Melanin is effective in protecting fast and slow growing fungi from various types of ionizing radiation.

Melanin is a ubiquitous pigment with unique physicochemical properties. The resistance of melanized fungi to cosmic and terrestrial ionizing radiation suggests that melanin also plays a pivotal role in radioprotection. In this study, we compared the effects of densely-ionizing deuterons and sparsely-ionizing X-rays on two microscopic fungi capable of melanogenesis. We utilized the fast-growing pathogenic basiodiomycete forming an induced DOPA-melanin, Cryptococcus neoformans (CN); and the slow-growing environmental rock-inhabiting ascomycete synthesizing a constitutive DHN-melanin, Cryomyces antarcticus (CA); melanized and non-melanized counterparts were compared. CA was more resistant to deuterons than CN, and similar resistance was observed for X-rays. Melanin afforded protection against high-dose (1.5 kGy) deuterons for both CN and CA (p-values < 10-4 ). For X-rays (0.3 kGy), melanin protected CA (p-values < 10-4 ) and probably CN. Deuterons increased XTT activity in melanized strains of both species, while the activity in non-melanized cells remained stable or decreased. For ATP levels the reverse occurred: it decreased in melanized strains, but not in non-melanized ones, after deuteron exposure. For both XTT and ATP, which reflect the metabolic activity of the cells, larger and more statistically-significant differences as a function of melanization status occurred in CN. Our data show, for the first time, that melanin protected both fast-growing and slow-growing fungi from high doses of deuterons under physiological conditions. These observations may give clues for creating melanin-based radioprotectors.

Treatment of early and established Cryptococcus neoformans infection with radiolabeled antibodies in immunocompetent mice.

We investigated the utility of radioimmunotherapy (RIT) in early and established cryptococcal infection in immunocompetent mice. RIT with (213)Bi-18B7 antibody completely eliminated fungus from mouse lungs and brains for early infection, while (188)Re-18B7 significantly reduced CFU in the lungs or both lungs and brains during early and established infection, respectively. The results point to the independence of RIT of the immune status of the host, which is encouraging for translation of this strategy into the clinic.

Radioimmunotherapy is more effective than antifungal treatment in experimental cryptococcal infection.

Radioimmunotherapy (RIT) prolongs the survival of mice infected with Cryptococcus neoformans. To compare the efficacy of RIT with that of amphotericin B, we infected AJ/Cr mice intravenously with either nonmelanized or melanized C. neoformans cells. Infected mice were either left untreated or treated 24 h after infection with (213)Bi-18B7 antibody, amphotericin B, or both. Melanization before infection did not increase resistance of C. neoformans to RIT in vivo. (213)Bi-18B7 treatment almost completely eliminated colony-forming units from the lung and brain, whereas amphotericin B did not decrease the number of colony-forming units. We conclude that RIT is more effective than amphotericin B against systemic infection with C. neoformans.

An algorithm for the sedation of patients with obstructive sleep apnea by non-anesthesiologists.

OBJECTIVE: Patients with obstructive sleep apnea (OSA) are at risk for adverse events when moderate sedation is administered by nurse protocols (NAMS) under the guidance of non-anesthesiologists. An algorithm was applied for the appropriate section of patients to receive NAMS and the application of continuous positive airway pressure (CPAP). METHODS: An algorithm was developed for patients with OSA who were scheduled for gastroenterology, radiology, and cardiology procedures using NAMS. Those with normal airways and without contraindications for NAMS were classified as CPAP-independent (CPAP-I; not routinely used) or CPAP-dependent (CPAP-D; always used). CPAP machines were brought in by CPAP-D patients or supplied by the hospital and set at a patient's routine setting or 10 cm H2O if not known. CPAP-D patients for procedures for which CPAP could not be applied were done under anesthesia care. We retrospectively examined this program for the 2008-2018 period. RESULTS: Since the inception of this protocol in 2008, 803 patients with OSA safely underwent procedures using either personal CPAP or CPAP provided by the hospital. CONCLUSIONS: Patients with OSA can safely have NAMS for procedures when CPAP is applied based on a protocol that considers airway evaluation, the procedure, and whether there is dependence upon CPAP.

Radioimmunotherapy is effective against high-inoculum Cryptococcus neoformans infection in mice and does not select for radiation-resistant cryptococcal cells.

We investigated the utility of radioimmunotherapy (RIT) in the treatment of experimental cryptococcal infection with high-inoculum and the possibility of RIT treatment selecting for fungal cells with radiation-resistant phenotypes. RIT reduced mortality in high-burden infections, and we found no evidence for the development of radiation-resistant cells.

