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Treatment of early and established Cryptococcus neoformans infection with 1

radiolabeled antibodies in immunocompetent mice 2

Running title: RIT of C. neoformans in immunocompetent mice 3

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Zewei Jiang1, Ruth A. Bryan

1, Alfred Morgenstern

2, Frank Bruchertseifer

2, Arturo 5

Casadevall3,4

, Ekaterina Dadachova1,3*

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Departments of 1Nuclear Medicine,

3Microbiology and Immunology and

4Medicine, Albert 7

Einstein College of Medicine, Bronx, NY, USA; 2European Commission, Joint Research 8

Centre, Institute for Transuranium Elements, Karlsruhe, Germany 9

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*Author for correspondence: Ekaterina Dadachova, PhD, Albert Einstein College of Medicine, 11

Department of Nuclear Medicine, 1695A Eastchester Rd., Bronx, NY 10461; Ph: 718-405-12

8485; FAX: 718-405- 8457; E-mail: ekaterina.dadachova@einstein.yu.edu 13

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Word count: abstract – 50, text - 1,000. 16

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Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00473-11 AAC Accepts, published online ahead of print on 17 October 2011

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ABSTRACT 24

We investigated the utility of radioimmunotherapy in early and established cryptococcal 25

infection in immunocompetent mice. RIT with 213

Bi-18B7 antibody completely eliminated 26

fungus from mice lungs and brains for early infection, while 188

Re-18B7 significantly reduced 27

CFUs in the lungs or both lungs and brains during early and established infection, respectively. 28

The results point to the independence of RIT on the immune status of the host which is 29

encouraging for translation of this strategy into the clinic. 30

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Key words: C. neoformans, radioimmunotherapy, established infection, C57BL6 mice, 213-32

Bismuth, 188-Rhenium 33

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The inability of immunocompromised individuals to mount an effective immune response to 47

fungal infections reduces the efficacy of standard antifungal therapies. For example, 48

Cryptococcus neoformans (CN) infections cause life threatening meningoencephalitis 49

primarily in immunocompromised patients, causing more deaths than tuberculosis among 50

AIDS patients in the developing world (9). Our laboratory is developing a radioimmunotherapy 51

(RIT) approach as a novel treatments for infectious diseases (7). The RIT relies on the 52

antigen-binding characteristics of antibodies to deliver cytotoxic radiation to target cells 53

(12). Radiolabeled monoclonal antibodies (mAbs) Zevalin® and Bexxar® are FDA-54

approved for untreated, refractory and recurrent lymphomas. Several years ago we 55

demonstrated the potential efficacy of RIT against infectious diseases by showing 56

prolonged survival in mice systemically infected with CN and treated post-infection with 57

radiolabeled 18B7 mAb to CN polysaccharide capsule (8). Subsequently, we applied RIT 58

against bacteria and HIV (7). This approach showed little toxicity (6), and work has 59

begun to uncover the radiobiological and immune mechanisms of RIT (5, 1). We 60

previously tested the ability of 213

Bi-18B7 to kill more virulent H99 strain in AJ/Cr mice, and 61

found that it significantly reduced the fungal burden in both lungs and brains 24 hours post 62

treatment (5). Very recently, we demonstrated that RIT was more efficient than standard 63

antifungal therapy in treating systemic CN infection in mice (3). 64

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Until now, all studies of RIT of systemic CN infection were performed in A/JCr mice because 66

they are highly susceptible to IV infection, possibly due to partial complement deficiency (10) 67

and RIT was also always administered 24 hrs post-infection with CN. However, this reliance 68

on one model led to concerns about its general applicability in other hosts and whether RIT 69

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would retain efficacy in settings of more established infection. Since RIT relies primarily on 70

the killing of the microbes by cytocidal radiation - it would be important to demonstrate its 71

independence of the host immune status. In addition, demonstrating the efficacy of RIT 72

towards the established infections characterized by the high fungal load would be beneficial for 73

its future applications in clinical practice. Experimental and natural infection may not be 74

comparable but experimental infection is the basis for all available models and 48 hr is 75

sufficient for an organism that replicates every 2-4 hr in vivo. In this study we aimed to 76

investigate whether: 1) CN systemic infection in immunocompetent C57BL6 mice is amenable 77

to RIT at 24 hrs; 2) established CN systemic infection at 48 hrs could be treated with RIT in 78

the same mouse strain. 79

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We hypothesized that at 24 hrs after infection both alpha-emitter 213-Bismuth (213

