In vivo Evaluation of Free and Chelated Accelerator-produced Actinium- 225 - Radiation Dosimetry and Toxicity Results.

BACKGROUND AND OBJECTIVE: The demand for the alpha-emitting radionuclide Actinium-225 (225Ac) for use in radionuclide therapy is growing. Producing 225Ac using high energy linear accelerators, cyclotrons or photoinduction could increase its supply. One potential problem with accelerator produced 225Ac using Thorium-232 targets is the presence in final product of 0.1-0.3% by activity of the long-lived 227Ac impurity at the end of irradiation. It is important to comprehensively evaluate the behavior of accelerator-produced 225Ac in vivo before using it in pre-clinical and clinical applications. METHODS: Biodistribution of accelerator-produced 225Ac in acetate (free) and DOTA complex forms was performed in male and female CD-1 mice. The biodistribution data was used for radiation dosimetry calculations. The toxicity studies of free 225Ac were conducted in CD-1 mice at 1.036 and 2.035 kBq/g body weight. Blood counts, body weight and post-mortem histology were evaluated. RESULTS: In both genders, there was a pronounced uptake of free 225Ac in the liver when compared to 225Ac-DOTA which resulted in 200 and 50 times higher liver radiation dose for free 225Ac in male and female mice, respectively. 227Ac contribution to radiation dose delivered by 225Ac was calculated to be negligible. Mice given free 225Ac did not lose weight, had only transient effect on their blood counts and showed no histological damage to the liver and bone marrow. CONCLUSION: Our biodistribution/dosimetry/toxicity study of accelerator-produced 225Ac demonstrated the patterns very similar to 229Th-derived 225Ac. We conclude that accelerator-produced 225Ac is suitable for the developmental work of targeted radionuclide therapy.

A Radiolabeled Fully Human Antibody to Human Aspartyl (Asparaginyl) ß-Hydroxylase Is a Promising Agent for Imaging and Therapy of Metastatic Breast Cancer.

There is a need for novel effective and safe therapies for metastatic breast cancer based on targeting tumor-specific molecular markers of cancer. Human aspartyl (asparaginyl) ß-hydroxylase (HAAH) is a highly conserved enzyme that hydroxylates epidermal growth factor-like domains in transformation-associated proteins and is overexpressed in a variety of cancers, including breast cancer. A fully human monoclonal antibody (mAb) PAN-622 has been developed to HAAH. In this study, they describe the development of PAN-622 mAb as an agent for imaging and radioimmunotherapy of metastatic breast cancer. PAN-622 was conjugated to several ligands such as DOTA, CHXA″, and DTPA to enable subsequent radiolabeling and its immunoreactivity was evaluated by an HAAH-specific enzyme-linked immunosorbent assay and binding to the HAAH-positive cells. As a result, DTPA-PAN-622 was chosen to investigate biodistribution in healthy CD-1 female mice and 4T1 mammary tumor-bearing BALB/c mice. The 111In-DTPA-pan622 mAb concentrated in the primary tumors and to some degree in lung metastases as shown by SPECT/CT and Cherenkov imaging. A pilot therapy study with 213Bi-DTPA-PAN-622 demonstrated a significant effect on the primary tumor. The authors concluded that human mAb PAN-622 to HAAH is a promising reagent for development of imaging and possible therapeutic agents for the treatment of metastatic breast cancer.

Targeted therapy of osteosarcoma with radiolabeled monoclonal antibody to an insulin-like growth factor-2 receptor (IGF2R).

