antibody conjugates.

The invention also provides a formulation of a diagnostic agent. The preparation is used for diagnosing one or more fungal infections by using the antibody or antigen binding fragment of the invention. The diagnostic agent may be provided by a kit. The kit may include instructions for diagnosing one or more fungal infections. Kits described herein are also provided according to the invention.

If desired, mixtures of antibodies according to the invention may be used for diagnosis or therapy, e.g.mixtures of two or more antibodies recognizing different epitopes of a fungal stress protein, and/or mixtures of antibodies of different kinds, e.g.mixtures of IgG and IgM recognizing the same or different epitopes of the invention.

The contents of each reference discussed herein, including the references cited therein, are incorporated by reference in their entirety.

The invention will be further apparent from the following description which shows by way of example only specific embodiments of the compositions of the invention and experiments thereon.

Detailed Description

Experiment of

The experiments described below investigated the antifungal effects of antibodies against hsp90 antigen from candida albicans in combination with antifungal agents such as amphotericin B or fluconazole. The results show that in some cases the combined use of an antibody and an antifungal agent results in an enhanced antifungal effect compared to the use of either compound alone. Experiments prove that the amphotericin B and an antibody against Candida albicans hsp90 have extremely surprising strong synergistic effects on various common fungal pathogens which are difficult to solve. The synergistic effect has very important significance for clinically treating fungal infection. A preliminary clinical trial involving four Candida infected patients demonstrated the effectiveness of the present invention in humans.

Materials and methods

The strain is as follows:

the experiment was carried out using a non-Aspergillus yeast strain (Table 1) spread on Sabouraud's dextran agar (Oxoid, Basingstoke, UK) and incubated at 37 ℃ for 24 hours. Strains were identified using the API 20C system (BioMerieux, MarcyL' Etoile, France). If desired, the identification can be confirmed by morphological microscopic examination on corn meal agar (Oxoid).

Isolates of all species of Aspergillus (Table 1) were cultured on Sabouraud dextran agar (Oxoid, Basingstoke, UK) for 24 hours at 35 ℃.

TABLE 1 sources of the strains
		Bacterial strain	Reference to the literature
1. Candida albicans (fulminant type) 2 Candida albicans (fluconazole resistant) 3 krusei candida krusei (FA/157)4 Candida tropicalis	B.M.J.，1985， 290：746-748Opportunistic Pathogens，1997， 1：27-31Int J Systemic Bacteriol 1996， 46: 35-40 national fungal pathogen Collection

(NCPF #3111)5 Candida parapsilosis (NCPF #3104)6 Torulopsis glabrata (NCPF #3240)7 Aspergillus fumigatus (NCPF #2109)8 Aspergillus flavus 9 Aspergillus niger

National fungal pathogen Collection clinical isolates of the national fungal pathogen Collection, identification of clinical isolates by canonical morphology, identification by canonical morphology

Inoculum:

for non-Aspergillus, a suspension was prepared by suspending individual colonies (1 mm. or more in diameter) in 5 ml of sterilized 0.85% saline to form 1X 10⁴The concentration of cells/ml is obtained by counting with a haemocytometer counting grid. For Aspergillus, see below.

Antifungal agents:

amphotericin B was purchased as a lyophilized powder from Sigma (Poole, Dorset) and used for intravenous injection (Fungizone). Fluconazole is supplied by Pfizer as an intravenous solvent (Diflucan). Amphotericin B was dissolved in dimethyl sulfoxide at a concentration of 1.2mg/ml, and fluconazole was also dissolved in 0.85% physiological saline at a concentration of 1.2 mg/ml. The stock solution was stored at-70 ℃ until use. Abelcet (liposomal amphotericin B) used in clinical studies was produced by Bristol-Meyers Squid (USA) and formulated according to the manufacturer's instructions.

Antibody:

previous DNA sequences for an antibody specific for an antigenic determinant of candida albicans hsp90, as disclosed in british patent 2240979 and EP patent 0406029, were genetically engineered by codon optimization for expression in e.coli (Operon Technologies Inc, Alameda, CA, usa) and inserted into e.coli expression vectors. The amino acid sequence of the anti-hsp 90 antibody of the invention comprises SEQ ID NO: 2 (including the heavy chain, light chain and spacer domains). The antibodies of the invention recognize a polypeptide comprising SEQ ID NO: 1, or an antigenic determinant thereof.

Anti-hsp 90 antibodies were expressed in E.coli hosts and purified by affinity chromatography and imidazole exchange columns to a purity of up to 95%. Standard molecular biology methods are used (see, e.g., Harlow and Lane, supra; Sambrook J. et al, 1989, molecular cloning: A Laboratory Manual: (Amersham pharmacia))Molecular cloning: laboratory manual) Second edition, Cold spring harbor laboratory Press,cold spring harbor, new york; sambrook j. and Russell d., 2001, Molecular Cloning: a Laboratory Manual (Molecular cloning: laboratory manual) Third edition, cold spring harbor laboratory press, cold spring harbor).

The preparation of the mycograb (rtm) formulation is as follows: vials containing 10 mg of pure anti-hsp 90 antibody, 150 mg of pharmaceutical grade (Ph Eur) urea and 174 mg of L-arginine (PhEur) were redissolved in 5 ml of water.

Analyzing the culture solution:

RPMI broth was prepared by adding 0.3 g glutamine per liter of RPMI1640 broth (Sigma R7880) and then buffering with 34.6 g/liter morpholinopropanesulfonic acid (MOPS) to adjust ph to 7.0.

