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BackgroundAddressing prosthetic joint infections poses a significant challenge within orthopedic surgery, marked by elevated morbidity and mortality rates. The presence of biofilms and infections attributed to Staphylococcus aureus (S. aureus) further complicates the scenario.ObjectiveTo investigate the potential of radioimmunotherapy as an innovative intervention to tackle biofilm-associated infections.MethodsOur methodology involved employing specific monoclonal antibodies 4497-IgG1, designed for targeting wall teichoic acids found on S. aureus and its biofilm. These antibodies were linked with radionuclides actinium-225 (225Ac) and lutetium-177 (177Lu) using DOTA as a chelator. Following this, we evaluated the susceptibility of S. aureus and its biofilm to radioimmunotherapy in vitro, assessing bacterial viability and metabolic activity via colony-forming unit enumeration and xylenol tetrazolium assays.ResultsBoth [225Ac]4497-IgG1 and [177Lu]4497-IgG1 exhibited a noteworthy dose-dependent reduction in S. aureus in planktonic cultures and biofilms over a 96-h exposure period, compared to non-specific antibody control groups. Specifically, doses of 7.4 kBq and 7.4 MBq of [225Ac]4497-IgG1 and [177Lu]4497-IgG1 resulted in a four-log reduction in planktonic bacterial counts. Within biofilms, 14.8 kBq of [225Ac]4497-IgG1 and 14.8 Mbq [177Lu]4497-IgG1 led to reductions of two and four logs, respectively.ConclusionOur findings underscore the effectiveness of [225Ac]4497-IgG1 and [177Lu]4497-IgG1 antibodies in exerting dose-dependent bactericidal effects against planktonic S. aureus and biofilms in vitro. This suggests that radioimmunotherapy might serve as a promising targeted treatment approach for combating S. aureus and its biofilm.

Novel therapeutic approaches are much needed for the treatment of osteosarcoma. Targeted radionuclide therapy (TRT) and radioimmunotherapy (RIT) are promising approaches that deliver therapeutic radiation precisely to the tumor site. We have previously developed a fully human antibody, named IF3, that binds to insulin-like growth factor 2 receptor (IGF2R). IF3 was used in TRT to effectively inhibit tumor growth in osteosarcoma preclinical models. However, IF3’s relatively short half-life in mice raised the need for improvement. We generated an Fc-engineered version of IF3, termed IF3δ, with amino acid substitutions known to enhance antibody half-life in human serum. In this study, we confirmed the specific binding of IF3δ to IGF2R with nanomolar affinity, similar to wild-type IF3. Additionally, IF3δ demonstrated binding to human and mouse neonatal Fc receptors (FcRn), indicating the potential for FcRn-mediated endocytosis and recycling. Biodistribution studies in mice showed a higher accumulation of IF3δ in the spleen and bone than wild-type IF3, likely attributed to abnormal spleen expression of IGF2R in mice. Therefore, the pharmacokinetics data from mouse xenograft models may not precisely reflect their behavior in canine and human patients. However, the findings suggest both IF3 and IF3δ as promising options for the RIT of osteosarcoma.

Background: Osteosarcoma (OS) represents the most common primary bone tumor in humans and in companion dogs, being practically phenotypically identical. There is a need for effective treatments to extend the survival of patients with OS. Here, we examine the dosimetry in beagle dogs and cross-reactivity with human tissues of a novel human antibody, IF3, that targets the insulin growth factor receptor type 2 (IGF2R), which is overexpressed on OS cells, making it a candidate for radioimmunotherapy of OS. Methods: [89Zr]Zr-DFO-IF3 was injected into three healthy beagle dogs. PET/CT was conducted at 4, 24, 48, and 72 h. RAPID analysis was used to determine the dosimetry of [177Lu]Lu-CHXA”-IF3 for a clinical trial in companion dogs with OS. IF3 antibody was biotinylated, and a multitude of human tissues were assessed with immunohistochemistry. Results: PET/CT revealed that only the liver, bone marrow, and adrenal glands had high uptake. Clearance was initially through renal and hepatobiliary excretion in the first 72 h followed by primarily physical decay. RAPID analysis showed bone marrow to be the dose-limiting organ with a therapeutic range for 177Lu calculated to be 0.487–0.583 GBq. Immunohistochemistry demonstrated the absence of IGF2R expression on the surface of healthy human cells, thus suggesting that radioimmunotherapy with [177Lu]Lu-CHXA”-IF3 will be well tolerated. Conclusions: Image-based dosimetry has defined a safe therapeutic range for canine clinical trials, while immunohistochemistry has suggested that the antibody will not cross-react with healthy human tissues.