Evaluation of novel highly specific antibodies to cancer testis antigen Centrin-1 for radioimmunoimaging and radioimmunotherapy of pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) accounts for >90% of pancreatic malignancies, and has median survival of <6 months. There is an urgent need for diagnostic and therapeutic options for PDAC. Centrin1 (CETN1) is a novel member of Cancer/Testis Antigens, with a 25-fold increase of CETN1 gene expression in PDX from PDAC patients. The absence of selective anti-CETN1 antibodies is hampering CETN1 use for diagnosis and therapy. Here we report the generation of highly specific for CETN1 antibodies and their evaluation for radioimmunoimaging and radioimmunotherapy (RIT) of experimental PDAC. METHODS: The antibodies to CETN1 were generated via mice immunization with immunogenic peptide distinguishing CETN1 from CETN2. Patient tumor microarrays were used to evaluate the binding of the immune serum to PDAC versus normal pancreas. The antibodies were tested for their preferential binding to CETN1 over CETN2 by ELISA. Mice bearing PDAC MiaPaCa2 xenografts were imaged with microSPECT/CT and treated with 213 Bi- and 177 Lu-labeled antibodies to CETN1. RESULTS: Immune serum bind to 50% PDAC cases on patient tumor microarrays with no specific binding to normal pancreas. Antibodies demonstrated preferential binding to CETN1 versus CETN2. Antibody 69-11 localized to PDAC xenografts in mice in vivo and ex vivo. RIT of PDAC xenografts with 213 Bi-labeled antibodies was effective, safe, and CETN1-specific. CONCLUSIONS: The results demonstrate the ability of these novel antibodies to detect CETN1 both in vitro and in vivo; as well, the RIT treatment of experimental PDAC when radiolabeled with 213 Bi is highly efficient and safe. Further evaluation of these novel reagents for diagnosis and treatment of PDAC is warranted.

Radiofungicidal effects of external gamma radiation and antibody-targeted beta and alpha radiation on Cryptococcus neoformans.

We evaluated the clonogenic survival, membrane permeability, metabolic activity (XTT reduction), and apoptosis (FLICA binding) of Cryptococcus neoformans cells subjected to gamma rays from an external source, and beta and alpha particles delivered to fungal cells by capsule-specific antibody. We found that gamma, beta, and alpha radiation affected cells through different pathways.

Finite-element model of interaction between fungal polysaccharide and monoclonal antibody in the capsule of Cryptococcus neoformans.

Many microorganisms such as bacteria and fungi possess so-called capsules made of polysaccharides which protect these microorganisms from environmental insults and host immune defenses. The polysaccharide capsule of Cryptococcus neoformans, a human pathogenic yeast, is capable of self-assembly, composed mostly of glucuronoxylomannan (GXM), a polysaccharide with a molecular weight of approximately 2,000,000, and has several layers with different densities. The objective of this study was to model pore-hindered diffusion and binding of the GXM-specific antibody within the C. neoformans capsule. Using the finite-element method (FEM), we created a model which represents the in vivo binding of a GXM-specific antibody to a C. neoformans cell taking into account the intravenous infusion time of antibody, antibody diffusion through capsular pores, and Michaelis-Menten kinetics of antibody binding to capsular GXM. The model predicted rapid diffusion of antibody to all regions of the capsule where the pore size was greater than the Stokes diameter of the antibody. Binding occurred primarily at intermediate regions of the capsule. The GXM concentration in each capsular region was the principal determinant of the steady-state antibody-GXM complex concentration, while the forward binding rate constant influenced the rate of complex formation in each region. The concentration profiles predicted by the model closely matched experimental immunofluorescence data. Inclusion of different antibody isotypes (IgG, IgA, and IgM) into the modeling algorithm resulted in similar complex formation in the outer capsular regions, but different depths of binding at the inner regions. These results have implications for the development of new antibody-based therapies.

Comparative evaluation of capsular polysaccharide-specific IgM and IgG antibodies and F(ab')2 and Fab fragments as delivery vehicles for radioimmunotherapy of fungal infection.