Broth microdilution experiments:

both stock solutions were diluted two-fold in RPMI broth (amphotericin B was diluted from 40 to 0.024 mg/ml, fluconazole was diluted from 400 to 0.4 mg/ml). 100 μ l of inoculum suspension diluted 1: 10 (corresponding to 1X 10)³Colony forming units) were added to the microtiter plates. Then 50. mu.l of antifungal agent and 50. mu.l of antibody were added thereto. The antibody may be in stock solution (0.4 mg/ml) or in 1/10 or 1/100 dilutions. In the absence of antibody, the volume of 50 microliters was made up with RPMI. The total volume per well was 200 microliters. The final concentration of antibody in the experiment was 100. mu.g/ml (stock), 10. mu.g/ml (1/10 antibody), or 1. mu.g/ml (1/100 antibody).

The plates were incubated overnight at 37 ℃ and the Minimum Inhibitory Concentration (MIC) was defined as the lowest concentration that inhibited growth.

Colonies were counted in culture wells with macroscopic reduction in yeast growth. Results are expressed as colony forming units per ml broth (cfu/ml).

Study of Aspergillus:

isolates of A.fumigatus, A.flavus and A.niger were prepared from RPMI1640 medium. InoculumThe final concentration of the suspension was 1X 10 per ml⁴Conidia, then added to flat bottom microtiter plates at 100 μ l/well, respectively. Amphotericin B was serially double diluted (concentrations ranging from 250 to 0.75 μ g/ml) and added to the corresponding titration wells. Mycograb was added to each well to a final concentration of 100 microgram/ml in the formulation buffer. The series control for each isolate contained formulation buffer only. The assay plates were then incubated at 35 ℃/200rpm for 48 hours, with the Minimum Inhibitory Concentration (MIC) value for each isolate being determined in titration wells with sterile growth.

Animal cooperativity:

30 CD1 mice (approximately 25 g of each body weight) were inoculated by injection with 100. mu.l of a fulminant Candida albicans strain (equivalent to 1.5X 10)⁷cfc), mice were divided into three groups 2 hours later and injected:

(A) a first group: 100 μ l of 10 mM ammonium acetate solution (AAT, pH9) followed by 100 μ l of amphotericin B formulated in 5% (w/v) glucose solution, corresponding to 0.6 mg/kg;

(B) second group: 100 microliters of a 10 millimolar ammonium acetate solution (pH9) containing 500 micrograms of anti-hsp 90 antibody, followed by 100 microliters of amphotericin B formulated in 5% (w/v) glucose solution, equivalent to 0.6 mg/kg;

(C) third group: 100 microliters of a 10 millimolar ammonium acetate solution (pH9) containing 50 micrograms of anti-hsp 90 antibody was followed by 100 microliters of amphotericin B formulated in 5% (w/v) glucose solution, corresponding to 0.6 mg/kg.

Animals were culled 48 hours later and yeast counts of liver, spleen and kidney tissues were performed on Sabrow plates.

Clinical study:

open label safety and pharmacokinetic studies of anti-hsp 90 antibody (in the form of Mycograb, supra) were performed in both the manchester healthcare center of manchester, uk and the Wythenshawe hospital, with four candida-infected patients attending. The candida pyogenic disease sign detection is carried out on the patient and comprises the following steps: carrying out candida albicans positive culture on a plurality of parts or deep wound surfaces; high temperature or variable temperature (exothermic) detection; high pulse rate (tachycardia) and high white blood cell count (WBC).

Following conventional Abelcet (liposomal amphotericin B) and/or fluconazole treatment, patients were then given various doses of Mycogarb, including alternative experimental doses (0.1 mg/kg) and therapeutic doses of 1 mg/kg. Patients were monitored for signs of clinical and laboratory infection (monitored laboratory parameters include blood chemistry, hematology and clotting factors) and Mycograb levels in patient serum and urine were measured.

Results

In vitro experiments to examine the effect of the combination of anti-candida albicans hsp90 antibody (antibody) and antifungal agent are shown in tables 2 to 18. The results of the animal experiments are shown in Table 19.

In vitro experiments:

composition comprising antibody and fluconazole:

table 2 shows the Minimum Inhibitory Concentrations (MICs) of fluconazole against the fungal pathogens tested, with or without different dilutions of anti-hsp 90 antibody, the MICs being evaluated in broth microdilution experiments. In the presence of the antibody as a stock solution or in a 10-fold dilution, the Minimal Inhibitory Concentration (MIC) of the candida albicans fulminans strain decreased by 4-fold (1.56-0.39. mu.g/ml fluconazole), while the antibody diluted 100-fold decreased the Minimal Inhibitory Concentration (MIC) of fluconazole by 2-fold.

In the presence of the antibody as a stock solution, a slight decrease in the Minimum Inhibitory Concentration (MIC) of fluconazole-resistant candida albicans and candida krusei was observed. When the antibodies were diluted 1: 10 or 1: 100, no effect was observed on the Minimum Inhibitory Concentration (MIC) of fluconazole of these strains, compared with the antibody-free samples.

Anti-candida albicans hsp90 antibody had no discernable effect on fluconazole Minimum Inhibitory Concentration (MIC) for the remaining strains, such as candida tropicalis, candida parapsilosis, and torulopsis glabrata.

TABLE 2 Minimum Inhibitory Concentration (MIC) for fluconazole
						MIC (microgram/ml)
Fluconazole Antibody-free antibodies	Fluconazole Stock solution antibody [100μg/ml]	Fluconazole 1/10 antibodies [10μg/ml]	Fluconazole 1/100 antibodies [1μg/ml]
				Candida albicans outbreak type strain Candida albicansCandida krusei-resistant Candida krusei-Candida tropicalis Candida parapsilosis		1.56251003.1251.566.25	0.3912.5503.1251.566.25	0.39251003.1251.566.25	0.78251003.1251.566.25

Further quantitative experiments on the number of viable cell colonies for each fungal strain at different concentrations of fluconazole, different dilutions of anti-candida albicans hsp90 antibody are shown in table 2.