ABSTRACT Purpose Despite the availability of new drugs, many patients with acute myeloid leukemia (AML) do not achieve remission and outcomes remain poor. Venetoclax is a promising new therapy approved for use in combination with a hypomethylating agent or with low‐dose cytarabine for the treatment of newly diagnosed older AML patients or those ineligible for intensive chemotherapy. 225Actinium‐lintuzumab (225Ac‐lintuzumab) is a clinical stage radioimmunotherapy targeting CD33 that has shown evidence of single‐agent activity in relapsed/refractory AML. Increased expression of MCL‐1 is a mediator of resistance to venetoclax in cancer. Experimental design Here we investigated the potential for 225Ac‐lintuzumab‐directed DNA damage to suppress MCL‐1 levels as a possible mechanism of reversing resistance to venetoclax in two preclinical in vivo models of AML. Results We demonstrated that 225Ac‐lintuzumab in combination with venetoclax induced a synergistic increase in tumor cell killing compared to treatment with either drug alone in venetoclax‐resistant AML cell lines through both an induction of double‐stranded DNA breaks (DSBs) and depletion of MCL‐1 protein levels. Further, this combination led to significant tumor growth control and prolonged survival benefit in venetoclax‐resistant in vivo AML models. Conclusions There results suggest that the combination of 225Ac‐lintuzumab with venetoclax is a promising therapeutic strategy for the treatment of patients with venetoclax‐resistant AML. Clinical trial of this combination therapy (NCT03867682) is currently ongoing.

Melanized fungi have been isolated from some of the harshest radioactive environments, and their ability to thrive in these locations is in part due to the pigment melanin. Melanin imparts a selective advantage to fungi by providing a physical shield, a chemical shield, and possibly a signaling mechanism. In previous work we demonstrated that protracted exposure of the melanized yeast Exophiala dermatitidis to mixed alpha-, beta-, and gamma-emitting radiation resulted in an adapted strain able to mount a unique response to ionizing radiation in the environment in a melanin-dependent fashion. By exploring the genome and transcriptome of this adapted melanized strain relative to a non-irradiated control we determined the altered response was transcriptomic in nature, as whole genome sequencing revealed limited variation. Transcriptomic analysis indicated that of the adapted isolates analyzed, two lineages existed: one like the naïve, non-adapted strain, and one with a unique transcriptomic signature that exhibited downregulation of metabolic processes, and upregulation of translation-associated genes. Analysis of differential gene expression in the adapted strain showed an overlap in response between the control conditions and reactive oxygen species conditions, whereas exposure to an alpha particle source resulted in a robust downregulation of metabolic processes and upregulation of DNA replication and repair genes, and RNA metabolic processes. This suggest previous exposure to radiation primes the fungus to respond to subsequent exposures in a unique way. By exploring this unique response, we have expanded our knowledge of how melanized fungi interact with and respond to ionizing radiation in their environment.

COVID-19 pandemic has heightened the interest toward diagnosis and treatment of infectious diseases. Nuclear medicine with its powerful scintigraphic, single photon emission computer tomography (SPECT) and positron emission tomography (PET) imaging modalities has always played an important role in diagnosis of infections and distinguishing them from the sterile inflammation. In addition to the clinically available radiopharmaceuticals there has been a decades-long effort to develop more specific imaging agents with some examples being radiolabeled antibiotics and antimicrobial peptides for bacterial imaging, radiolabeled anti-fungals for fungal infections imaging, radiolabeled pathogen-specific antibodies and molecular engineered constructs. In this opinion piece, we would like to discuss some examples of the work published in the last decade on developing nuclear imaging agents for bacterial, fungal, and viral infections in order to generate more interest among nuclear medicine community toward conducting clinical trials of these novel probes, as well as toward developing novel radiotracers for imaging infections.

Implant-associated Staphylococcus aureus infections are difficult to treat because of biofilm formation. Bacteria in a biofilm are often insensitive to antibiotics and host immunity. Monoclonal antibodies (mAbs) could provide an alternative approach to improve the diagnosis and potential treatment of biofilm-related infections. Here, we show that mAbs targeting common surface components of S. aureus can recognize clinically relevant biofilm types. The mAbs were also shown to bind a collection of clinical isolates derived from different biofilm-associated infections (endocarditis, prosthetic joint, catheter). We identify two groups of antibodies: one group that uniquely binds S. aureus in biofilm state and one that recognizes S. aureus in both biofilm and planktonic state. Furthermore, we show that a mAb recognizing wall teichoic acid (clone 4497) specifically localizes to a subcutaneously implanted pre-colonized catheter in mice. In conclusion, we demonstrate the capacity of several human mAbs to detect S. aureus biofilms in vitro and in vivo.