PURPOSE: The applicability of radioimmunotherapy with organism-specific monoclonal antibodies to treatment of infectious disease in experimental models has been recently shown for fungal, bacterial, and viral infections. To identify the best delivery vehicle for radioimmunotherapy of human pathogenic fungus Cryptococcus neoformans (CN), we have done comparative evaluation of capsular polysaccharide-specific antibodies with IgG1 and IgM isotypes and F(ab')2 and Fab fragments. EXPERIMENTAL DESIGN: 18B7 IgG1 and 13F1 IgM and their isotype-matching controls were radiolabeled with 188Re, and their binding to 24067 and H99 CN strains was evaluated by doing Scatchard and kinetics analyses. The doses delivered during in vitro radioimmunotherapy were estimated using a cellular dosimetry algorithm. The biodistribution of 188Re-labeled 18B7 and 13F1 and of 111In-labeled 18B7 and its F(ab')2 and Fab fragments was done in A/JCr mice systemically infected with 24067 CN strain. RESULTS: 18B7 IgG1 showed superior to 13F1 IgM binding to 24067 CN (Ka=1.7x10(9) mol/L(-1) and 5.4x10(7) mol/L(-1), respectively). Substantial killing of 24067 and H99 CN cells was achieved with 1 microCi 188Re-18B7 (55 cGy dose), whereas no killing was observed for 1 microCi 188Re-13F1 (2 cGy dose). In vivo 188Re-18B7 localized specifically in the lungs of CN-infected mice, whereas uptake of 188Re-13F1 was nonspecific. 111In-F(ab')2 fragments showed higher uptake in the lungs and lower in the liver at the 48-h time point in comparison with intact 111In-18B7. CONCLUSIONS: Comparative evaluation of IgG and IgM and of F(ab')2 and Fab fragments as potential delivery vehicles for radioimmunotherapy of cryptococcal infection strongly suggests that affinity for the target antigen is an important prerequisite for successful targeting of infection in vivo and that in vitro affinity measurements may predict the in vivo efficacy of candidate monoclonal antibodies.

Morphological changes in melanized and non-melanized Cryptococcus neoformans cells post exposure to sparsely and densely ionizing radiation demonstrate protective effect of melanin.

There is a need for novel and effective prophylactic treatments and radioprotective materials to protect civilians and military personnel from ionizing radiation in contaminated environments. Melanin, a naturally occurring, ubiquitous pigment, has been shown to confer radioresistance, acting as a potential radioprotective agent. We have demonstrated that melanized Cryptococcus neoformans (CN) cells had improved survival post ionizing irradiation than non-melanized ones. The goal of this study was to identify morphological changes in melanized and non-melanized CN cells following irradiation with densely-ionizing deuterons and alpha particles relative to sparsely-ionizing gamma radiation. We observed significant differences between the melanized and non-melanized CN cellular ultrastructure following irradiation. Melanized CN cells were relatively resistant to mid and max-dose levels of alpha particles and deuterons irradiation. Following irradiation the capsule was stripped, but the cell wall was intact and structural integrity was maintained. At the maximum dose, cytoplasmic vacuolization, and mitochondrial swelling started to occur. In contrast, the non-melanized CN strain was sensitive to the mid-dose radiation. Non-melanized cells presented two morphologies: small condensed, and swollen, lacking structural integrity. This morphological investigation provides the first direct evidence of the radioprotective properties of melanin in CN cells subjected to high RBE and high LET ionizing radiation.

Survival and redox activity of Friedmanniomyces endolithicus, an Antarctic endemic black meristematic fungus, after gamma rays exposure.

Despite living organisms are not exposed to acute ionizing radiation under natural conditions, some exhibit a high radiation resistance. Understanding this phenomenon is important for assessing the impact of radiation-related accidents, occupational exposures and space missions. In this context, in this study we analyzed the effect of gamma rays on the Antarctic cryptoendolithic melanized fungus Friedmanniomyces endolithicus CCFEE 5208 and demonstrated its resistance to acute doses of gamma radiation (up to 400 Gy), accompanied by increase in metabolic activity.

The effect of protracted X-ray exposure on cell survival and metabolic activity of fast and slow growing fungi capable of melanogenesis.

The aim of this study was to analyse how protracted exposure to X-rays delivered at low dose rates of 0.0032-0.052 kGy h-1 affects the survival and metabolic activity of two microfungi capable of melanogenesis: fast-growing Cryptococcus neoformans (CN) and slow-growing Cryomyces antarcticus (CA). Melanized CN and CA cells survived the protracted exposure better than non-melanized ones, which was consistent with previous reports on the radioprotective role of melanin in these fungi after high dose rate exposures. The survival data were described by the linear quadratic dose response model. The XTT metabolic profiles were practically identical for melanized CN and CA with activity dose-dependent increasing: no changes in the activity of the non-melanized CN and CA were recorded by this assay. In contrast, the MTT assay, which measures the intracellular energy-related processes, recorded an increase in activity of non-melanized CN and CA cells, but not in their melanized counterparts. This could reflect intensive repair processes initiated by the non-melanized cells post exposure. This study suggests that differences in radiation responses between melanized and non-melanized fungal cells occur over a wide range of radiation dose rates.