INTRODUCTION: Osteosarcoma overall survival has plateaued around 70%, without meaningful improvements in over 30years. Outcomes for patients with overt metastatic disease at presentation or who relapse are dismal. In this study we investigated a novel osteosarcoma therapy utilizing radioimmunotherapy (RIT) targeted to IGF2R, which is widely expressed in OS. METHODS: Binding efficiency of the Rhenium-188(188Re)-labeled IGF2R-specific monoclonal antibody (mAb) to IGF2R on OS17 OS cells was assessed with Scatchard plot analysis. Biodistribution studies were performed in heterotopic murine osteosarcoma xenografts. Tumor growth was compared over a 24-day period post-treatment between mice randomized to receive 188Re-labeled IGF2R-specific murine mAb MEM-238 (188Re-MEM-238) or one of three controls: 188Re-labeled isotype control mAb, unlabeled MEM-238, or no treatment. RESULTS: Results demonstrate that the radioimmunoconjugate had a high binding constant to IGF2R. Both 188Re-MEM-238 and the isotype control had similar initial distribution in normal tissue. After 48h 188Re-MEM-238 exhibited a 1.8 fold selective uptake within tumor compared to the isotype control (p=0.057). Over 24days, the tumor growth ratio was suppressed in animals treated with RIT compared to unlabeled and untreated controls (p=0.005) as demonstrated by a 38% reduction of IGF2R expressing osteosarcoma cells in the RIT group (p=0.002). CONCLUSIONS: In conclusion, given the lack of new effective therapies in osteosarcoma, additional investigation into this target is warranted. ADVANCES IN KNOWLEDGE: High expression of IGF2R on osteosarcoma tumors, paired with the specificity and in vivo anti-cancer activity of 188Re-labeled IGF2R-specific mAb suggests that IGF2R may represent a novel therapeutic target in the treatment of osteosarcoma. IMPLICATIONS FOR PATIENT CARE: This targeted approach offers the benefits of being independent of a specific pathway, a resistance mechanism, and/or an inherent biologic tumor trait and therefore is relevant to all OS tumors that express IGF2R.

Beta emitters rhenium-188 and lutetium-177 are equally effective in radioimmunotherapy of HPV-positive experimental cervical cancer.

Cervical cancer caused by the infection with the human papillomavirus (HPV) remains the fourth leading killer of women worldwide. Therefore, more efficacious treatments are needed. We are developing radioimmunotherapy (RIT) of HPV-positive cervical cancers by targeting E6 and E7 viral oncoproteins expressed by the cancer cells with the radiolabeled monoclonal antibodies (mAbs). To investigate the influence of different radionuclides on the RIT efficacy-we performed RIT of experimental cervical cancer with Rhenium-188 ((188) Re) and Lutetium-177 ((177) Lu)-labeled mAb C1P5 to E6. The biodistribution of (188) Re- and (177) Lu-labeled C1P5 was performed in nude female mice bearing CasKi cervical cancer xenografts and the radiation dosimetry calculations for the tumors and organs were carried out. For RIT the mice were treated with 7.4 MBq of either (188) Re-C1P5 or (177) Lu-C1P5 or left untreated, and observed for their tumor size for 28 days. The levels of (188) Re- and (177) Lu-C1P5 mAbs-induced double-strand breaks in CasKi tumors were compared on days 5 and 10 post treatment by staining with anti-gamma H2AX antibody. The radiation doses to the heart and lungs were similar for both (177) Lu-C1P5 and (188) Re-C1P5. The dose to the liver was five times higher for (177) Lu-C1P5. The doses to the tumor were 259 and 181 cGy for (177) Lu-C1P5 and (188) Re-C1P5, respectively. RIT with either (177) Lu-C1P5 or (188) Re-C1P5 was equally effective in inhibiting tumor growth when each was compared to the untreated controls (P = 0.001). On day 5 there was a pronounced staining for gamma H2AX foci in (177) Lu-C1P5 group only and on day 10 it was observed in both (177) Lu-C1P5 and (188) Re-C1P5 groups. (188) Re- and (177) Lu-labeled mAbs were equally effective in arresting the growth of CasKi cervical tumors. Thus, both of these radionuclides are candidates for the clinical trials of this approach in patients with advanced, recurrent or metastatic cervical cancer.

Effect of naive and radiolabeled rhTRAIL on the cervical cancer xenografts in mice.

BACKGROUND: There is a need for novel treatments of advanced cervical cancer. We investigated the utility of recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL), a molecule capable of inducing apoptosis in cancer cells, for the therapy of CasKi cervical cancer xenografts in nude mice. RESULTS: CasKi cells proved to be sensitive in vitro to rhTRAIL with an IC50 of 120 ng/ml. (125)I-tagged rhTRAIL specifically accumulated in CasKi tumors in mice with the highest uptake of 9.4% ID/g at 2 h post-injection. Both naive and 200 µCi (188)Re-tagged rhTRAIL administered in the amount of 0.35 mg/kg body weight significantly retarded CasKi tumor growth to the same extent in mice without the side effects of cisplatin chemotherapeutic control. CONCLUSION: rhTRAIL is a promising novel agent for treatment of advanced cervical cancer.

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.

Melanoma stem cells in experimental melanoma are killed by radioimmunotherapy.