Radiation damage is associated with inflammation and immunity in the intestinal mucosa, including gut microbiota. Melanin has a unique capacity to coordinate a biological reaction in response to environmental stimuli, such as radiation exposure. Thus, melanin and melanized microbes have potential to be used for mitigation of injury induced by radiation. The purpose of the current study is to examine the safety of these agents for future targeting gut microbiome to prevent radiation-induced injury. We administered mice with soluble allomelanin and observed its effect on the intestinal physiology and body weight. We then established a melanized bacterial strain in probiotic E. coli Nissle. We measured the body weight of the mice treated with melanized E. coli Nissle. We showed the enhanced bacterial abundance and colonization of the melanized bacteria E. coli Nissle in the intestine. Melanized E. coli Nissle colonized the colon in less than 3 h and showed consistent colonization over 24 h post one oral gavage. We did not find significant changes of bodyweight in the mice treated with melanized bacteria. We did not observe any inflammation in the intestine. These results demonstrate the safety of soluble melanin and melanin-producing bacteria and will support the future studies to treat radiation-induced injuries and restore dysbiosis.

Nearly all cases of cervical cancer are initiated by persistent infection with high-risk strains of human papillomavirus (hr-HPV). When hr-HPV integrates into the host genome, the constitutive expression of oncogenic HPV proteins E6 and E7 function to disrupt p53 and retinoblastoma regulation of cell cycle, respectively, to favor malignant transformation. HPV E6 and E7 are oncogenes found in over 99% of cervical cancer, they are also expressed in pre-neoplastic stages making these viral oncoproteins attractive therapeutic targets. Monoclonal antibodies (mAbs) represent a novel potential approach against the actions of hr-HPV E6 and E7 oncoproteins.In this report, we describe the utilization of anti-HPV E6 and HPV E7 mAbs in an experimental murine model of human cervical cancer tumors. We used differential dosing strategies of mAbs C1P5 (anti-HPV 16 E6) and TVG701Y (anti-HPV E7) administered via intraperitoneal or intratumoral injections. We compared mAbs to the action of chemotherapeutic agent Cisplatin and demonstrated the capacity of mAbs to significantly inhibit tumor growth. Furthermore, we investigated the contribution of the immune system and found increased complement deposition in both C1P5 and TVG701Y treated tumors compared to irrelevant mAb therapy. Taken together, the results suggest that anti-HPV E6 and E7 mAbs exert inhibition of tumor growth in a viral-specific manner and stimulate an immune response that could be exploited for an additional treatment options for patients. Keywords: cervical cancer, monoclonal antibodies, HPV E6 and E7, immunotherapy, nude mice

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) accounts for >90% of pancreatic malignancies, and has median survival of

Objective: Positron emission tomography (PET) imaging is a powerful non-invasive method to determine the in vivo behavior of biomolecules. Determining biodistribution and pharmacokinetic (PK) properties of targeted therapeutics can enable a better understanding of in vivo drug mechanisms such as tumor uptake, off target accumulation and clearance. Zirconium-89 (89Zr) is a readily available tetravalent PET-enabling radiometal that has been used to evaluate the biodistribution and PK of monoclonal antibodies. In the current study, we performed in vitro and in vivo characterization of 89Zr-lintuzumab, a radiolabeled anti-CD33 antibody, as a model to evaluate the in vivo binding properties in preclinical models of AML. Methods: Lintuzumab was conjugated to p-SCN-Bn-deferoxamine (DFO) and labeled with 89Zr using a 5:1 µCi:µg specific activity at 37 °C for 1h. The biological activity of 89Zr-lintuzumab was evaluated in a panel of CD33 positive cells using flow cytometry. Fox Chase SCID mice were injected with 2 × 106 OCI-AML3 cells into the right flank. After 12 days, a cohort of mice (n = 4) were injected with 89Zr-lintuzumab via tail vein. PET/CT scans of mice were acquired on days 1, 2, 3 and 7 post 89Zr-lintuzumab injection. To demonstrate 89Zr-lintuzumab specific binding to CD33 expressing tumors in vivo, a blocking study was performed. This cohort of mice (n = 4) was injected with native lintuzumab and 24 h later 89Zr-lintuzumab was administered. This group was imaged 3 and 7 days after injection of 89Zr-lintuzumab. A full ex vivo biodistribution study on both cohorts was performed on day 7. The results from the PET image and ex vivo biodistribution studies were compared. Results: Lintuzumab was successfully radiolabeled with 89Zr resulting in a 99% radiochemical yield. The 89Zr-lintuzumab radioconjugate specifically binds CD33 positive cells in a similar manner to native lintuzumab as observed by flow cytometry. PET imaging revealed high accumulation of 89Zr-lintuzumab in OCI-AML3 tumors within 24h post-injection of the radioconjugate. The 89Zr-lintuzumab high tumor uptake remains for up to 7 days. Tumor analysis of the PET data using volume of interest (VOI) showed significant blocking of 89Zr-lintuzumab in the group pre-treated with native lintuzumab (pre-blocked group), thus indicating specific targeting of CD33 on OCI-AML3 cells in vivo. The tumor uptake findings from the PET imaging study are in agreement with those from the ex vivo biodistribution results. Conclusions: PET imaging of 89Zr-lintuzumab shows high specific uptake in CD33 positive human OCI-AML3 tumors. The results from the image study agree with the observations from the ex vivo biodistribution study. Our findings collectively suggest that PET imaging using 89Zr-lintuzumab could be a powerful drug development tool to evaluate binding properties of anti-CD33 monoclonal antibodies in preclinical cancer models.