Melanin, Radiation, and Energy Transduction in Fungi.

Melanin pigments are found in many diverse fungal species, where they serve a variety of functions that promote fitness and cell survival. Melanotic fungi inhabit some of the most extreme habitats on earth such as the damaged nuclear reactor at Chernobyl and the highlands of Antarctica, both of which are high-radiation environments. Melanotic fungi migrate toward radioactive sources, which appear to enhance their growth. This phenomenon, combined with the known capacities of melanin to absorb a broad spectrum of electromagnetic radiation and transduce this radiation into other forms of energy, raises the possibility that melanin also functions in harvesting such energy for biological usage. The ability of melanotic fungi to harness electromagnetic radiation for physiological processes has enormous implications for biological energy flows in the biosphere and for exobiology, since it provides new mechanisms for survival in extraterrestrial conditions. Whereas some features of the way melanin-related energy transduction works can be discerned by linking various observations and circumstantial data, the mechanistic details remain to be discovered.

Radiolabeled melanin-binding peptides are safe and effective in treatment of human pigmented melanoma in a mouse model of disease.

The incidence of melanoma is rising, and therapeutic options for metastatic melanoma are limited. We report the results of experimental melanoma therapy with 188-Rhenium-labeled melanin-binding decapeptide ((188)RE-HYNIC-4B4) and a comprehensive safety evaluation of this treatment. (188)RE-HYNIC- 4B4 bound only to nonviable eumelanotic MNT1 and pheomelanotic SK-28-MEL human melanoma cells in vitro, as determined by immunofluorescence, which is consistent with the inaccessibility of intracellular melanin in live cells, and suggests specificity for tumors with a significant amount of extracellular melanin. Administration of 1 mCi (188)RE-HYNIC-4B4 to MNT1 tumor-bearing mice significantly slowed tumor growth, with the therapeutic effect being a result of specific binding to tumor melanin, as irrelevant (188)RE-labeled decapeptide did not produce therapeutic gain. Repeated doses of (188)RE-HYNIC-4B4 had a more profound effect on tumor growth than a single dose. Treatment of tumors with 0.3-0.4 cm diameter was more effective than of larger ones (0.5-0.7 cm). There was no difference in uptake of (188)REHYNIC- 4B4 in melanized tissues of black C57BL6 mice and no histologically apparent damage to these tissues in comparison with white BALB/C mice. Treatment of C57BL6 mice with (188)RE-HYNIC-4B4 did not change their behavior, as established by SHIRPA protocol, and did not cause damage to neurons and glial cells. These results indicate that radiolabeled melanin-binding peptides are efficient and safe in treatment of melanoma and could be potentially useful against this tumor.

A fully human antibody to gp41 selectively eliminates HIV-infected cells that transmigrated across a model human blood brain barrier.

OBJECTIVE: Many HIV patients on combined antiretroviral therapy exhibit HIV-associated neurocognitive disorders because the brain becomes a viral reservoir. There is a need for therapeutics that can enter the central nervous system (CNS) and eradicate the virus. DESIGN: Radiolabeled human mAb 2556 to HIV gp41 selectively kills HIV-infected cells in vivo and in vitro. Here we tested the ability of 213Bi-2556 to cross a tissue culture model of the human blood brain barrier and kill HIV-infected peripheral blood mononuclear cells (PBMCs) and monocytes on the CNS side of the barrier. METHODS: 2556 mAb isoelectric point was determined with isoelectric focusing. The ability of radiolabeled 2556 to penetrate through the barrier was studied by adding it to the upper chamber of the barriers and its penetration into the CNS side was followed for 5 h. To assess the ability of Bi-2556 to kill the HIV-infected cells on the CNS side of barrier, the HIV-infected and uninfected PBMCs and monocytes were allowed to transmigrate across the barriers overnight followed by application of Bi-2556 or control mAb Bi-1418 to the top of the barrier. Killing of cells was measured by TUNEL and Trypan blue assays. The barriers were examined by confocal microscopy for overt damage. RESULTS: The isoelectric point of Bi-2556 was 9.6 enabling its penetration through the barrier by transcytosis. Bi-2556 killed significantly more transmigrated HIV-infected cells in comparison to Bi-1418 and uninfected cells. No overt damage to barriers was observed. CONCLUSION: We demonstrated that Bi-2556 mAb crossed an in-vitro human blood brain barrier and specifically killed transmigrated HIV-infected PBMCs and monocytes without overt damage to the barrier.

