JAC Advance Access originally published online on February 25, 2008
Journal of Antimicrobial Chemotherapy 2008 61(5):1132-1139; doi:10.1093/jac/dkn075
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Original research |
Efungumab and caspofungin: pre-clinical data supporting synergy
1 NeuTec Pharma Ltd, a wholly owned subsidiary of Novartis Pharma AG, Williams House, Lloyd Street North, Manchester M15 6SE, UK 2 The University of Manchester, 2nd Floor, Clinical Sciences Building, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK 3 Electron Microscopy Unit, Ground Floor, Clinical Sciences Building, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK
* Corresponding author. Tel: +44-161-232-2900; Fax: +44-161-232-2901; E-mail: james.burnie{at}neutecpharma.com
Received 21 November 2007; returned 8 January 2008; revised 23 January 2008; accepted 2 February 2008
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Abstract |
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Objectives: Heat shock protein 90 (hsp90) is targeted by the humoral response in invasive candidiasis. This paper tests for synergy between caspofungin and efungumab—a human antibody fragment against hsp90.
Methods: The MIC-0, MIC-2 values and FICI were determined for a range of yeasts against efungumab and caspofungin. These yeasts were injected intravenously into mice with: 100 µL of saline plus 100 µL of formulation buffer; 100 µL of caspofungin (1 or 4 mg/kg) plus 100 µL of formulation buffer; or 100 µL of caspofungin (1 or 4 mg/kg) plus 100 µL of efungumab 2 mg/kg. Yeast counts were determined for kidney, liver and spleen. Electron microscopy was performed on efungumab-stained Candida grown with and without caspofungin.
Results: The FICIs of efungumab and caspofungin at MIC-0 and MIC-2, respectively, were: fluconazole-susceptible Candida albicans: 0.5, 0.52; fluconazole-resistant C. albicans, Candida tropicalis and Candida krusei: 0.5, 0.5; Candida parapsilosis: 2, 0.5; Candida glabrata: 0.26, 0.28; and Candida guilliermondii: 2, 0.27. A statistically significant reduction in colony counts or increase in the number of negative biopsies (P < 0.05) was seen in mice on combination therapy at 1 mg/kg caspofungin for the renal biopsies of C. glabrata, liver biopsies of fluconazole-resistant C. albicans, C. krusei and C. guilliermondii and spleen biopsies of C. guilliermondii, and at 4 mg/kg for the renal biopsies of C. tropicalis, the liver biopsies of C. parapsilosis and the spleen biopsies of C. guilliermondii and C. glabrata. Electron microscopy confirmed extracellular hsp90 up-regulated by growth in caspofungin.
Conclusions: Efungumab increased the susceptibility of Candida to caspofungin.
Keywords: hsp90 , combination therapies , antibodies , echinocandins , yeasts
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Introduction |
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Invasive candidiasis remains a costly, life-threatening disease with a high attributable mortality rate, despite advances in the formulation and diversification of therapeutic agents.1 There is a growing medical need for new agents and therapeutic strategies, especially combination therapy.2 Conventional antifungal therapies target the fungus directly and leave the host exposed to potentially toxic molecules such as proteinases, hydrolases and heat shock proteins released by the fungus.3 Their neutralization by antibody would be a further way of improving clinical outcome, especially if it included a molecule critical to fungal pathogenesis and the development of resistance to currently licensed antifungal agents.4
Previous work established a correlation between recovery from invasive candidiasis and the production of antibody to heat shock protein 90 (hsp90).5,6 Efungumab (Mycograb®) has been developed as a human recombinant antibody fragment against hsp90 that incorporates the dominant paratope found in patients who recovered from invasive candidiasis, mimicking this humoral immune response.7 The presence of extracellular hsp90 has been demonstrated in Candida albicans and Cryptococcus neoformans, in Campylobacter jejuni, where it acts as a binding protein for surface lipoprotein JlpA, and in dengue, where it interacts with the viral envelope glycoprotein.8–11 In cancer, antibody-mediated neutralization of hsp90 has blocked metastasis formation and impacted on the functioning of the multiple molecules involved in tumour progression.12,13