The survival of Candida albicans (burst type) was not reduced by the addition of antibody stock or 1: 100 dilution at the different concentrations of fluconazole tested (Table 3).

TABLE 3 colony count of fluconazole by Candida albicans (burst type) (in cfc/ml)
					Fluconazole concentration (μ g/ml)
0.09	0.19	0.39
			Antibody-free stock solution [ 100. mu.g/ml ]]1/100 antibody [ 1. mu.g/ml]		3.6×1053.6×1061×106	1×1051.3×1052.6×104	5×1041.3×1045.3×104
(control 6X 106cfc/ml)

For fluconazole-resistant candida albicans strains, a 2-fold decrease in colony survival was observed at a fluconazole concentration of 12.5 μ g/ml in the presence of antibody as stock solution (table 4). In the presence of the antibody as a stock solution at low concentrations of fluconazole, the survival rate of the same strain was slightly reduced, whereas at a dilution of the antibody of 1: 100, there was no visible effect.

TABLE 4 colony count of Fluconazole by Candida albicans Fluconazole-resistant strains (in cfc/ml)
						Fluconazole concentration (μ g/ml)
1.56	3.12	6.25	12.5
				Antibody-free stock solution [ 100. mu.g/ml ]]1/100 antibody [ 1. mu.g/ml]		3×1072×1063×107	1.3×1074.3×1051.1×107	3×1065.6×1041.1×107	6×1066×1036.3×106
(control 2.6X 10)7cfc/ml)

The antibody stock or dilution was not significantly antifungal to candida krusei within the range of fluconazole concentrations tested (table 5).

TABLE 5 colony counts of Fluconazole by Candida krusei (in cfc/ml)
				Fluconazole concentration (μ g/ml)
25	50
		Antibody-free stock solution [ 100. mu.g/ml ]]1/100 antibody [ 1. mu.g/ml]		3.2×1078.3×1061.3×106	1.6×1076×1061.6×106
(control 1X 10)7cfc/ml)

The concentration of each fluconazole tested had no significant effect on the survival of candida tropicalis, regardless of the presence of the antibody (table 6).

TABLE 6 colony count of fluconazole by Candida tropicalis (in cfc/ml)
					Fluconazole concentration (μ g/ml)
0.09	0.19	0.39
			Antibody-free stock solution [ 100. mu.g/ml ]]1/100 antibody [ 1. mu.g/ml]		5×1057×1051×105	6×1036.3×1046×103	6×1029×1022×103
(control 1.6X 106cfc/ml)

Table 7 shows that the presence or absence of the antibody had no effect on colony survival of Torulopsis glabrata at each concentration of fluconazole tested.

TABLE 7 colony count of fluconazole by Torulopsis glabrata (in cfc/ml)
					Fluconazole concentration (μ g/ml)
0.39	0.78	1.56
			Antibody-free antibody stock solution		2×1071.5×107	1×1071.2×107	6×1049.3×105

[100μg/ml]1/100 antibody [ 1. mu.g/ml]	2.3×107	1.9×107	2×105
				(control 4.3X 107cfc/ml)

Table 8 shows that the presence or absence of the antibody has no significant effect on the colony survival rate of Candida parapsilosis at different concentrations of fluconazole detected.

TABLE 8 near smoothing	Colony count of Fluconazole by Candida (in cfc/ml)
						Fluconazole concentration (μ g/ml)
0.78	1.56	3.13	6.25
				Antibody-free stock solution [ 100. mu.g/ml ]]1/100 antibody [ 1. mu.g/ml]		7×1068.6×1064×105	5.6×1062.3×1063×106	2.6×1061.6×1062.3×106	3×1061.6×1065×106
(control 1X 10)7cfc/ml)

Composition of antibody and amphotericin B:

table 9 shows the Minimum Inhibitory Concentrations (MIC) of amphotericin B against the fungal pathogens tested in the absence of antibody or anti-Candida albicans hsp90 antibody at various dilutions, as measured by broth microdilution.

In comparison with the results for fluconazole (see tables 2-8 above), all strains tested here showed at least a 4-fold decrease in the Minimum Inhibitory Concentration (MIC) of amphotericin B when undiluted antibody was added to the culture broth (table 9). In addition, in all the strains tested, the Minimum Inhibitory Concentration (MIC) of amphotericin B was reduced at least 2-fold (final concentration of antibody 1. mu.g/ml) even when 100-fold dilution of the antibody was added to the culture broth.

The most significant effect observed with the antibody and amphotericin B containing compositions when lowering the Minimum Inhibitory Concentration (MIC) of amphotericin B was the fluconazole resistant candida albicans strain. The antibody stock solution reduced the Minimum Inhibitory Concentration (MIC) of amphotericin B by 10-fold, even 100-fold

The antibody, diluted by a factor of two, also reduced the Minimum Inhibitory Concentration (MIC) of amphotericin B by approximately 25% (table 9).

TABLE 9 Minimum Inhibitory Concentration (MIC) of amphotericin B
						MIC (microgram/ml)
Amphotericin Antibody-free antibodies	Amphotericin Stock solution antibody [100μg/ml]	Amphotericin 1/10 antibodies [10μg/ml]	Amphotericin 1/100 antibodies [1μg/ml]
				Candida albicans outbreak type Candida albicans fluconazole drug-resistant Candida krusei Candida tropicalis Candida glabrata Candida tropicalis		0.1560.3120.6250.0780.0390.625	0.0390.0390.1560.019＜0.0090.156	0.0390.0780.3120.039＜0.0090.313	0.0780.0780.3120.0390.0190.313

The results of detailed experiments conducted to quantify the number of viable colonies of cells from each fungal strain at different concentrations of amphotericin and different dilutions of anti-candida albicans hsp90 antibody are shown in table 9.

