Toward developing a universal treatment for fungal disease using radioimmunotherapy targeting common fungal antigens

Abstract

Background: Previously, we demonstrated the ability of radiolabeled antibodies recognizing the cryptococcal polysaccharide capsule to kill Cryptococcus neoformans both in vitro and in infected mice. This approach, known as radioimmunotherapy (RIT), uses the exquisite ability of antibodies to bind antigens to deliver microbicidal radiation. To create RIT reagents which would be efficacious against all major medically important fungi, we have selected monoclonal antibodies (mAbs) to common surface fungal antigens such as heat shock protein 60 (HSP60), which is found on the surface of diverse fungi; beta (1,3)-glucan, which is a major constituent of fungal cell walls; ceramide which is found at the cell surface, and melanin, a polymer present in the fungal cell wall.

Methods: MAbs 4E12, an IgG2a to fungal HSP60; 2G8, an IgG2b to beta-(1,3)-glucan; and 6D2, an IgM to melanin, were labeled with the alpha particle emitting radionuclide 213-Bismuth ((213)Bi) using the chelator CHXA". B11, an IgM antibody to glucosylceramide, was labeled with the beta emitter 188-Rhenium ((188)Re). Model organisms Cryptococcus neoformans and Candida albicans were used to assess the cytotoxicity of these compounds after exposure to either radiolabeled mAbs or controls.

Results: (213)Bi-mAbs to HSP60 and to the beta-(1,3)-glucan each reduced the viability of both fungi by 80-100%. The (213)Bi-6D2 mAb to melanin killed 22% of C. neoformans, but did not kill C. albicans. B11 mAb against fungal ceramide was effective against wild-type C. neoformans, but was unable to kill a mutant lacking the ceramide target. Unlabeled mAbs and radiolabeled irrelevant control mAbs caused no killing.

Conclusion: Our results suggest that it is feasible to develop RIT against fungal pathogens by targeting common antigens and such an approach could be developed against fungal diseases for which existing therapy is unsatisfactory.

Fig. 1

RIT of C. albicans and C. neoformans using ²¹³Bi-2G8 antibody recognizing glucan: a C. albicans, with the concentration of pseudohyphae reduced to 20%; b C. albicans, with 50% pseudohyphae; c C. neoformans. Each experiment was performed three times, the results shown are from one typical experiment, the CFUs for each antibody dose were plated in triplicate

Fig. 2

RIT with ²¹³Bi-4E12 antibody to Hsp60: a Western blot showing binding of 4E12 to mitochondrial and cell wall fractions of C. albicans and C. neoformans; b immunofluorescence of live H. capsulatum, C. albicans and C. neoformans cells with 4E12; c RIT of C. albicans; d RIT of C. neoformans. Each experiment was performed three times, the results shown are from one typical experiment, the CFUs for each antibody dose were plated in triplicate. Hc–H. capsulatum, Ca–C. albicans and Cn–C. neoformans

Fig. 3

RIT of C. neoformans with ¹⁸⁸Re-B11 antibody to glucosylceramide: a wild-type C. neoformans with ¹⁸⁸Re-B11; b glucosylceramide–depleted mutant Δgcs with ¹⁸⁸Re-B11. Each experiment was performed three times, the results shown are from one typical experiment, the CFU samples for each antibody dose were plated in triplicate

Fig. 4

RIT using ²¹³Bi-6D2 antibody to melanin: a C. albicans; b C. neoformans. Each experiment was performed three times, the results shown are from one typical experiment, the CFUs for each antibody dose were plated in triplicate

Fig. 5

Binding of the ²¹³Bi-2G8, ²¹³Bi-4E12 and control ²¹³Bi-18B7 antibodies to C. neoformans and C. albicans cells and respective killing of the cells. Each experiment was performed three times, the results shown are from one typical experiment, triplicates were used for binding measurements and for CFUs. CN C. neoformans, CA C. albicans