Background: Osteosarcoma (OS) has an overall patient survival rate of ~70% with no significant improvements in the last two decades, and novel effective treatments are needed. OS in companion dogs is phenotypically close to human OS, which makes a comparative oncology approach to developing new treatments for OS very attractive. We have recently created a novel human antibody, IF3 to IGF2R, which binds to this receptor on both human and canine OS tumors. Here, we evaluated the efficacy and safety of radioimmunotherapy with 177Lu-labeled IF3 of mice bearing canine-patient-derived tumors and performed canine and human dosimetry calculations. Methods: Biodistribution and microSPECT/CT imaging with 111In-IF3 was performed in mice bearing canine OS Gracie tumors, and canine and human dosimetry calculations were performed based on these results. RIT of Gracie-tumor-bearing mice was completed with 177Lu-IF3. Results: Biodistribution and imaging showed a high uptake of 111In-IF3 in the tumor and spleen. Dosimetry identified the tumor, spleen and pancreas as the organs with the highest uptake. RIT was very effective in abrogating tumor growth in mice with some spleen-associated toxicity. Conclusions: These results demonstrate that RIT with 177Lu-IF3 targeting IGF2R on experimental canine OS tumors effectively decreases tumor growth. However, because of the limitations of murine models, careful evaluation of the possible toxicity of this treatment should be performed via nuclear imaging and image-based dosimetry in healthy dogs before clinical trials in companion dogs with OS can be attempted.

Invasive fungal infections (IFI) cause devastating morbidity and mortality, with the number of IFIs more than tripling since 1979. Our laboratories were the first to demonstrate that radiolabeled microorganism-specific monoclonal antibodies are highly effective for treatment of experimental fungal, bacterial and viral infections. Later we proposed to utilize surface expressed pan-antigens shared by major IFI-causing pathogens such as beta-glucans as RIT targets. Here we evaluated in vivo RIT targeting beta-glucan in Blastomyces dermatitidis which causes serious infections in immunocompromised and immunocompetent individuals and in companion dogs. B. dermatitidis cells were treated with the 400-2 antibody to (1→3)-β-glucans radiolabeled with the beta-emitter 177Lutetium (177Lu) and alpha-emitter 213Bismuth (213Bi) and the efficacy of cell kill was determined by colony forming units (CFUs). To determine the antigen-specific localization of the 400-2 antibody in vivo, C57BL6 mice were infected intratracheally with 2 × 105B. dermatitidis cells and given 111In-400-2 antibody 24 h later. To evaluate the killing of B. dermatitidis cells with RIT, intratracheally infected mice were treated with 150 μCi 213Bi-400-2 and their lungs analyzed for CFUs 96 h post-infection. 213Bi-400-2 proved to be more effective in killing B. dermatitidis cells in vitro than 177Lu-400-2. Three times more 111In-400-2 accumulated in the lungs of infected mice, than in the non-infected ones. 213Bi-400-2 lowered the fungal burden in the lungs of infected mice more than 2 logs in comparison with non-treated infected controls. In conclusion, our results demonstrate the ability of an anti-(1-3)-beta-D-glucan antibody armed with an alpha-emitter 213Bi to selectively kill B. dermatitidis cells in vitro and in vivo. These first in vivo results of the effectiveness of RIT targeting pan-antigens on fungal pathogens warrant further investigation.

Daratumumab is an anti-CD38 directed monoclonal antibody approved for the treatment of multiple myeloma (MM) and functions primarily via Fc-mediated effector mechanisms such as complement-dependent cytotoxicity (CDC), antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis, and T-cell activation. However, not all patients respond to daratumumab therapy and management of MM remains challenging. Radioimmunotherapy with alpha particle-emitting radionuclides represents a promising approach to significantly enhance the potency of therapeutic antibodies in cancer treatment. Here we report the results of mechanistic and feasibility studies using daratumumab radiolabeled with an alpha-emitter 225Actinium for therapy of MM. CD38-positivelymphoma Daudi cell line and MM cell lines KMS-28BM and KMS-28PE were treated in vitro with 225Ac-daratumumab. 225Ac-daratumumab Fc-functional properties were assessed with C1q binding and ADCC assays. The pharmacokinetics and tumor uptake of 111In-daratumumab in Daudi tumor-bearing severe combined immunodeficiency (SCID) mice were measured with microSPECT/CT. The therapeutic effects of 225Ac-daratumumab on Daudi and KSM28BM tumors in mice and treatment side effects were evaluated for 50 days posttreatment. The safety of 225Ac-labeled antimurine CD38 mAb in immunocompetent mice was also evaluated. 225Ac-daratumumab efficiently and specifically killed CD38-positive tumor cells in vitro, while its complement binding and ADCC functions remained unaltered. MicroSPECT/CT imaging demonstrated fast clearance of the radiolabeled daratumumab from the circulation and tissues, but prolonged retention in the tumor up to 10 days. Therapy and safety experiments with 225Ac-daratumumab showed a significant increase in the antitumor potency in comparison to naked antibody without any significant side effects. Our results highlight the potential of targeting alpha-emitters to tumors as a therapeutic approach and suggest that 225Ac-daratumumab may be a promising therapeutic strategy for the treatment of hematologic malignancies.