Table 10 shows the survival of the exploded Candida albicans strain in the presence or absence of antibodies when cultured with amphotericin B. The antibody significantly reduced the number of surviving colonies (at least 10-fold) in all amphotericin concentrations tested. For example, the survival rate of Candida albicans (burst type) was 0.2% at a 100-fold dilution of the antibody, compared to the survival rate of the strain in the absence of the antibody, at a concentration of 0.078. mu.g/ml amphotericin B. Amphotericin B was present at a concentration of 0.078 μ g/ml and the inhibitory effect of the antibody at 100 dilution was equivalent to the survival of the strain in amphotericin B (no antibody) at 0.156 μ g/ml. Thus, even at relatively low concentrations of antibody, the amount of amphotericin B required to achieve a characteristic mortality rate for the strain can be reduced.

TABLE 10 colony counts of outbreak Candida albicans on amphotericin B (in cfc/ml)

	Amphotericin B concentration (μ g/ml)
					0.019	0.039	0.078	0.156
	Antibody-free stock solution [ 100. mu.g/ml ]]1/10 antibody [ 10. mu.g/ml]1/100 antibody [ 1. mu.g/ml]	1×1075.3×1055×1056.6×106	1.6×1076×1031×1043.2×105	4.1×1053×1023.0×1028.0×102					1.3×1034.3×1023×1011×102
(control 1X 10)7cfc/ml)

Table 11 shows the colony survival rate of candida albicans (fluconazole resistant strain) at different amphotericin B concentrations and different antibody dilutions. At low concentrations of antifungal agent, there was no significant effect of antibody or amphotericin B. However, when amphotericin B levels were close to the minimum inhibitory dose of the antifungal (see table 9 above), the antibodies had a significant effect on colony survival. For example, at a concentration of 0.078. mu.g/ml amphotericin B, the survival rate of the strain was 0.1% at 100-fold dilution of the antibody, as compared to the survival rate of the strain without the antibody.

TABLE 11 colony counts of Candida albicans Fluconazole resistant strains for amphotericin B (in cfc/ml)
					Amphotericin B concentration (μ g/ml)
0.019	0.039	0.078
			Antibody-free stock solution [ 100. mu.g/ml ]]1/10 antibody [ 10. mu.g/ml]1/100 antibody [ 1. mu.g/ml]		4.6×1065.3×1052.2×1063.4×106	4.3×1063.0×1036.3×1041.6×105	6×1063×1031.6×1035.3×103
(control 1.6X 107cfc/ml)

Colony survival of C.krusei in the presence of amphotericin B and varying concentrations of antibody

Shown in table 12. Under the conditions of high amphotericin B concentrations tested, the antibodies were found to be very effective against this strain. Even at 100-fold dilution, when the concentration of amphotericin B was 0.312 μ g/ml, the number of colonies of candida krusei detectable compared to that without antibody was 0.01%.

TABLE 12 colony counts of Candida krusei on amphotericin B (in cfc/ml)
					Amphotericin B concentration (μ g/ml)
0.078	0.156	0.312
			Antibody-free stock solution [ 100. mu.g/ml ]]1/10 antibody [ 10. mu.g/ml]1/100 antibody [ 1. mu.g/ml]		1×1071×1066.3×1061.6×107	1.7×1071.76×1051.6×1051.53×106	9×105＜102＜1021.3×102
(control 1X 10)7cfc/ml)

For all concentrations of amphotericin B detected (range 0.019-0.156 μ g/ml), antibodies were observed to reduce colony survival of candida tropicalis strains (table 13). This effect is enhanced at high concentrations of antibody and amphotericin B.

TABLE 13 colony counts of Candida tropicalis on amphotericin B (in cfc/ml)
						Amphotericin B concentration (μ g/ml)
0.019	0.039	0.078	0.156
				Antibody-free stock solution [ 100. mu.g/ml ]]1/10 antibody [ 10. mu.g/ml]1/100 antibody [ 1. mu.g/ml]		1.3×1061.1×1041×1041.1×106	1×1062.3×1043.4×1042×104	2.6×1052×1024×1022.6×103	6.6×10303×1010

(control 1.6X 106cfc/ml)

Table 14 shows the survival rate of Torulopsis glabrata in the presence of various concentrations of amphotericin B and antibody. At all amphotericin B concentrations tested, a significant inhibitory effect of the antibody on the growth of torulopsis glabrata was observed. For example, when the concentration of amphotericin B was 0.009 μ g/ml, the inhibition rate of the growth of the strain by diluting the antibody 100 times was 99.2%, the concentration of amphotericin B was 99.99% at 0.019 μ g/ml, and the concentration of amphotericin B was 99.91% at 0.039 μ g/ml.

TABLE 14 colony counts of Torulopsis glabrata for amphotericin B (in cfc/ml)
					Amphotericin B concentration (μ g/ml)
0.009	0.019	0.039
			Antibody-free stock solution [ 100. mu.g/ml ]]1/10 antibody [ 10. mu.g/ml]1/100 antibody [ 1. mu.g/ml]		1.1×1078.6×1039×1059×104	9.6×1068.3×1026.3×1031×103	1.4×1052.6×1022.3×1021.3×102
(control 1X 10)7cfc/ml)

The survival rates of the fungal Candida parapsilosis at different levels of antibody and amphotericin B are shown in Table 15. For all concentrations of amphotericin B tested, antibody stocks were observed to reduce the survival of the strain. When the antibody is at a low concentration, the effect is lower than that of the other strains tested.