INTRODUCTION: In spite of recently approved B-RAF inhibitors and immunomodulating antibodies, metastatic melanoma has poor prognosis and novel treatments are needed. Melanoma stem cells (MSC) have been implicated in the resistance of this tumor to chemotherapy. Recently we demonstrated in a Phase I clinical trial in patients with metastatic melanoma that radioimmunotherapy (RIT) with 188-Rhenium((188)Re)-6D2 antibody to melanin was a safe and effective modality. Here we investigated the interaction of MSC with RIT as a possible mechanism for RIT efficacy. METHODS: Mice bearing A2058 melanoma xenografts were treated with either 1.5 mCi (188)Re-6D2 antibody, saline, unlabeled 6D2 antibody or (188)Re-labeled non-specific IgM. RESULTS: On Day 28 post-treatment the tumor size in the RIT group was 4-times less than in controls (P<0.001). The tumors were analyzed by immunohistochemistry and FACS for two MSC markers--chemoresistance mediator ABCB5 and H3K4 demethylase JARID1B. There were no significant differences between RIT and control groups in percentage of ABCB5 or JARID1B-positive cells in the tumor population. Our results demonstrate that unlike chemotherapy, which kills tumor cells but leaves behind MSC leading to recurrence, RIT kills MSC at the same rate as the rest of tumor cells. CONCLUSIONS: These results have two main implications for melanoma treatment and possibly other cancers. First, the susceptibility of ABCB5+ and JARID1B+cells to RIT in melanoma might be indicative of their susceptibility to antibody-targeted radiation in other cancers where they are present as well. Second, specifically targeting cancer stem cells with radiolabeled antibodies to ABCB5 or JARID1B might help to completely eradicate cancer stem cells in various cancers.

Compton scattering by internal shields based on melanin-containing mushrooms provides protection of gastrointestinal tract from ionizing radiation.

There is a need for radioprotectors that protect normal tissues from ionizing radiation in patients receiving high doses of radiation and during nuclear emergencies. We investigated the possibility of creating an efficient oral radioprotector based on the natural pigment melanin that would act as an internal shield and protect the tissues via Compton scattering followed by free radical scavenging. CD-1 mice were fed melanin-containing black edible mushrooms Auricularia auricila-judae before 9 Gy total body irradiation. The location of the mushrooms in the body before irradiation was determined by in vivo fluorescent imaging. Black mushrooms protected 80% of mice from the lethal dose, while control mice or those given melanin-devoid mushrooms died from gastrointestinal syndrome. The crypts of mice given black mushrooms showed less apoptosis and more cell division than those in control mice, and their white blood cell and platelet counts were restored at 45 days to preradiation levels. The role of melanin in radioprotection was proven by the fact that mice given white mushrooms supplemented with melanin survived at the same rate as mice given black mushrooms. The ability of melanin-containing mushrooms to provide remarkable protection against radiation suggests that they could be developed into oral radioprotectors.

Pre-clinical evaluation of a 213Bi-labeled 2556 antibody to HIV-1 gp41 glycoprotein in HIV-1 mouse models as a reagent for HIV eradication.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

In vitro and in vivo evaluation of melanin-binding decapeptide 4B4 radiolabeled with 177Lu, 166Ho, and 153Sm radiolanthanides for the purpose of targeted radionuclide therapy of melanoma.

Melanoma is a malignancy with increasing incidence. Although primary tumors that are localized to the skin can be successfully treated by surgical removal, there is no satisfactory treatment for metastatic melanoma, a condition that has currently an estimated 5-year survival of just 6%. During the last decade, ß- or α-emitter-radiolabeled peptides that bind to different receptors on a variety of tumors have been investigated as potential therapeutic agents in both the preclinical and clinical settings with encouraging results. A recent study demonstrated that 188-Rhenium ((188)Re)-labeled, via HYNIC ligand, fungal melanin-binding decapeptide 4B4 was effective against experimental MNT1 human melanoma and was safe to normal melanized tissues. The availability of radiolanthanides with diverse nuclear emission schemes and half-lives provides an opportunity to expand the repertoire of peptides for radionuclide therapy of melanoma. The melanin-binding decapeptide 4B4 was radiolabeled with (177)Lu, (166)Ho, and (153)Sm via a DO3A chelate. The stability studies of Ln*-DO3A-4B4 in phosphate-buffered saline, serum, and a hydroxyapatite assay demonstrated that (177)Lu-labeled peptide was more stable than (166)Ho- and (153)Sm-labeled peptides, most likely because of the smallest ionic radius of the former allowing for better complexation with DO3A. Binding of Ln*-DO3A-4B4 to the lysed highly melanized MNT1 melanoma cells demonstrated the specificity of peptides binding to melanin. In vivo biodistribution data for (177)Lu-DO3A-4B4 given by intraperitoneal administration to lightly pigmented human metastatic A2058 melanoma-bearing mice demonstrated very high uptake in the kidneys and low tumor uptake. Intravenous administration did not improve the tumor uptake. The plausible explanation of low tumor uptake of (177)Lu-DO3A-4B4 could be its decreased ability to bind to melanin during in vitro binding studies in comparison with (188)Re-HYNIC-4B4, exacerbated by the very fast clearance from the blood and the kidneys "sink" effect.