Background: With the limited options available for therapy to treat invasive fungal infections (IFI), radioimmunotherapy (RIT) can potentially offer an effective alternative treatment. Microorganism-specific monoclonal antibodies have shown promising results in the experimental treatment of fungal, bacterial, and viral infections, including our recent and encouraging results from treating mice infected with Blastomyces dermatitidis with 213Bi-labeled antibody 400-2 to (1→3)-β-glucan. In this work, we performed a safety study of 213Bi-400-2 antibody in healthy dogs as a prelude for a clinical trial in companion dogs with acquired invasive fungal infections and later on in human patients with IFI. Methods: Three female beagle dogs (≈6.1 kg body weight) were treated intravenously with 155.3, 142.5, or 133.2 MBq of 213Bi-400-2 given as three subfractions over an 8 h period. RBC, WBC, platelet, and blood serum biochemistry parameters were measured periodically for 6 months post injection. Results: No significant acute or long-term side effects were observed after RIT injections; only a few parameters were mildly and transiently outside reference change value limits, and a transient atypical morphology was observed in the circulating lymphocyte population of two dogs. Conclusions: These results demonstrate the safety of systemic 213Bi-400-2 administration in dogs and provide encouragement to pursue evaluation of RIT of IFI in companion dogs.

Melanoma is a cancer with increasing incidence and there is a need for alternatives to immunotherapy within effective approaches to treatment of metastatic melanoma. We performed comparative radioimmunotherapy (RIT) of experimental B16-F10 melanoma with novel humanized IgG to melanin h8C3 labeled with a beta emitter, 177Lu, and an alpha-emitter, 213Bi, as well as biodistribution, microSPECT/CT imaging, and mouse and human dosimetry calculations. microSPECT/CT imaging showed that a humanized antibody that targets “free” melanin in the tumor microenvironment had high tumor uptake in B16F10 murine melanoma in C57Bl/6 mice, with little to no uptake in naturally melanized tissues. Extrapolation of the mouse dosimetry data to an adult human demonstrated that doses delivered to major organs and the whole body by 177Lu-h8C3 would be approximately two times higher than those delivered by 213Bi-h8C3, while the doses to the tumor would be almost similar. RIT results indicated that 213Bi-h8C3 was more effective in slowing down the tumor growth than 177Lu-h8C3, while both radiolabeled antibodies did not produce significant hematologic or systemic side effects. We concluded that h8C3 antibody labeled with 213Bi is a promising reagent for translation into a clinical trial in patients with metastatic melanoma.

Eliminating virally infected cells is an essential component of any HIV eradication strategy. Radioimmunotherapy (RIT), a clinically established method for killing cells using radiolabeled antibodies, was recently applied to target HIV-1 gp41 antigen expressed on the surface of infect-ed cells. Since gp41 expression by infected cells is likely down-regulated in patients on an-tiretroviral therapy (ART), we evaluated the ability of RIT to kill ART-treated infected cells us-ing both in vitro models and lymphocytes isolated from HIV-infected subjects. Human peripheral blood mononuclear cells (PBMCs) were infected with HIV and cultured in the presence of two clinically relevant ART combinations. Scatchard analysis of the 2556 human monoclonal anti-body to HIV gp41 binding to the infected and ART-treated cells demonstrated sufficient residual expression of gp41 on the cell surface to warrant subsequent RIT. This is the first time the quantification of gp41 post-ART is being reported. Cells were then treated with Bismuth-213-labeled 2556 antibody. conjugated to the human monoclonal antibody 2556, which binds to HIV gp41. Cell survival was quantified by Trypan blue and residual viremia by p24 ELISA. Cell surface gp41 expression was assessed by Scatchard analysis. The experiments were repeated using PBMCs isolated from blood specimens obtained from 15 HIV-infected individuals: ten on ART and five ART-naive. We found that 213Bi-2556 killed ART-treated infected PBMCs and reduced viral production to undetectable levels. ART and RIT co-treatment was more effective at reducing viral load in vitro than either therapy alone, indicating that gp41 expression under ART was sufficient to allow 213Bi-2556 to deliver cytocidal doses of radiation to infected cells. This study provides proof of concept that 213Bi-2556 may represent an innovative and effective targeting method for killing HIV-infected cells treated with ART, and supports continued development of 213Bi-2556 for co-administration with ART towards an HIV eradication strategy.