Bi, 46 min 81

physical half-life) and beta-emitter 188-Rhenium (188

Re, 16.9 hr half-life) should be capable of 82

killing fungal cells in infected C57BL6 mice when carried to the sites of the infection by the 83

mAb 18B7. We also hypothesized that 188

Re which has more radioactive atoms per unit of 84

activity than 213

Bi will be a more effective choice of a radionuclide for treatment of 48 hr 85

infection which is characterized by higher microbial burden. 86

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CN strain 24067 was obtained from ATCC (Manassas, VA) and the cells grown as in (3). 88

Animal experiments followed guidelines of Albert Einstein College of Medicine Institute for 89

Animal Studies. Groups of 5-8 C57BL6 female 8 to 10 weeks old mice (Jackson Laboratories) 90

were infected IV via tail vein with 106 CN cells and were left untreated or treated IP with: 100 91

µCi 213

Bi-18B7 24 hr post-infection; 100 µCi 188

Re-18B7 24 hr post-infection; 100 µCi 188

Re-92

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18B7 48 hrs post-infection. Radiolabeling of 18B7 mAb with 213

Bi or with 188

Re was 93

performed as in (5). The total amount of 18B7 mAb per mouse was 30 µg which has been 94

shown to have no effect on infection burden in AJ/Cr mice (5, 8). Mice were monitored for 95

their survival for 75 days, humanely sacrificed, their brains and lungs removed, divided in half, 96

and one half was stained and analyzed histologically for signs of inflammation and possible 97

radiation scarring (hematoxylin and eosin (H&E)) and presence of CN cells (Gomori 98

Methenamine-Silver Nitrate stain (GMS)). The remaining tissue was disrupted, diluted, and 99

plated for CFUs. Differences in CFUs between the groups were analyzed by Student’s t-test for 100

unpaired data. P values of <0.05 were considered significant. 101

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None of the mice in the study including untreated infected controls died during the 75 days of 103

observation pointing to the development of an indolent chronic infection. There was 104

approximately equal microbial burden in the lungs and the brains of the untreated mice (Fig. 105

1a) that was confirmed by the GMS staining (Fig. 1b). These data are in concordance with the 106

previous studies on CN infection of C57BL6 mice (11, 14) and in contrast with the data in 107

AJ/Cr mice in which the fungal load in the lungs is usually several orders of magnitude higher 108

than in the brain (8). Treatment of mice with 188

Re-18B7 mAb 24 hr post-infection produced 109

one log reduction in the CFUs in lungs (P=0.04) and none – in the brains (P=0.07) (Fig. 1a). 110

Administration of 213

Bi-18B7 24 hr post-infection completely eliminated fungal cells from both 111

the lungs and the brains (the detection limit of plating assay was 50 CFUs) (Fig. 1a) which 112

was confirmed by GMS staining (Fig. 1e). We observed the same elimination of the fungal 113

burden from the lungs and brains of AJ/Cr mice infected with 105 CN cells and treated with 114

213Bi-18B7 (3). For established infection at 48 hrs

188Re-18B7 mAb was at least as effective in 115

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decreasing CFUs in the lungs (P=0.03) as at 24 hr and also significantly reduced the fungal 116

burden in the brains (P=0.02) (Fig. 1a). The latter fact might reflect the increased permeability 117

of the blood-brain-barrier with the onset of infection which facilitates the influx of the 118

radiolabeled antibody into the brain. According to Shi et al (13), in an IV mouse model, at 24 119

hrs there is little penetration by CN through the brain microvasculature, with only 8% of the 120

cells in the brain parenchyma, and the rest still in the capillaries. Therefore, at 24 hours, most 121

of the cells would be accessible to the radiolabeled mAbs and are either completely eliminated 122

by 213

Bi-18B7 which prevents any further penetration into the brain, or are only partially 123

eliminated by less powerful 188

Re-18B7 with the remaining cells continue to penetrate into the 124

brain. If the treatment with 188

Re-18B7 is administered at 48 hrs post infection when the BBB 125

became more permeable for the mAb - 188

Re-18B7 can “chase” CN directly in the brain which 126

results in decreasing CFUs in this organ (Fig. 1). Thus, the decrease is brain CFUs post RIT 127

can be attributed to its direct antifungal effects. The pathology of the lungs and brains of 128