Efungumab and other hsp90 inhibitors, such as geldanamycin, have an antifungal effect in vitro and, in the case of efungumab, in an animal model of fungal infection.7,14–16 Geldanamycin can block the development of resistance to caspofungin and fluconazole in Aspergillus terreus, Saccharomyces cerevisiae and C. albicans, respectively.14–16 In a randomized, double-blind study in patients with invasive candidiasis, the combination of efungumab and lipid-associated amphotericin B was superior to monotherapy with lipid-associated amphotericin B, demonstrating a higher clinical response rate [86% versus 52% (P < 0.001)], a higher mycological response rate [89% versus 54% (P < 0.001)], a faster rate of culture-confirmed clearance (hazard ratio, 2.3; 95% CI, 1.4–3.8; P = 0.001) and less Candida-attributable mortality [18% versus 4% (P = 0.025)].17
Echinocandins have recently become popular as an alternative to amphotericin B in the treatment of fungal infection. A systematic review of comparative studies in fungal infections, primarily those due to Candida, has reported an overall treatment success rate of 52.6% in caspofungin-treated patients and 44.7% for amphotericin B and lipid-associated amphotericin B-treated patients.18 Caspofungin demonstrated a more favourable safety profile than amphotericin B, with significantly fewer discontinuations due to drug toxicity (odds ratio, 0.25; 95% CI, 0.07–0.85) and significantly less nephrotoxicity, hypokalaemia and fever.18 These findings support the use of caspofungin as an alternative treatment to amphotericin B, but underline the need to improve clinical outcome. Resistance to caspofungin has been increasingly reported and hsp90 is implicated in this process.14,15,19 This raises the question as to whether the addition of efungumab to caspofungin therapy will increase susceptibility and lead to an improvement in the overall treatment success rates. This paper examines this hypothesis at a pre-clinical level.
The testing of yeasts for susceptibility to efungumab by microdilution, after 48 h incubation at 37°C in RPMI 1640 broth, used an endpoint of minimum inhibitory concentration (MIC-0), defined as the lowest concentration with no visible growth consistent with the CLSI (formerly NCCLS) document M27-A2.20 For caspofungin, the recommended endpoint criterion is a prominent reduction in growth (MIC-2 or 
50% inhibition) relative to control.21 The study reported here tested both endpoints and assessed the combination of caspofungin and efungumab in the same mouse model used previously to delineate the synergy between efungumab and amphotericin B.7 Efficacy in mice was measured 48 h after the intravenous injection of the yeasts by the reduction in mean colony counts from kidney, liver and spleen or the number of negative biopsies as appropriate. The effect of caspofungin on extracellular hsp90 was characterized by electron microscopy using C. albicans grown in a concentration of caspofungin less than the MIC-0 as this had previously been shown to exert a maximum cell wall effect.22
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Yeast strains used
The yeasts examined were: a fluconazole-susceptible outbreak strain of C. albicans,23 a fluconazole-resistant (MIC = 64 mg/L) strain of C. albicans24 and a fluconazole-resistant (MIC = 100 mg/L) strain of Candida krusei (FA/157),25 Candida tropicalis (NCPF 3111), Candida parapsilosis (NCPF 3104), Candida glabrata (NCPF 3240) and Candida guilliermondii (clinical isolate from the intensive care unit, Rottenrow Hospital, Glasgow, UK). For short-term storage, isolates were grown on Sabouraud dextrose agar slopes (Oxoid, Basingstoke, UK) and kept at room temperature. NCPF strains were obtained from the National Collection of Pathogenic Fungi, held by the Mycology Reference Laboratory, South West HPA, Mertle Rd, Bristol, UK. For long-term storage, yeasts were kept at –70°C in a suspension of Sabouraud broth with glycerol (9:1, v/v). Before use, the yeasts were plated onto Sabouraud dextrose agar and incubated overnight at 37°C. One colony was then placed in 10 mL Sabouraud dextrose broth and grown for 6 h, with shaking at 37°C; 1 mL was added to 250 mL Sabouraud dextrose broth and grown overnight, with shaking at 37°C. The cells were washed twice in saline, the concentration determined by haemocytometer and confirmed by plating dilutions onto Sabouraud dextrose agar.
Efungumab (Novartis AG, Basel, Switzerland) powder is a polyhistidine-tagged, purified human recombinant anti-hsp90 single-chain antibody fragment (Mycograb®), reconstituted by the addition of sterile water to a final concentration of 2 mg/mL. Caspofungin (Merck, Sharp & Dohme Ltd, Hoddesdon, UK) was dissolved in sterile water. Further drug dilutions were prepared in standard RPMI 1640 medium (Sigma-Aldrich, Poole, UK).