(1) Background: Monoclonal antibodies are used in the treatment of multiple conditions including cancer, autoimmune disorders, and infectious diseases. One of the initial steps in the selection of an antibody candidate for further pre-clinical development is determining its pharmacokinetics in small animal models. The use of mass spectrometry and other techniques to determine the fate of these antibodies is laborious and expensive. Here we describe a straightforward and highly reproducible methodology for utilizing radiolabeled antibodies for pharmacokinetics studies. (2) Methods: Commercially available bifunctional linker CHXA„ and 111Indium radionuclide were used. A melanin-specific chimeric antibody A1 and an isotype matching irrelevant control A2 were conjugated with the CHXA„, and then radiolabeled with 111In. The biodistribution was performed at 4 and 24 h time points in melanoma tumor-bearing and healthy C57BL/6 female mice. (3) The biodistribution of the melanin-binding antibody showed the significant uptake in the tumor, which increased with time, and very low uptake in healthy melanin-containing tissues such as the retina of the eye and melanized skin. This biodistribution pattern in healthy tissues was very close to that of the isotype matching control antibody. (4) Conclusions: The biodistribution experiment allows us to assess the pharmacokinetics of both antibodies side by side and to make a conclusion regarding the suitability of specific antibodies for further development.

Microbial population responses to combined effects of chronic irradiation and other stressors (chemical contaminants, other sub-optimal conditions) are important for ecosystem functioning and bioremediation in radionuclide-contaminated areas. Quantitative mathematical modeling can improve our understanding of these phenomena. To identify general patterns of microbial responses to multiple stressors in radioactive environments, we analyzed three data sets on: (1) bacteria isolated from soil contaminated by nuclear waste at the Hanford site (USA); (2) fungi isolated from the Chernobyl nuclear-power plant (Ukraine) buildings after the accident; (3) yeast subjected to continuous γ-irradiation in the laboratory, where radiation dose rate and cell removal rate were independently varied. We applied generalized linear mixed-effects models to describe the first two data sets, whereas the third data set was amenable to mechanistic modeling using differential equations. Machine learning and information-theoretic approaches were used to select the best-supported formalism(s) among biologically-plausible alternatives. Our analysis suggests the following: (1) Both radionuclides and co-occurring chemical contaminants (e.g. NO2) are important for explaining microbial responses to radioactive contamination. (2) Radionuclides may produce non-monotonic dose responses: stimulation of microbial growth at low concentrations vs. inhibition at higher ones. (3) The extinction-defining critical radiation dose rate is dramatically lowered by additional stressors. (4) Reproduction suppression by radiation can be more important for determining the critical dose rate, than radiation-induced cell mortality. In conclusion, the modeling approaches used here on three diverse data sets provide insight into explaining and predicting multi-stressor effects on microbial communities: (1) the most severe effects (e.g. extinction) on microbial populations may occur when unfavorable environmental conditions (e.g. fluctuations of temperature and/or nutrient levels) coincide with radioactive contamination; (2) an organism's radioresistance and bioremediation efficiency in rich laboratory media may be insufficient to carry out radionuclide bioremediation in the field--robustness against multiple stressors is needed.

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 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

Any strategy for curing HIV infection must include a method to eliminate viral-infected cells. Based on our earlier proof-of-principle results targeting HIV-1 infected cells with radiolabeled antibody (mAb) to gp41 viral antigen, we embarked on identifying a suitable candidate mAb for preclinical development.Among the several human mAbs to gp41 tested, mAb 2556 was found to have high affinity, reactivity with multimeric forms of gp41 present on both the surface of virus particles and cells expressing HIV-1 Env, and recognition of a highly conserved epitope of gp41 shared by all HIV-1 subtypes. Also, mAb 2556 was the best in competition with HIV-1+ serum antibodies, which is an extremely important consideration for efficacy in the treatment of HIV patients. When radiolabeled with alpha-emitting radionuclide 213-Bismuth ((213)Bi) - (213)Bi-2556 efficiently and specifically killed ACH-2 human lymphocytes chronically infected with HIV-1, and HIV-1 infected human peripheral blood mononuclear cells (hPBMCs). The number of binding sites for (213)Bi-2556 on the surface of the infected cells was >10(6). The in vivo experiments were performed in two HIV-1 mouse models--splenic and intraperitoneal. In both models, the decrease in HIV-1 infected hPBMCs from the spleens and peritoneum, respectively, was dose-dependent with the most pronounced killing of hPBMCs observed in the 100 µCi (213)Bi-2556 group (P = 0.01). Measurement of the blood platelet counts and gross pathology of the treated mice demonstrated the lack of toxicity for (213)Bi-2556.We describe the preclinical development of a novel radiolabeled mAb reagent that could potentially be part of an HIV eradication strategy that is ready for translation into the clinic as the next step in its development. As viral antigens are very different from "self" human antigens - this approach promises high selectivity, increased efficacy and low toxicity, especially in comparison to immunotoxins.