188Re-18B7 mAb-treated mice showed focal nature of the remaining fungal load (Fig. 1c,d). 129

The ability of a single injection of 188

Re-labeled mAb to significantly decrease microbial 130

burden in established infection is encouraging taking into consideration that: 1) there are at 131

least 3 orders of magnitude more fungal cells in the organs at 48 hrs post-infection in 132

comparison with 24 hr; 2) multi-day courses of antifungals such as amphotericin B are required 133

to achieve the same results (4). It might be possible that repeat administrations of 213

Bi-18B7 134

mAb would result in successful treatment of an established infection. This is the 1st study 135

where the CFUs in brain and lungs were compared in the long term survivors between 213

Bi- 136

and. 188

Re – labeled mAbs. While the two isotopes are similar in terms of prolonging mouse 137

survival (8), they are different in regard to the mechanism of CN killing. 213

Bi-18B7 is 138

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extremely lethal to CN with only one or two “hits” needed for killing a cell leading to a loss of 139

metabolic activity in treated cells, whereas 188

Re has to deliver hundreds of “hits” needed for 140

killing (1). Thus, it is possible that 188

Re-18B7 is decreasing the CFUs enough to allow 141

survival, but not completely eliminating the organisms. Lastly, analysis of the H&E stained 142

tissues demonstrated no evidence of radiation fibrosis in the lungs and the brains (not shown), 143

consistent with previous observations (2, 6). 144

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In conclusion, RIT was effective against early and established infection in immunocompetent 146

C57BL6 mice. Previously the efficacy of RIT was shown in complement deficient AJ/Cr mice. 147

The results point to the independence of RIT on the immune status of the host which is 148

encouraging for translation of this strategy into the clinic for treatment of cancer and organ 149

transplant patients with opportunistic infections. 150

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ACKNOWLEDGEMENTS 152

E. Dadachova is a Sylvia and Robert S. Olnick Faculty Scholar in Cancer Research and is 153

supported by the NIH grant AI60507. A. Casadevall is supported by the following NIH grants: 154

AI33774-11, HL59842-07, AI33142-11, AI52733-02, and GM 07142-01. A. Morgenstern and 155

F. Bruchertseifer are supported by European Commission. 156

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FIGURE LEGENDS 159

Fig. 1. RIT of C57BL6 mice infected IV with 106 CN cells: a) CFUs in the brains and the 160

lungs of RIT-treated and control mice. Mice were treated IP with either: 100 µCi 213

Bi-18B7 161

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24 hr post-infection; 100 µCi 188

Re-18B7 24 hr post-infection; 100 µCi 188

Re-18B7 48 hrs 162

post-infection; or left untreated and sacrificed 75 days post-treatment. Detection limit of the 163

method was 50 CFUs. No CFUs were found in the brains and lungs of mice treated with 100 164

µCi 213

Bi-18B7 which are presented in the graph as 40 CFUs/organ. The asterisks show the 165

groups in which the CFUs were significantly different from the untreated controls; b) - e) – 166

histology of the brains (left panels) and lungs (right panels) of RIT-treated and control mice. 167

Organs were stained with GMS. CN cells stained with GMS appear black on the images. b) 168

untreated mouse; c) mouse treated with 100 µCi 188

Re-18B7 24 hr post-infection; d) mouse 169

treated with 100 µCi 188

Re-18B7 48 hrs post-infection; e) mouse treated with 100 µCi 213

Bi-170

18B7 24 hr post-infection. The size bar is 100 µm. 171

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REFERENCES 174

1. Bryan, R. A., X. Huang, A. Morgenstern, F. Bruchertseifer, A. Casadevall, and E. 175

Dadachova. 2008. Radio-fungicidal effects of external gamma radiation and antibody-176

targeted beta and alpha radiation on Cryptococcus neoformans. Antimicrob. Agents 177

Chemother. 52: 2232-2235 178

2. Bryan R.A., Jiang Z., Huang X., Morgenstern A., Bruchertseifer F., Sellers R., 179

Casadevall A., Dadachova E. 2009. Radioimmunotherapy is effective against a high 180

infection burden of Cryptococcus neoformans in mice and does not select for radiation-181

resistant phenotypes in cryptococcal cells. Antimicrob. Agents Chemother. 53:1679-82 182

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