Effects of radiation type and delivery mode on a radioresistant eukaryote Cryptococcus neoformans.

INTRODUCTION: Most research on radioresistant fungi, particularly on human pathogens such as Cryptococcus neoformans, involves sparsely-ionizing radiation. Consequently, fungal responses to densely-ionizing radiation, which can be harnessed to treat life-threatening fungal infections, remain incompletely understood. METHODS: We addressed this issue by quantifying and comparing the effects of densely-ionizing α-particles (delivered either by external beam or by (213)Bi-labeled monoclonal antibodies), and sparsely-ionizing (137)Cs γ-rays, on Cryptococcus neoformans. RESULTS: The best-fit linear-quadratic parameters for clonogenic survival were the following: α = 0.24 × 10(-2) Gy(-1) for γ-rays and 1.07 × 10(-2) Gy(-1) for external-beam α-particles, and ß = 1.44 × 10(-5) Gy(-2) for both radiation types. Fungal cell killing by radiolabeled antibodies was consistent with predictions based on the α-particle dose to the cell nucleus and the linear-quadratic parameters for external-beam α-particles. The estimated RBE (for α-particles vs. γ-rays) at low doses was 4.47 for the initial portion of the α-particle track, and 7.66 for the Bragg peak. Non-radiological antibody effects accounted for up to 23% of cell death. CONCLUSIONS: These results quantify the degree of C. neoformans resistance to densely-ionizing radiations, and show how this resistance can be overcome with fungus-specific radiolabeled antibodies.

Susceptibility of the human pathogenic fungi Cryptococcus neoformans and Histoplasma capsulatum to gamma-radiation versus radioimmunotherapy with alpha- and beta-emitting radioisotopes.

UNLABELLED: Fungal diseases are difficult to treat in immunosuppressed patients and, consequently, new approaches to therapy are urgently needed. One novel strategy is to use radioimmunotherapy (RIT) with fungal-binding monoclonal antibodies (mAbs) labeled with radionuclides. However, many fungi manifest extreme resistance to gamma-radiation, such that the doses of several thousand gray are required for 90% cell killing, whereas for mammalian cells the lethal dose is only a few gray. We compared the susceptibility of human pathogenic fungi Cryptococcus neoformans (CN) and Histoplasma capsulatum (HC) to external gamma-radiation and to the organism-specific mAbs 18B7 and 9C7, respectively, conjugated to (213)Bi and (188)Re radionuclides. METHODS: CN and HC cells were irradiated with up to 8,000 Gy ((137)Cs source, 30 Gy/min). RIT of CN with (213)Bi- and (188)Re-labeled specific mAb and of HC with (188)Re-labeled specific mAb used 0-1.2 MBq per 10(5) microbial cells. After irradiation or RIT, the cells were plated for colony-forming units (CFUs). Cellular dosimetry calculations were performed, and the pathway of cell death after irradiation was evaluated by flow cytometry. RESULTS: Both CN and HC proved to be extremely resistant to gamma-radiation such that significant killing was observed only for doses of >4,000 Gy. In contrast, these cells were much more susceptible to killing by radiation delivered with a specific mAb, such that a 2-logarithm reduction in colony numbers was achieved by incubating them with (213)Bi- and (188)Re-labeled mAb 18B7 or with (188)Re-9C7 mAb. Dosimetry calculations showed that RIT was approximately 1,000-fold more efficient in killing CN and approximately 100-fold more efficient in killing HC than gamma-radiation. Both gamma-radiation and RIT caused cell death via an apoptotic-like pathway with a higher percentage of apoptosis observed in RIT-treated cells. CONCLUSION: Conjugating a radioactive isotope to a fungal-specific antibody converted an immunoglobulin with no antifungal activity into a microbicidal molecule. RIT of fungal cells using specific antibodies labeled with alpha- and beta-emitting radioisotopes was significantly more efficient in killing CN and HC than gamma-radiation when based on the mean absorbed dose to the cell. These results strongly support the concept of using RIT as an antimicrobial modality.

Mathematical modeling predicts enhanced growth of X-ray irradiated pigmented fungi.