TABLE 15 colony counts of Candida tropicalis on amphotericin B (in cfc/ml)
					Amphotericin B concentration (μ g/ml)
0.156	0.313	0.626

Antibody-free stock solution [ 100. mu.g/ml ]]1/10 antibody [ 10. mu.g/ml]1/100 antibody [ 1. mu.g/ml]	1.46×1071.3×1051.03×1071.8×107	3×1063×1041.76×1052.9×105	6.3×1036.0×1026.0×1033.3×103
				(control 1X 10)7cfc/ml)

Composition containing antibody but no antifungal agent:

in a further experiment, the effect of different concentrations of anti-candida albicans hsp90 antibody (without antifungal agent) on different fungal strains (as used in tables 2-15 above) was examined. The results shown in Table 16 indicate that for most of the strains tested, the antibodies themselves had no effect on the survival of these strains. However, for the strains Torulopsis glabrata, Candida tropicalis and Candida parapsilosis, it was observed that antibodies themselves reduced their partial survival rate.

TABLE 16 influence of the antibody on the growth of the yeasts by itself (expressed as cfc/ml)
							Antibody Candida albicans (μ g/ml)	Candida albicans	Candida krusei	Torulopsis glabrata	Candida tropicalis	Candida parapsilosis	(burst type) (Fluconazole resistance)
00.3130.6251.252.5	1.2×1075.6×1063.3×1065.0×1065.3×106	1×1076×1065.3×1065.6×1066.3×106	3.3×1071.6×1069.3×1066.6×1064.3×106	1.3×1071.2×1071×1076.6×1066×106	1×1066.0×1053.3×1053.6×1059×105	7.0×1062.6×1043.0×1041.6×1046.6×13

All aspergillus experiments:

the Minimum Inhibitory Concentration (MIC) of Aspergillus fumigatus for amphotericin B was 2.5. mu.g/ml. After addition of Mycograb, the MIC became 0.125. mu.g/ml (2-fold decrease). The Minimum Inhibitory Concentration (MIC) of Aspergillus flavus for amphotericin B was 2.5. mu.g/ml. After addition of 100. mu.g/ml Mycograb, the MIC became 0.125. mu.g/ml (2-fold decrease). The Minimum Inhibitory Concentration (MIC) of A.niger for amphotericin B was 2.5. mu.g/ml. After addition of 100. mu.g/ml Mycograb, the MIC became 0.125. mu.g/ml (2-fold decrease).

Summary of in vitro test results

The results in tables 2-16 show that anti-candida albicans hsp90 antibody can inhibit the growth of certain fungi by itself, and that surprisingly high antifungal activity was observed for all strains tested when the antibody was used in combination with amphotericin B. This surprising effect between antibody and amphotericin B was not found in other antifungal agents tested; the use of fluconazole in combination with an antibody does not produce obvious and potentially useful results.

Using a cut-off criterion with a 4-fold difference in MIC, the data in Table 2 show that fluconazole was effective against only fulminant Candida albicans when used in combination with either a stock solution of antibody (final concentration 100. mu.g/ml) or a 10-fold dilution of antibody (final concentration 10. mu.g/ml). However, when the same criteria were applied, it can be seen from table 9 that amphotericin B was effective against all strains tested when used in combination with antibody stock or 10-fold dilutions. It can therefore be concluded that there is a strong synergy between amphotericin B and the anti-candida albicans hsp90 antibody when measured by an improvement in the MIC.

For experiments quantifying fungal colonies treated with different antifungal agents and antibodies (see tables 3-8 and 10-15 above), another different cut-off criterion, defined as a 2 log (100 fold) drop in viable colonies, can be cited to test the possible effectiveness of the combination therapy.

A summary of the efficacy of the combination therapy of fluconazole and anti-candida albicans hsp90 antibody is shown in table 17. Here, the lowest concentration of fluconazole (or the highest concentration used in the experiment) that can function as desired is shown and the cut-off criterion suggests that the survival rate of the fungus may be reduced by at least 100-fold. The results show that when fluconazole and the antibody stock solution are used together, only the fluconazole-resistant strain of candida albicans can produce obvious effect.

TABLE 17 summary of in vitro experiments with fluconazole
					Fluconazole (μg/ml)	Antibody stock solution [100μg/ml]	Watch (A)

Candida albicans outbreak type Candida albicans fluconazole drug-resistant type Candida krusei candida tropicalis Torulopsis glabrata candida parapsilosis	0.3912.5500.391.566.25	-+----	345678
				+ denotes that the cutoff criterion is met; -indicating that the cut-off criterion is not fulfilled; by cut-off criterion is meant that colony counts are reduced by at least 100-fold

A summary of the efficacy of amphotericin B in combination with anti-Candida albicans hsp90 antibody is shown in Table 18. It can be seen that the antibody stock solution meets the cut-off criteria for all tested strains (100-fold reduction in fungal colony growth), that the 10-fold dilution of the antibody pair (burst and fluconazole-resistant) candida albicans, candida krusei and torulopsis glabrata and that the 100-fold dilution of the antibody pair (burst) candida albicans and torulopsis glabrata meet the cut-off criteria.

Notably, the synergistic effect between amphotericin B and the antibody was observed not only against fluconazole-sensitive candida albicans strains, but also against fluconazole-resistant candida albicans strains and yeasts such as candida krusei and torulopsis glabrata, which are themselves resistant to fluconazole.