Antifungal susceptibility testing
The MICs of caspofungin and efungumab were determined by broth microdilution according to the CLSI (formerly NCCLS) document M27-A2 (2002).20 Caspofungin and efungumab concentrations were tested alone and in combination by MIC endpoints and chequerboard titrations. An inoculum suspension was prepared from individual colonies (diameter 1 mm) in RPMI 1640 with glutamine broth medium, buffered to pH 7.0 with morpholinepropanesulphonic acid. The suspension was adjusted to 0.5 McFarland standard and further diluted 1:50 then 1:20 in RPMI medium, producing a final inoculum of 103 cfu/mL. The MIC plates were incubated at 37°C for 48 h. For caspofungin and efungumab, either alone or in combination, the endpoint was determined as the lowest concentration to produce optically clear wells (no growth, MIC-0) or a cell count of 
5% of the control well, as determined by plating out onto Sabouraud agar plates. The MIC-2 was also determined and taken as the concentration resulting in a prominent decrease in turbidity (50% growth inhibition) compared with the growth control. The fractional inhibitory concentration (FIC) for each drug was calculated by dividing the MIC in the presence of the second drug by the MIC in its absence, with the sum of the two FICs giving the FIC index (FICI). The interaction was defined as synergistic if the FICI was 0.5, no interaction if >0.5–4.0 and antagonistic if >4.0.
Mouse model of Candida infection
Yeasts were grown overnight in Sabouraud dextrose broth at 37°C, washed in saline and injected as a 0.1 mL bolus into the lateral tail vein of 25 g female CD1 mice (Charles River). After preliminary dose ranging experiments, the following inocula were used, in colony forming units: 6 x 106 (C. tropicalis), 9 x 106 (C. krusei), 1x107 (fluconazole-susceptible and fluconazole-resistant strains of C. albicans and C. guilliermondii), 3 x 107 (C. glabrata) and 2 x 108 (C. parapsilosis). Two hours after infection, randomized groups of 10 animals were injected intravenously with: (i) 100 µL of saline and 100 µL of efungumab formulation buffer (200 mM arginine, 500 mM urea corrected to pH 9.5 by HCl); (ii) 100 µL of caspofungin (1 or 4 mg/kg) plus 100 µL of efungumab formulation buffer; or (iii) 100 µL of caspofungin (1 or 4 mg/kg) plus 100 µL of 2 mg/kg efungumab. Mice were culled at 48 h and yeast cell counts determined for kidney, liver and spleen. Organs were aseptically removed, weighed and processed in sterile saline using the Seward Stomacher 80 Lab System. After processing, samples were diluted and plated onto Sabouraud dextrose agar and incubated overnight at 37°C.
Animal work was carried out under British Home Office Licenses 40/02321 and 40/0543 to the standards for experimentation and care set down in the UK Animals (Scientific Procedures) Act of 1986 and the Code of Practice for the Housing and Care of Animals used in Scientific Procedures 1989.
If 60% of the organ biopsies were negative (<1 cfu/mg of tissue), statistical analysis was performed by Fisher's exact test, comparing the number of negative biopsies between the test and control groups. If organ biopsies were culture-positive, groups were compared by analysis of variance followed by Scheffe's test for multiple comparisons. The results were expressed as a mean ± SD. A P value of <0.05 was considered significant for all statistical determinations. All of the above analyses were performed on SPSS versions 10.1 and 13.0 software for Windows, http://www.spss.com and Graph Pad InStat Version 3.06 for Windows, Graph Pad Software, CA, USA, http://www.graphpad.com.
Fluconazole-susceptible C. albicans was inoculated into YPD broth medium with buffer, 0.125 or 0.25 mg/L caspofungin and incubated overnight at 30°C. Cells were washed three times with phosphate-buffered saline (PBS) buffer and suspended to an OD600 of 0.5–0.6. Cells were fixed in 0.5% glutaraldehyde and 4% formaldehyde for 120 min at room temperature and washed in 0.5 M NH4Cl for 60 min, dehydrated through a graded series of alcohol, centrifuged in fresh resin in embedding capsules and polymerized at 60°C overnight. Samples were sectioned and placed on electron microscopy grids. Sections were incubated with efungumab at 64 mg/L, blocked with 1% bovine serum albumin and incubated with an anti-polyhistidine antibody IgG secondary antibody (Sigma-Aldrich) for 1 h and subsequently stained with 10 nM colloidal gold-conjugated anti-mouse IgG (British BioCell International) for 45 min. Sections were washed in PBS three times for 5 min, followed by distilled water and then examined under the electron microscope. Control cells were included, for which the step of incubation with efungumab was replaced by incubation with PBS buffer.