Melanin-covered nanoparticles for protection of bone marrow during radiation therapy of cancer.

PURPOSE: Protection of bone marrow against radiotoxicity during radioimmunotherapy and in some cases external beam radiation therapy such as hemi-body irradiation would permit administration of significantly higher doses to tumors, resulting in increased efficacy and safety of treatment. Melanin, a naturally occurring pigment, possesses radioprotective properties. We hypothesized that melanin, which is insoluble, could be delivered to the bone marrow by intravenously administrated melanin-covered nanoparticles (MNs) because of the human body's "self-sieving" ability, protecting it against ionizing radiation. METHODS AND MATERIALS: The synthesis of MNs was performed via enzymatic polymerization of 3,4-dihydroxyphenylalanine and/or 5-S-cysteinyl-3,4-dihydroxyphenylalanine on the surface of 20-nm plain silica nanoparticles. The biodistribution of radiolabeled MNs in mice was done at 3 and 24 h. Healthy CD-1 mice (Charles River Laboratories International, Inc., Wilmington, MA) or melanoma tumor-bearing nude mice were given MNs intravenously, 50 mg/kg of body weight, 3 h before either whole-body exposure to 125 cGy or treatment with 1 mCi of (188)Re-labeled 6D2 melanin-binding antibody. RESULTS: Polymerization of melanin precursors on the surface of silica nanoparticles resulted in formation of a 15-nm-thick melanin layer as confirmed by light scattering, transmission electron microscopy, and immunofluorescence. The biodistribution after intravenous administration showed than MN uptake in bone marrow was 0.3% and 0.2% of injected dose per gram at 3 and 24 h, respectively, whereas pre-injection with pluronic acid increased the uptake to 6% and 3% of injected dose per gram, respectively. Systemic MN administration reduced hematologic toxicity in mice treated with external radiation or radioimmunotherapy, whereas no tumor protection by MNs was observed. CONCLUSIONS: MNs or similar structures provide a novel approach to protection of bone marrow from ionizing radiation based on prevention of free radical formation by melanin.

Radioimmunotherapy of experimental human metastatic melanoma with melanin-binding antibodies and in combination with dacarbazine.

PURPOSE: Melanin has emerged as an attractive target for radioimmunotherapy (RIT) of melanoma, and a radiolabeled monoclonal antibody (mAb) 6D2 to melanin is currently in clinical evaluation. We investigated two approaches to improve the targeting of radiation to tumors using melanin-binding mAbs: (a) the use of an additional mAb to melanin could provide information on whether using antibodies to melanin can serve as a general approach to development of therapeutics for melanoma, and (b) as melanin targeting involves the antibody binding to extracellular melanin released from necrotic melanoma cells, we hypothesized that the administration of a chemotherapeutic agent followed by RIT would facilitate the delivery of radiation to the tumors due to the increased presence of free melanin. EXPERIMENTAL DESIGN: We evaluated the therapeutic efficacy of two melanin-binding IgM mAbs labeled with (188)Re (6D2 and 11B11). We compared the efficacy of RIT with (188)Re-6D2 to chemotherapy with dacarbazine (DTIC) and to combined chemotherapy and RIT in human metastatic melanoma-bearing nude mice. RESULTS: Therapeutic efficacy of (188)Re-labeled 6D2 and 11B11 was comparable despite differences in their affinity and binding site numbers. Comparison of chemotherapy with DTIC and RIT revealed that RIT was more effective in slowing tumor growth in mice. Administration of DTIC followed by RIT was more effective than either modality alone. CONCLUSIONS: These results provide encouragement for the development of RIT for melanoma with melanin-binding mAbs and suggest that combining chemotherapy and RIT may be a promising approach for the treatment of metastatic melanoma.