Ionizing radiation is known for its cytotoxic and mutagenic properties. However, recent evidence suggests that chronic sub-lethal irradiation stimulates the growth of melanin-pigmented (melanized) fungi, supporting the hypothesis that interactions between melanin and ionizing photons generate energy useful for fungal growth, and/or regulate growth-promoting genes. There are no quantitative models of how fungal proliferation is affected by ionizing photon energy, dose rate, and presence versus absence of melanin on the same genetic background. Here we present such a model, which we test using experimental data on melanin-modulated radiation-induced proliferation enhancement in the fungus Cryptococcus neoformans, exposed to two different peak energies (150 and 320 kVp) over a wide range of X-ray dose rates. Our analysis demonstrates that radiation-induced proliferation enhancement in C. neoformans behaves as a binary "on/off" phenomenon, which is triggered by dose rates <0.002 mGy/h, and stays in the "on" position. A competing dose rate-dependent growth inhibition becomes apparent at dose rates >5000 mGy/h. Proliferation enhancement of irradiated cells compared with unirradiated controls occurs at both X-ray peak energies, but its magnitude is modulated by X-ray peak energy and cell melanization. At dose rates <5000 mGy/h, both melanized and non-melanized cells exposed to 150 kVp X-rays, and non-melanized cells exposed to 320 kVp X-rays, all exhibit the same proliferation enhancement: on average, chronic irradiation stimulates each founder cell to produce 100 (95% CI: 83, 116) extra descendants over 48 hours. Interactions between melanin and 320 kVp X-rays result in a significant (2-tailed p-value = 4.8 × 10(-5)) additional increase in the number of radiation-induced descendants per founder cell: by 55 (95% CI: 29, 81). These results show that both melanin-dependent and melanin-independent mechanisms are involved in radiation-induced fungal growth enhancement, and implicate direct and/or indirect interactions of melanin with high energy ionizing photons as an important pro-proliferative factor.

Treatment of experimental pancreatic cancer with 213-Bismuth-labeled chimeric antibody to single-strand DNA.

BACKGROUND: Novel approaches to treatment of pancreatic cancer (PCa) are urgently needed. A chimeric monoclonal antibody (mAb) chTNT3 binds to single-strand DNA (ssDNA) and RNA released from the non-viable cells in fast growing tumors. Here the authors investigated whether radioimmunotherapy (RIT) using chTNT3 mAb radiolabeled with 213-Bismuth ((213)Bi) could be effective in treatment of experimental PCa. METHODS: Two human PCa cell lines, Panc1 and MiaPaCa-2, were used for in vitro experiments. The xenografts in mice were established using MiaPaCa-2 cells. Therapy compared (213)Bi-chTNT3 (700 µCi) to gemcitabine or cisplatin, untreated controls and 'cold' chTNT3. RESULTS: RIT abrogated the tumors growth while tumors in control groups grew aggressively. Chemotherapy was less effective than RIT and toxic to mice while RIT did not have any side effects. CONCLUSIONS: RIT with (213)Bi-chTNT3 was safe and effective in the treatment of experimental PCa in comparison with chemotherapy. This makes α-RIT targeting ssDNA a promising modality for further development.

Complement-dependent cytotoxicity of an antibody to melanin in radioimmunotherapy of metastatic melanoma.

AIM: Novel treatments for metastatic melanoma are urgently needed. MATERIALS & METHODS: We developed radioimmunotherapy of metastatic melanoma using 6D2 monoclonal antibody (mAb) to melanin with encouraging therapeutic results, preclinically and in patients. RESULTS: We observed tumor suppression with the unlabeled 6D2 mAb and investigated its tumoricidal mechanisms. In melanoma tumor-bearing mice, we detected more complement-C3 deposition in the tumors from 188-rhenium-labeled 6D2 mAb-treated mice when compared with untreated controls. 6D2 and isotype-control mAb TEPC caused suppression of tumor growth in A2058 melanoma tumor-bearing mice. Tumors of mice treated with the unlabeled 6D2 mAb were infiltrated with more lymphocytes compared with controls. In vitro antibody-dependent cell-mediated cytotoxicity did not contribute to the tumor-suppressive effect of 6D2 mAb, while 6D2 mAb demonstrated a strong effect on initiating complement-dependent cytotoxicity. CONCLUSION: We concluded that 6D2 mAb mediated complement-dependent cytotoxicity, resulting in killing of the tumor cells and suppression of tumor growth. These observations will help to improve the treatment protocols of radioimmunotherapy, as well as immunotherapy.