TABLE 18 summary of in vitro experiments with amphotericin B
							Amphotericin (μg/ml)	Antibodies Stock solution [100μg/ml]	Antibodies 1/10 [10μg/ml]	Antibodies 1/100 [1μg/ml]	Watch (A)
Candida albicans outbreak type candida albicans fluconazole resistant form	0.0390.039	++	++	+-	1011

Candida krusei Candida tropicalis Torulopsis glabrata Candida parapsilosis	0.1560.0190.0090.156	++++	+-+-	--+-	12131415
						+ denotes that the cutoff criterion is met; -indicating that the cut-off criterion is not fulfilled; by cut-off criterion is meant that colony counts are reduced by at least 100-fold

All aspergillus experimental results show a synergistic effect between amphotericin B and Mycograb on the most common aspergillus.

(2) Animal experiments:

mice infected with the outbreak of candida albicans strain were divided into three groups, treated with amphotericin B alone (first group), amphotericin B and 500 micrograms of anti-hsp 90 antibody (second group), and amphotericin B and 50 micrograms of anti-hsp 90 antibody (third group). The results of yeast colony counts for various tissues of the mice after 48 hours of treatment are shown in Table 19. The results show that the yeast numbers of animals treated with amphotericin B and 500 micrograms of antibody (second group) are significantly reduced (by at least one order of magnitude) compared to animals treated with amphotericin B alone (first group). The yeast count was also reduced in animals treated with amphotericin B and 50 micrograms of antibody (third group) compared to animals treated with amphotericin B alone (first group). Thus, in vivo data confirmed in vitro data demonstrating a synergistic effect between the anti-hsp 90 antibody and the antifungal agent amphotericin B in the treatment of fungal infections.

TABLE 19 colony counts of Candida albicans (burst type) in the treated group mouse tissues
					Colonies (cfc/ml, log 10. + -. standard deviation)
First group	Second group	Third group
			Kidney, liver and spleen		6.80±0.9164.26±1.424.18±1.18	4.42±1.283.22±0.0283.07±0.089	4.35±1.373.83±1.003.94±1.25

(3) Clinical study:

patients with candida infection at position 4 and who were not responsive to conventional antifungal therapy were given a combination therapy of an antifungal agent and an anti-hsp 90 antibody in the form of Mycograb (see above) and their disease status was monitored.

Patient 1 was initially diagnosed with acute pancreatitis and had postoperative Adult Respiratory Distress Syndrome (ARDS), requiring ventilation. The patients have positive extracellular culture of Candida albicans in pancreatic gland beds. The patient's white blood cell count (WBC) is very high (78.4), although this count varies greatly and may not be solely due to sepsis alone. Treatment was initially with 3mg/kg Abelcet.

5 days after the initiation of Abelcet treatment, patient was given an additional 1 first dose of a 0.1mg/kg test dose of Mycogarb (day 1). On day 3, the patient was given a clinical dose of 1mg/kg of Mycogarb. Patients were discontinued from Abelcet and Mycograb therapy on day 3 after administration of clinical doses of Mycograb for a variety of reasons, such as low platelet count for at least 4 days. However, after 6 days (day 9) patient 1 had additional Candida albicans growth in the ascites and was given fluconazole treatment (400 mg). The following day (day 10), the patient was given the last two clinical doses of Mycogarb, each dose being 1 mg/kg.

Despite the fact that patient 1 had been treated for 7 days of Abelcet, the patient had Candida albicans growth at the tracheostoma site and was tachycardia before the patient was treated with Mycogab at the clinical dose from day 3. The combination treatment of Abelcet and Mycograb given on day 3 resulted in no candida albicans growth for the following 5 days. No changes in blood chemistry, hematology and coagulation factors associated with Mycograb were observed during Mycograb treatment. The combination treatment with fluconazole and Mycograb for subsequent relapse (two doses on day 10) was less successful than expected for the synergistic results of in vitro experiments (see, e.g., tables 2 and 17), but eventually the patient did recover from candidiasis.

Serum levels of Mycograb at different time intervals after administration of Mycograb therapy for patient 1 are listed in table 20. The test dose given on day 1 did not reach the serum test level. The 1mg/kg dose administered on day 3 and day 10 reached serum levels comparable to the synergy with amphotericin B in vitro experiments (see tables 9 and 18). After administration of the second dose on day 10, serum levels of Mycograb increased, suggesting accumulation of Mycograb in certain tissues after the first dose. Mycograb at a dose of 1.0mg/kg was detectable in urine (data not shown).

TABLE 20 Mycograb levels in patient 1 serum (expressed in. mu.g/ml)
					Time (hours)	Day 1	Day 3	Day 10	Day 10
0.1mg/kg	1.0mg/kg	1.0mg/kg body weight First agent	1.0mg/kg body weight Second agent
				00.51.02.04.06.08.012.024.048.0		0000000000	04.02.52.51.0-0000	03.01.20.50.30.10: then administering a second dose	03.01.41.00.40.10

Patient 2 was diagnosed with small bowel stenosis due to adhesions and had adult respiratory distress syndrome requiring ventilation. Multiple sites of patients included ascites with Candida albicans growth, and infection-related fluctuating body temperatures (35.8-38.2 ℃), elevated white blood cell counts (11.4), and occasional tachycardia (110). Patients were initially treated with 3mg/kg of Abelcet.

Patient 2 still had fluctuating body temperature retention, elevated white blood cell counts and occasional tachycardia 4 days after the onset of Abelcet treatment. The patient was given a test dose of 0.1mg/kg of Mycogarb (day 1). The following day, the patient was given a 1mg/kg clinical dose of Mycogarb (day 2). To complete a 5 day treatment course, the patient is given the last additional dose of Abelcet on day 2. After 2 days (day 4), the patient received the last 2 clinical doses of Mycograb.