In vitro evaluation, biodistribution and scintigraphic imaging in mice of radiolabeled anthrax toxins.

INTRODUCTION: There is a lot of interest towards creating therapies and vaccines for Bacillus anthracis, a bacterium which causes anthrax in humans and which spores can be made into potent biological weapons. Systemic injection of lethal factor (LF), edema factor (EF) and protective antigen (PA) in mice produces toxicity, and this protocol is commonly used to investigate the efficacy of specific antibodies in passive protection and vaccine studies. Availability of toxins labeled with imageable radioisotopes would allow to demonstrate their tissue distribution after intravenous injection at toxin concentration that are below pharmacologically significant to avoid masking by toxic effects. METHODS: LF, EF and PA were radiolabeled with (188)Re and (99m)Tc, and their performance in vitro was evaluated by macrophages and Chinese hamster ovary cells toxicity assays and by binding to macrophages. Scintigraphic imaging and biodistribution of intravenously (IV) injected (99m)Tc-and (123)I-labeled toxins was performed in BALB/c mice. RESULTS: Radiolabeled toxins preserved their biological activity. Scatchard-type analysis of the binding of radiolabeled PA to the J774.16 macrophage-like cells revealed 6.6 x 10(4) binding sites per cell with a dissociation constant of 6.7 nM. Comparative scintigraphic imaging of mice injected intravenously with either (99m)Tc-or (123)I-labeled PA, EF and LF toxins demonstrated similar biodistribution patterns with early localization of radioactivity in the liver, spleen, intestines and excretion through kidneys. The finding of renal excretion shortly after IV injection strongly suggests that toxins are rapidly degraded which could contribute to the variability of mouse toxigenic assays. Biodistribution studies confirmed that all three toxins concentrated in the liver and the presence of high levels of radioactivity again implied rapid degradation in vivo. CONCLUSIONS: The availability of (188)Re and (99m)Tc-labeled PA, LF and EF toxins allowed us to confirm the number of PA binding sites per cell, to provide an estimate of the dissociation constant of PA for its receptor and to demonstrate tissue distribution of toxins in mice after intravenous injection.

Treating cancer as an infectious disease--viral antigens as novel targets for treatment and potential prevention of tumors of viral etiology.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

Phage display library derived peptides that bind to human tumor melanin as potential vehicles for targeted radionuclide therapy of metastatic melanoma.

Metastatic melanoma remains an incurable disease, and there is a great need for novel therapeutic modalities. We have recently identified melanin as a target for radionuclide therapy of melanoma and demonstrated the feasibility of this approach using a 188-rhenium ( (188)Re)-radiolabeled melanin-binding decapeptide to fungal melanin known as 4B4. Although the results indicated that radiolabeled melanin-binding decapeptide had activity against melanoma, that peptide also manifested high kidney uptake and this might become a concern during clinical trials. We hypothesized that by identifying peptides with different amino acid composition against tumor melanin we might be able to decrease their kidney uptake. Using the Heptapeptide Ph.D.-7 Phage Display Library, we identified three heptapeptides that bind to human tumor melanin. These peptides were radiolabeled with (188)Re via HYNIC ligand, and their comprehensive biodistribution in A2058 human metastatic melanoma tumor-bearing nude mice was compared to that of (188)Re-4B4 decapeptide. While tumor uptake of heptapeptides was quite similar to that of (188)Re-4B4 decapeptide, there was dramatically less uptake in the kidneys at both 3 h (6% ID/g vs 38%) and 24 h (2% ID/g vs 15%) postinjection. Administration of one of the generated heptapeptides, (188)Re-HYNIC-AsnProAsnTrpGlyProArg, to A2058 human metastatic melanoma-bearing nude mice resulted in significant retardation of the tumor growth. Immunofluorescence showed that in spite of their relatively small size heptapeptides were not able to penetrate through the membranes of viable melanoma cells and bound only to extracellular melanin, which provides assurance that they will be safe to healthy melanin-containing tissues during radionuclide therapy. Thus, these heptapeptides appear to have potentially significant advantages for targeted therapy of melanoma relative to existing melanin-binding peptides.

Computational model predicts effective delivery of 188-Re-labeled melanin-binding antibody to metastatic melanoma tumors with wide range of melanin concentrations.