Mortality associated with invasive aspergillosis (IA) remains high, partly because of delayed diagnosis. Detection of microbial exoantigens, released in serum and other body fluids during infection, may help timely diagnosis. In course of IA, Aspergillus galactomannan (GM), a well established polysaccharide biomarker, is released in body fluids including urine. Urine is an abundant, safely collected specimen, well-suited for point-of-care (POC) testing, which could play an increasing role in screening for early disease. Our main objective was to demonstrate GM antigenuria as a clinically relevant biological phenomenon in IA and establish proof-of-concept that it could be translated to POC diagnosis. Utilizing a novel IgM monoclonal antibody (MAb476) that recognizes GM-like antigens from Aspergillus and other molds, we demonstrated antigenuria in an experimental animal IA model (guinea pig), as well as in human patients. In addition, we investigated the chemical nature of the urinary excreted antigen in human samples, characterized antigen detection in urine by immunoassays, described a putative assay inhibitor in urine, and indicated means of alleviation of the inhibition. We also designed and used a lateral flow immunochromatographic assay to detect urinary excreted antigen in a limited number of IA patient urine samples. In this study, we establish that POC diagnosis of IA based on urinary GM detection is feasible. Prospective studies will be necessary to establish the performance characteristics of an optimized device and define its optimal clinical use.

Certain fungi thrive in highly radioactive environments including the defunct Chernobyl nuclear reactor. Cryptococcus neoformans (C. neoformans), which uses L-3,4-dihydroxyphenylalanine (L-DOPA) to produce melanin, was used here to investigate how gamma radiation under aqueous aerobic conditions affects the properties of melanin, with the aim of gaining insight into its radioprotective role. Exposure of melanized fungal cell in aqueous suspensions to doses of γ-radiation capable of killing 50 to 80% of the cells did not lead to a detectable loss of melanin integrity according to EPR spectra of melanin radicals. Moreover, upon UV-visible (Xe-lamp) illumination of melanized cells, the increase in radical population was unchanged after γ-irradiation. Gamma-irradiation of frozen cell suspensions and storage of samples for several days at 77 K however, produced melanin modification noted by a reduced radical population and reduced photoresponse. More direct evidence for structural modification of melanin came from the detection of soluble products with absorbance maxima near 260 nm in supernatants collected after γ-irradiation of cells and cell-free melanin. These products, which include thiobarbituric acid (TBA)-reactive aldehydes, were also generated by Fenton reagent treatment of cells and cell-free melanin. In an assay of melanin integrity based on the metal (Bi(+3)) binding capacity of cells, no detectable loss in binding was detected after γ-irradiation. Our results show that melanin in C. neoformans cells is susceptible to some damage by hydroxyl radical formed in lethal radioactive aqueous environments and serves a protective role in melanized fungi that involves sacrificial breakdown.

There is an obvious and urgent need for novel approaches to treat infectious diseases. The use of monoclonal antibodies in therapy of infectious diseases is now experiencing renewed interest. During the last 5 years radioimmunotherapy (RIT), a modality previously developed only for cancer treatment, has been successfully adapted for the treatment of experimental fungal, bacterial, and viral infections. As our model organism for studying the efficacy, mechanisms, potential toxicity, and radioresistance to RIT, as well as for comparison of RIT with the existing antimicrobial therapies we have chosen the encapsulated yeast Cryptococcus neoformans (CN). The success of RIT approach in laboratory studies provides encouragement for feasibility of therapeutically targeting microbes with labeled antibodies. In addition, the creation of “panantibodies” for RIT which would recognize antigens shared by the whole class of pathogens such as fungi, for example, would facilitate the introduction of RIT into the clinic.

Melanin, a high-molecular weight pigment that is ubiquitous in nature, protects melanized microorganisms against high doses of ionizing radiation. However, the physics of melanin interaction with ionizing radiation is unknown.We rationally designed melanins from either 5-S-cysteinyl-DOPA, L-cysteine/L-DOPA, or L-DOPA with diverse structures as shown by elemental analysis and HPLC. Sulfur-containing melanins had higher predicted attenuation coefficients than non-sulfur-containing melanins. All synthetic melanins displayed strong electron paramagnetic resonance (2.14.10(18), 7.09.10(18), and 9.05.10(17) spins/g, respectively), with sulfur-containing melanins demonstrating more complex spectra and higher numbers of stable free radicals. There was no change in the quality or quantity of the stable free radicals after high-dose (30,000 cGy), high-energy ((137)Cs, 661.6 keV) irradiation, indicating a high degree of radical stability as well as a robust resistance to the ionizing effects of gamma irradiation. The rationally designed melanins protected mammalian cells against ionizing radiation of different energies.We propose that due to melanin's numerous aromatic oligomers containing multiple pi-electron system, a generated Compton recoil electron gradually loses energy while passing through the pigment, until its energy is sufficiently low that it can be trapped by stable free radicals present in the pigment. Controlled dissipation of high-energy recoil electrons by melanin prevents secondary ionizations and the generation of damaging free radical species.