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Susceptibility testing
Five out of seven strains using the MIC-0 endpoint (Table 1) and six out of seven strains using the MIC-2 endpoint (Table 2) demonstrated synergy, whereas the remaining strains showed indifference with no evidence of antagonism. At the MIC-0, the indifference of C. parapsilosis and C. guilliermondii to the drug combination was due to the lack of susceptibility to caspofungin (MIC-0 >64 mg/L). When the MIC-2 was taken as the endpoint, synergy was demonstrated for both of these isolates.
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Animal testing
The activities of caspofungin and efungumab in the murine model of invasive candidiasis are summarized below (Table 3).
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C. albicans (fluconazole-resistant). Caspofungin at 1 and 4 mg/kg sterilized the kidney and spleen. At 4 mg/kg, it cleared the liver, but not at 1 mg/kg. In combination, 1 mg/kg caspofungin and 2 mg/kg efungumab sterilized the liver, demonstrating synergy compared with caspofungin monotherapy at the lower dose (P = 0.0011).
C. albicans (fluconazole-susceptible). Caspofungin at 1 and 4 mg/kg cleared the kidney [P = 0.0002 (1 mg/kg), P < 0.0001 (4 mg/kg) compared with untreated control]. Caspofungin at 1 mg/kg had no effect on the colony counts from liver biopsies, which were moderately reduced by the addition of efungumab and infection was cleared with 4 mg/kg caspofungin (P < 0.0001). Increasing the dose of caspofungin moderately reduced the colony counts from the spleen; the addition of efungumab increased the number of negative biopsies in the 4 mg/kg caspofungin group from 3 to 8 out of 10 mice. As a result, the analysis required was converted from one based on only a reduction in mean colony count to one based on organ sterilization.
C. krusei. Caspofungin at 1 mg/kg had little effect on the colony counts, whereas at 4 mg/kg it sterilized the kidney, but produced only a moderate reduction in the liver and spleen counts. The spleen counts only showed a statistically significant reduction (Scheffe's test) after the addition of efungumab at 2 mg/kg to caspofungin at 4 or 1 mg/kg. Synergy was also demonstrated by the liver counts which showed a significant reduction in colony counts (Scheffe's test) and an increase in the number of negative liver biopsies (P = 0.0325) when efungumab was added to 1 mg/kg caspofungin.
C. glabrata.
Caspofungin at 1 mg/kg produced only a marginal reduction in the mean colony counts. A statistically significant reduction in spleen counts was achieved with the addition of efungumab to 1 mg/kg caspofungin (Scheffe's test), and in the case of the kidney, there was a significant increase in the number of negative biopsies on combination therapy (P = 0.0198). At 4 mg/kg, caspofungin moderately reduced biopsy counts, but it required the addition of efungumab at 2 mg/kg to produce organ sterility at 60%. For the spleen biopsies, there was a statistically significant difference for combination therapy compared with 4 mg/kg caspofungin monotherapy (P = 0.0031).
C. tropicalis.
Caspofungin at 1 and 4 mg/kg sterilized liver and spleen biopsies (P = 0.001 and P < 0.0001, respectively). Caspofungin at 1 and 4 mg/kg produced a statistically significant reduction in the mean renal counts, which achieved sterility in 60% in the 4 mg/kg group after the addition of 2 mg/kg efungumab, a statistical significance difference compared with the monotherapy group (P = 0.0325).
C. parapsilosis. Caspofungin at 1 and 4 mg/kg produced a statistically significant reduction in colony counts in all three organs (Scheffe's test). The addition of efungumab to 4 mg/kg caspofungin increased the number of negative biopsies when compared with the control and monotherapy groups for both kidney (P = 0.0325) and liver (P = 0.0325) biopsies and further reduced the mean colony counts in the spleen biopsies.
C. guilliermondii. Caspofungin at 1 mg/kg significantly reduced the mean renal biopsy count but had no effect on the liver and spleen counts. The addition of efungumab at 2 mg/kg produced a statistically significant reduction in liver and spleen counts compared with low-dose caspofungin monotherapy (Scheffe's test) and significantly increased the number of negative renal biopsies compared with the untreated control group. At 4 mg/kg, caspofungin cleared the renal and liver biopsies, but an additional 2 mg/kg efungumab was required to clear the spleen biopsies (P = 0.0031).
In the presence of efungumab, gold particles bound to the outer layer of the yeast surface (Figure 1). More gold particles were observed clustering on the cell surface after growth in subinhibitory concentrations of 0.125 and 0.25 mg/L caspofungin, demonstrating the enhanced induction of extracellular hsp90 (Figure 2a and b, respectively). Control samples incubated with only buffer demonstrated no binding.