The patient is well tolerated by Mycograb. Clinical doses of Mycograb given on day 2 caused the body temperature to drop and stabilize (38.2-36.7 deg.C after receiving clinical doses of Mycograb on day 2, maintained at 36.7-37.4 deg.C until day 3), and the white blood cell count decreased (from 11.9 to 9.6). On day 4, the patient's clinical performance improved and there was no Candida albicans growth in ascites, blood culture or urine. During the treatment, there were no changes in blood chemistry, hematology and coagulation factors associated with Mycograb. Subsequent recovery was complicated by cellular sepsis, but this sepsis responded to antibiotics and the patient was fully convalescing.

The serum Mycograb concentrations of patient 2 at different time intervals after administration of Mycograb therapy are listed in table 21. The test dose given on day 1 did not reach the serum test level. The dose of 1.0mg/kg administered on day 2 did reach serum test levels comparable to the synergy with amphotericin B in vitro experiments (see tables 9 and 18). Mycograb at a dose of 1.0mg/kg was detectable in urine (data not shown).

TABLE 21 Mycograb levels in patient 2 serum (expressed in. mu.g/ml)
					Time (hours)	Day 1	Day 2	Day 4	Day 4
0.1mg/kg	1.0mg/kg	1.0mg/kg body weight First agent	1.0mg/kg body weight Second agent
				00.51.02.04.06.08.012.024.048.0		0000000000	01.50.50.30.100000	01.00.50.1000: then administering a second dose	01.00.50.50.100000

Patient 3 had a 6 week history of pancreatitis and resulted in 80% pancreatectomy. The patient has moderate elevated liver function Levels (LFT), may be associated with alcohol abuse, and is a carrier of methicillin-resistant Staphylococcus aureus (MRSA). Candida albicans grows in many parts of the patient, and intravenous fluconazole treatment is given. After 12 days, the response to fluconazole was lost and the patient was changed to 300mg Abelcet treatment. After 3 days, the patient was still hot (38.5 ℃), and Candida albicans was still growing in multiple locations (abdominal drain and gastroscopy tube), so a clinical dose of Mycogarb (1mg/kg) treatment was given on Abelcet basis (day 1).

On the same day as the dose of Mycograb, patient 3 had an acute onset of gram-negative septic shock (body temperature as high as 39.5 ℃, hypotensive), which may be caused by infection with pseudomonas aeruginosa, and growth of this bacterium was found in the pancreatic juice of the patient, although enterococcus faecalis also grown in blood culture of the patient. No further Mycograb treatment was given due to this incidence. The patients subsequently responded to antibiotic therapy (vancomycin and ceftazidime).

It is difficult to assess the effect of a single dose of Mycograb on patient 3 due to bacterial complications. Nevertheless, it was noted that the patient had discontinued Candida albicans growth 48 hours after Mycograb administration (e.g., from his gastroscopic tube and wound drainage), and had no fever on days 2 and 3. No changes in laboratory parameters (hematochemistry, hematology and coagulation factors) were observed in relation to Mycograb. On day 4, the patient relapsed to candida albicans while still on Abelcet treatment, but later recovered completely.

The serum Mycograb concentrations of patient 3 at different time points after administration of Mycograb therapy are listed in table 22. The single dose of 1.0mg/kg on day 1 gave serum test levels comparable to the synergy with amphotericin B in vitro experiments (see tables 9 and 18). Mycograb at a dose of 1.0mg/kg was detectable in urine (data not shown).

TABLE 22 Mycogarb levels in patient 3 serum
		Time (hours)	Day 1 1.0mg/kg body weight
00.51.02.04.06.08.012.024.048.0	02.51.51.20.100000

Patient 4 was diagnosed with candida albicans empyema, although the patient initially received in the intensive care unit (ITU) a lung abscess caused by streptococcus miers (isolated from blood culture). Two bronchial lavage specimens (right and left lungs) from the patient had Candida albicans growth. Candida albicans also grew in the two purulent breast fluid specimens after 3 days and 4 days. Abelcet treatment (5mg/kg) was started the following day. After 5 days from the beginning of Abelcet treatment, some clinical exacerbations were noted, followed by the morning (day 1) of high white blood cell counts (15.7) and regrowth of candida albicans in the intercostal drainage fluid.

Patient 4 was given Mycograb (1mg/kg body weight) at 8 am and 8 pm on day 1, respectively. In addition to a temporary increase in body temperature in the evening on day 1, the clinical symptoms of the patient improved. The pus chest fluid sample culture on day 3 did not grow candida albicans, and the patients were better day by day.

Patient 4 was well tolerated by Mycograb. No changes in laboratory parameters (hematochemistry, hematology and coagulation factors) were observed in relation to Mycograb. Thus, although the patient still had Candida albicans growth in the pleural effusion 6 days after initiation of Abelcet therapy and had a high white blood cell count just prior to receiving the first dose of Mycogarb (15.7), the patient subsequently stabilized and stopped Candida albicans growth.

The serum Mycograb concentrations of patient 4 at different time points after administration of Mycograb therapy are listed in table 23. The 1.0mg/kg dose given on day 1 reached serum levels comparable to the synergy with amphotericin B in vitro experiments (see tables 9 and 18). After the second dose on the first day, the concentration of Mycograb in serum increased, indicating that some tissue accumulation occurred after the first dose. Mycograb at a dose of 1.0mg/kg was detectable in urine (data not shown).