Metastatic melanoma is almost always deadly and new methods of treatment are urgently needed. Recently, we established the feasibility of radioimmunotherapy (RIT) for experimental melanoma in mice using a 188-rhenium (188Re)-labeled monoclonal antibody (mAb) 6D2 (IgM) to melanin. Our objective was to determine the effects of varying tumor melanin concentration and of different diffusivities and lymphatic clearance rates of the normal tissue, on the absorbed dose to the tumor in simulated therapy, in preparation for a clinical trial of RIT for melanoma. Using finite element analysis (FEA), we created a pharmacokinetic model that describes melanin-targeting RIT of a melanoma micrometastasis (1.3-mm radius) imbedded in normal tissue (14.3-mm radius). Our method incorporates antibody plasma kinetics, transcapillary transport, interstitial diffusion, and lymphatic clearance. Michaelis-Menten kinetics was used to model mAb binding to tumor melanin for melanin concentrations of 76, 7.6, 0.76, 0.076, and 0.0076 micromol/l. An absorbed dose was calculated, after accounting for direct and crossfire irradiation, on the basis of a 7.4-GBq intravenous dose of 188Re-6D2. The results showed that penetration of mAb into the tumor was inversely proportional to tumor melanin concentration. Decreased diffusivity and increased lymphatic clearance of the surrounding normal tissue decreased the dose to the tumor. The formation of mAb-melanin complex was remarkably similar within a 1000-fold range of melanin concentration, resulting in total doses of 2840, 2820, 2710, and 1990 cGy being delivered to tumors with melanin concentrations of 76, 7.6, 0.76, and 0.076 micromol/l, respectively. In conclusion, RIT of metastatic melanoma can be effective over a wide range of tumor melanin concentrations. The results can be useful in the design of a clinical trial of melanin-targeting RIT in patients with metastatic melanoma.

A monoclonal antibody to Bacillus anthracis protective antigen defines a neutralizing epitope in domain 1.

Antibody (Ab) responses to Bacillus anthracis toxins are protective, but relatively few protective monoclonal antibodies (MAbs) have been reported. Protective antigen (PA) is essential for the action of B. anthracis lethal toxin (LeTx) and edema toxin. In this study, we generated two MAbs to PA, MAbs 7.5G and 10F4. These MAbs did not compete for binding to PA, consistent with specificities for different epitopes. The MAbs were tested for their ability to protect a monolayer of cultured macrophages against toxin-mediated cytotoxicity. MAb 7.5G, the most-neutralizing MAb, bound to domain 1 of PA and reduced LeTx toxicity in BALB/c mice. Remarkably, MAb 7.5G provided protection without blocking the binding of PA or lethal factor or the formation of the PA heptamer complex. However, MAb 7.5G slowed the proteolytic digestion of PA by furin in vitro, suggesting a potential mechanism for Ab-mediated protection. These observations indicate that some Abs to domain 1 can contribute to host protection.

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.

Interaction of radiolabeled antibodies with fungal cells and components of the immune system in vitro and during radioimmunotherapy for experimental fungal infection.

BACKGROUND: The usefulness of radioimmunotherapy (RIT) for infectious diseases was recently demonstrated for several fungal and bacterial infections, but the mechanisms by which RIT is effective against microbes are uncertain. METHODS: We investigated the interaction between polysaccharide capsule-binding 18B7 monoclonal antibodies (MAbs) labeled with alpha-emitter 213Bi and Cryptococcus neoformans cells as well as between 213Bi-18B7 and components of immune system, both in vitro and in vivo. RESULTS: For 213Bi-18B7, the microbicidal effect was predominantly due to "direct-hit" killing, with some contribution from the "crossfire" effect. The efficacy of cell killing correlated with the binding capacity of the MAb to the capsule and was dependent on the MAb isotype. RIT also promoted the apoptosis-like death of fungal cells. Cooperation was observed in vitro between the antifungal activity of macrophages and RIT, suggesting the potential for synergistic action in vivo. RIT was associated with changes in concentration of the cytokines interleukin (IL)-2, IL-4, IL-10, tumor necrosis factor-alpha, and interferon-gamma, suggesting that the therapeutic effects of RIT may result from changes in the inflammatory response. CONCLUSIONS: The present results suggest that the antimicrobial efficacy of RIT involves killing through promotion of fungal cell apoptosis-like death, reduction in yeast capsule size, cooperation with macrophages, and modulation of the inflammatory response.