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There is growing evidence for the therapeutic activity of antibody against hsp90 in invasive candidiasis. Mice vaccination with an hsp90-expressing DNA vaccine demonstrated specific humoral immunity associated with protection against invasive candidiasis. This was also generated after immunization with peptides representing the epitopes LKVIRK or DEPAGE derived from hsp90.26–28 Efungumab has been designed to mimic the protective effect of hsp90 antibody as it represents the dominant sequence of the heavy chains of antibodies found in patients recovering from invasive candidiasis, binding to the LKVIRK epitope of hsp90. Use of efungumab in combination with lipid-associated amphotericin B in the clinic significantly improved both clinical response rates and culture-confirmed clearance of invasive candidiasis.17
Susceptibility testing demonstrated that the addition of efungumab improved the susceptibility to caspofungin of a variety of isolates that represent the most important species causing invasive candidiasis. This effect was demonstrated for concentrations of efungumab (0.25–8 mg/L MIC-0, 0.125–16 mg/L MIC-2) attainable during therapy and that have produced significant added benefit when used in combination with amphotericin B in pre-clinical and clinical studies.7,17
These data were mirrored in the mouse model, in which combination of efungumab with caspofungin provided statistically significantly greater fungal inhibition than 1 mg/kg caspofungin monotherapy as shown by liver biopsies from C. krusei- and C. guilliermondii-infected mice, in spleen biopsies for C. guilliermondii (Scheffe's test P < 0.05), in renal biopsies infected with C. glabrata and in liver biopsies from fluconazole-resistant C. albicans- and C. krusei-infected mice (Fisher's exact test P < 0.05). The enhanced activity of combination therapy (4 mg/kg caspofungin plus 2 mg/kg efungumab) compared with monotherapy with 4 mg/kg caspofungin achieved statistical significance against C. tropicalis in renal biopsies, against C. parapsilosis in liver biopsies and against C. guilliermondii and C. glabrata in spleen biopsies (Fisher's exact test P < 0.05). Previously, a similar pattern was seen with amphotericin B at 0.6 mg/kg, where the addition of efungumab at 2 mg/kg was needed for the complete resolution of infections caused by the fluconazole-resistant strains of C. albicans, C. krusei, C. glabrata and C. parapsilosis.7 These data support the hypothesis that the addition of efungumab to caspofungin could improve outcome in a way similar to that seen with lipid-associated amphotericin B therapy.17 Rapid improvement was seen in an 8-year-old child with peritonitis due to C. albicans treated with fluconazole, liposomal amphotericin B, 5-flucytosine and latterly caspofungin on the addition of efungumab therapy.29
Electron microscopy characterized a basal level of extracellular hsp90 expression that was increased by exposure to a sublethal concentration of caspofungin, similar to the effect seen with amphotericin B.9 The up-regulation of hsp90 has also been demonstrated by microarray analysis in S. cerevisiae following exposure to amphotericin B30 and in C. albicans by raising the temperature, by heavy metal stress using cadmium and by caspofungin exposure.31–33 Hsp90 inhibitors, such as geldanamycin and radiciol, have been shown to block the development of fluconazole resistance in C. albicans and caspofungin resistance in A. terreus.34
Biofilm formation resulting in increased resistance to amphotericin B and fluconazole was prevented by farnesol—an extracellular quorum-sensing molecule. This was associated with reduced hsp90 expression in C. albicans and a reduction in the transition from yeast to mycelia form.35,36 These observations are consistent with a model where hsp90 is pivotal to the survival response of yeast to low concentrations of an antifungal, the development of resistance specifically during biofilm formation and the ability of the yeast to invade tissue. Antifungal resistance has been linked to calcineurin, which is a calcium-activated protein phosphatase implicated in cell cycle progression, morphogenesis and virulence. Hsp90 binds to the catalytic subunit of calcineurin, keeping it stable and poised for activation.34
These data will form the scientific basis for a clinical study examining the hypothesis that the addition of efungumab to caspofungin therapy will improve outcome in invasive candidiasis similar to the improvement seen when efungumab was combined with lipid-associated amphotericin B.17
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This study was carried out as part of the ongoing drug development of efungumab funded through contracts of employment with NeuTec Pharma Ltd. R. Al-A. has a scholarship from the Government of Saudi Arabia.
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S. H., L. N., S. A., R. M. and J. B. are employees of Novartis AG. The employees own no stock in the company.
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References |
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