TABLE 23 Mycograb levels in patient 4 serum (expressed in. mu.g/ml)
			Time (hours)	Day 1
1.0mg/kg body weight First agent	1.0mg/kg body weight Second agent
		00.51.02.04.06.08.012.024.048.0		01.21.20.60.100-administration of a second agent-	8.02.51.41.20.60.30.1000

Conclusion

The data presented here clearly demonstrate that there is a surprising synergistic effect between the anti-candida albicans hsp90 antibody and the antifungal agent amphotericin B, which enhances antifungal activity against a number of pathologically important fungal strains. These results allow the application of a new, highly effective composition and a new antibody that can be combined into the composition for the treatment of fungal infections in humans and animals. The invention allows the use of lower or better results than the original dose, thus reducing the unwanted side effects.

Clinical significance of the invention includes: (i) the synergistic effect of amphotericin B and anti-hsp 90 antibodies in combination should be a particularly good therapeutic approach for the treatment of transmissible yeast infections. This will result in a reduction in mortality and morbidity from these infections. The results of the preliminary clinical trials provided herein demonstrate the efficacy of the present invention compared to existing treatments; (ii) amphotericin B is a toxic drug, particularly nephrotoxic. The synergy provided by the present invention means that low doses of amphotericin B can be used while maintaining its efficacy with a concomitant reduction in toxicity; and (iii) the attenuating effect of anti-hsp 90 antibodies allows the clinical exploration of high doses of amphotericin B and further helps to achieve better clinical results.

Sequence listing

<110> pharmaceutical Co Ltd for neuro technology

<120> treatment of fungal infections with beta glucan synthase inhibitors in combination with anti-HSP 90 antibodies

<130>N00/0101/PCT

<140>

<141>

<160>2

<170>PatentIn Vet.2.1

<210>1

<211>9

<212>PRT

<213> Candida

<400>1

Leu Lys Val Ile Arg Lys Asn Ile Val

1 5

<210>2

<211>248

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesized

<400>2

His Met Ala Glu Val Gin Leu Val Glu Ser Gly Ala Glu Val Lys Lys

1 5 10 15

Pro Gly Glu Set Leu Arg Ile Ser Cys Lys Gly Ser Gly Cys Ile Ile

20 25 30

Ser Ser Tyr Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu

35 40 45

Glu Trp Met Gly Lys Ile Asp Pro Gly Asp Ser Tyr Ile Asn Tyr Ser

50 55 60

Pro Ser Phe Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ash

65 70 75 80

Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met

85 90 95

Tyr Tyr Cys Ala Arg Gly Gly Arg Asp Phe Gly Asp Set Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Set Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln

130 135 140

Ser Pro Set Phe Leu Ser Ala Phe Val Gly Asp Arg Ile Thr Ile Thr

145 150 155 160

Cys Arg Ala Ser Ser Gly Ile Ser Arg Tyr Leu Ala Trp Tyr Gln Gln

165 170 175

Ala Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu

180 185 190

Gln Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu

195 200 205

Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr

210 215 220

Tyr Cys Gln His Leu Asn Set Tyr Pro Leu Thr Phe Gly Gly Gly Thr

225 230 235 240

Lys Val Asp Ile Lys Arg Ala Ala

245

Claims (17)

1. A method for targeting a polypeptide comprising SEQ ID NO: 1 or an antigen-binding fragment thereof.

2. A polypeptide comprising SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

3. The antibody or antigen-binding fragment of claim 1 or the antibody of claim 2, wherein the antibody or antigen-binding fragment is labeled with a detectable label.

4. The antibody or antigen-binding fragment of any one of claims 1 or 3, or the antibody of any one of claims 2 or 3, wherein said antibody or antigen-binding fragment is conjugated to an effector molecule.

5. Use of the antibody or antigen-binding fragment of any one of claims 1, 3 or 4 or the antibody of any one of claims 2 to 4 in the manufacture of a diagnostic agent for diagnosing one or more fungal infections.

6. The use of claim 5, wherein said diagnostic agent is provided in a kit.

7. The use of claim 6, wherein said kit comprises instructions for use in diagnosing one or more fungal infections.

8. A diagnostic kit as described in any one of claims 6 or 7.

9. A composition comprising the antibody or antigen-binding fragment thereof of claim 1 or 2, and an antifungal agent comprising at least one member selected from the group consisting of a polyene antifungal agent and an echinocandin antifungal agent.

10. A combined preparation comprising an antibody or antigen-binding fragment thereof according to claim 1 or 2, and an antifungal agent comprising at least one selected from the group consisting of a polyene antifungal agent and an echinocandin antifungal agent, for simultaneous, separate or sequential use in the treatment of a fungal infection.

11. A composition according to claim 9, or a combined preparation according to claim 10, wherein the polyene antifungal agent comprises amphotericin B or a derivative of amphotericin B.

12. A composition according to claim 9, or a combined preparation according to claim 10, wherein said echinocandin antifungal agent comprises LY 303366.

13. Use of a composition according to any one of claims 9, 11 or 12 for the treatment of a fungal infection.

14. The composition of claim 13, or the combined preparation according to any one of claims 10 to 12, wherein the fungal infection is caused by candida, cryptococcus, histoplasma, aspergillus, torulopsis, mucor, blastomyces, coccidioidomycosis or paracoccidioidomycosis.

15. The composition of claim 13 or 14, or the combined preparation of any of claims 10-12, or 14, wherein said fungal infection is resistant to said antifungal agent itself.

16. A composition according to any one of claims 9 to 15, or a combined preparation according to any one of claims 10 to 12, 14 or 15, for use in a method of treatment of fungal infections of the human or animal body.

17. A method of manufacture of a medicament for use in the treatment of fungal infections of the human or animal body characterised by the use of a composition according to any one of claims 9 to 16 or a combined preparation according to any one of claims 10 to 12 or 14 or 16.