Nearly 20% of human cancers worldwide have an infectious etiology with the most prominent examples being hepatitis B and C virus-associated hepatocellular carcinoma and human papilloma virus-associated cervical cancer. There is an urgent need to find new approaches to treatment and prevention of virus-associated cancers.Viral antigens have not been previously considered as targets for treatment or prevention of virus-associated cancers. We hypothesized that it was possible to treat experimental HPV16-associated cervical cancer (CC) and Hepatitis B-associated hepatocellular carcinoma (HCC) by targeting viral antigens expressed on cancer cells with radiolabeled antibodies to viral antigens. Treatment of experimental CC and HCC tumors with (188)Re-labeled mAbs to E6 and HBx viral proteins, respectively, resulted in significant and dose-dependent retardation of tumor growth in comparison with untreated mice or mice treated with unlabeled antibodies.This strategy is fundamentally different from the prior uses of radioimmunotherapy in oncology, which targeted tumor-associated human antigens and promises increased specificity and minimal toxicity of treatment. It also raises an exciting possibility to prevent virus-associated cancers in chronically infected patients by eliminating cells infected with oncogenic viruses before they transform into cancer.

BackgroundMelanin pigments are ubiquitous in nature. Melanized microorganisms are often the dominating species in certain extreme environments, such as soils contaminated with radionuclides, suggesting that the presence of melanin is beneficial in their life cycle. We hypothesized that ionizing radiation could change the electronic properties of melanin and might enhance the growth of melanized microorganisms.Methodology/principal findingsIonizing irradiation changed the electron spin resonance (ESR) signal of melanin, consistent with changes in electronic structure. Irradiated melanin manifested a 4-fold increase in its capacity to reduce NADH relative to non-irradiated melanin. HPLC analysis of melanin from fungi grown on different substrates revealed chemical complexity, dependence of melanin composition on the growth substrate and possible influence of melanin composition on its interaction with ionizing radiation. XTT/MTT assays showed increased metabolic activity of melanized C. neoformans cells relative to non-melanized cells, and exposure to ionizing radiation enhanced the electron-transfer properties of melanin in melanized cells. Melanized Wangiella dermatitidis and Cryptococcus neoformans cells exposed to ionizing radiation approximately 500 times higher than background grew significantly faster as indicated by higher CFUs, more dry weight biomass and 3-fold greater incorporation of (14)C-acetate than non-irradiated melanized cells or irradiated albino mutants. In addition, radiation enhanced the growth of melanized Cladosporium sphaerospermum cells under limited nutrients conditions.Conclusions/significanceExposure of melanin to ionizing radiation, and possibly other forms of electromagnetic radiation, changes its electronic properties. Melanized fungal cells manifested increased growth relative to non-melanized cells after exposure to ionizing radiation, raising intriguing questions about a potential role for melanin in energy capture and utilization.

The HIV epidemic is a major threat to health in the developing and western worlds. A modality that targets and kills HIV-1-infected cells could have a major impact on the treatment of acute exposure and the elimination of persistent reservoirs of infected cells. The aim of this proof-of-principle study was to demonstrate the efficacy of a therapeutic strategy of targeting and eliminating HIV-1-infected cells with radiolabeled antibodies specific to viral proteins in vitro and in vivo.Antibodies to HIV-1 envelope glycoproteins gp120 and gp41 labeled with radioisotopes bismuth 213 ((213)Bi) and rhenium 188 ((188)Re) selectively killed chronically HIV-1-infected human T cells and acutely HIV-1-infected human peripheral blood mononuclear cells (hPBMCs) in vitro. Treatment of severe combined immunodeficiency (SCID) mice harboring HIV-1-infected hPBMCs in their spleens with a (213)Bi- or (188)Re-labeled monoclonal antibody (mAb) to gp41 resulted in a 57% injected dose per gram uptake of radiolabeled mAb in the infected spleens and in a greater than 99% elimination of HIV-1-infected cells in a dose-dependent manner. The number of HIV-1-infected thymocytes decreased 2.5-fold in the human thymic implant grafts of SCID mice treated with the (188)Re-labeled antibody to gp41 compared with those treated with the (188)Re-control mAb. The treatment did not cause acute hematologic toxicity in the treated mice.The current study demonstrates the effectiveness of HIV-targeted radioimmunotherapy and may provide a novel treatment option in combination with highly active antiretroviral therapy for the eradication of HIV.