Targeted actinium-225 in vivo generators for therapy of ovarian cancer

Advanced ovarian cancer is largely incurable, but initially it is frequently confined to the i.p. space. We explored i.p. radioimmunotherapy in a mouse model of human ovarian cancer. Use of a targeted actinium-225 ((225)Ac) in vivo generator of alpha particles exploits the extreme, selective cytotoxicity of alpha particles, while providing a feasible half-life to enable delivery to tumor. (225)Ac chelated with 2-(p-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10 tetraacetic acid was conjugated to trastuzumab, an anti-HER-2/neu antibody. The radioimmunoconjugate was tested for immunoreactivity, internalization, and cytotoxicity using a human ovarian carcinoma cell line, SKOV3. (225)Ac-labeled trastuzumab retained immunoreactivity (50-90%), rapidly internalized into cells (50% at 2 h), and had an ED(50) of 1.3 nCi/ml after 4 days of incubation in vitro. i.p. administered (225)Ac- or (111)In-labeled trastuzumab behaved similarly with high tumor uptake [56-60% injected dose per gram (% ID/g) at 4 h, which increased to 65-70% ID/g at 24 h]. Tumor uptake was 3-5-fold higher than liver and spleen, the normal organs with the highest uptake. i.v. administration of (111)In-labeled trastuzumab produced slightly higher normal organ uptake compared with i.p.-administered (111)In-labeled trastuzumab. However, tumor uptake was low, 5%-26% ID/g. Therapy was examined with native trastuzumab and 220, 330, and 450 nCi of (225)Ac-labeled trastuzumab or (225)Ac-labeled control antibody at different dosing schedules. Therapy was initiated 9 days after tumor seeding. Groups of control mice and those administered native trastuzumab had median survivals of 33 and 37 or 44 days, respectively. Median survival was 52-126 days with (225)Ac-labeled trastuzumab at various doses and schedules, and 48-64 days for (225)Ac-labeled control the same schedules. Deaths from toxicity occurred with the highest activity levels. In conclusion, i.p. administration with a (225)Ac-labeled internalizing anti-HER-2/neu antibody can extend survival significantly in a nude mouse model of human ovarian cancer at levels that produce no apparent gross toxicity.     

Preclinical evaluation of the alpha-particle generator nuclide 225Ac for somatostatin receptor radiotherapy of neuroendocrine tumors.

PURPOSE: Peptide receptor radionuclide therapy (PRRT) using somatostatin analogues labeled with beta-particle-emitting isotopes such as 90Y or 177Lu has been a promising treatment strategy for metastasized neuroendocrine tumors. Although remission can be accomplished in a high percentage of neuroendocrine tumors, some tumors do not respond to this treatment. alpha-Emitting isotopes-such as the 10-day half-life alpha-emitting generator nuclide Actinum-225 (225Ac)-are characterized by extremely high cytotoxic activity on the cellular level, and may be superior in the treatment of neuroendocrine tumors not responding to PRRT using beta-emitting isotopes. EXPERIMENTAL DESIGN: Radiolabeling of 225Ac 1,4,7,10-tetra-azacylododecane N,N',N',N'-J-tetraacetic acid-Tyr3-octreotide (DOTATOC) was done at pH 5 (60 minutes at 70 degrees C) without further purification. Biodistribution in nude mice bearing AR42J rat pancreas neuroendocrine tumor xenografts were measured for up to 24 hours. Toxicity was tested by weight changes, retention variables (blood urea nitrogen and creatine), and histopathology in mice 7 months after treatment with 10 to 130 kBq (n = 4-5). Therapeutic efficacy was assessed by tumor weighing in animals treated 4 days after xenotransplantation and compared with 177Lu-DOTATOC as a reference. RESULTS: Activities up to 20 kBq had no significant toxic effects in mice. In contrast, activities higher than 30 kBq induced tubular necrosis. Biodistribution studies revealed that 225Ac-DOTATOC effectively accumulated in neuroendocrine xenograft tumors. 225Ac-DOTATOC activities were shown to be nontoxic (12-20 kBq), reduced the growth of neuroendocrine tumors, and showed improved efficacy compared with 177Lu-DOTATOC. CONCLUSIONS: 225Ac might be suitable to improve PRRT in neuroendocrine tumors.     

Evaluation of 225Ac for vascular targeted radioimmunotherapy of lung tumors

Several alpha particle emitting radioisotopes have been studied for use in radioimmunotherapy. Ac-225 has the potential advantages of a relatively long half life of 10 days, and a yield of 4 alpha emissions in its decay chain with a total energy release of approximately 28 MeV. A new, 12 coordination site chelating ligand, HEHA, has been chemically modified for coupling to targeting proteins without loss of chelating ability. HEHA was coupled with MAb 201B which binds to thrombomodulin and accumulates efficiently in murine lung. Ac-225 was bound to the HEHA-MAb 201B conjugate and injected into BALB/c mice bearing lung tumor colonies of EMT-6 mammary carcinoma. Biodistribution data at 1 and 4 h postinjection indicated that, as expected, 225Ac was delivered to lung efficiently (> 300% ID/g). The 225Ac was slowly released from the lung with an initial t1/2 = 49 h, and the released 225Ac accumulated in the liver. Injection of free HEHA was only partially successful in scavenging free 225Ac. In addition to the slow release of 225Ac from the chelate, data indicated that decay daughters of 225Ac were also released from the lung. Immediately after organ harvest, the level of 213Bi, the third alpha-decay daughter, was found to be deficient in the lungs and to be in excess in the kidney, relative to equilibrium values. Injected doses of 225Ac MAb 201B of 1.0 microCi, delivering a minimum calculated absorbed dose of about 6 Gy to the lungs, was effective in killing lung tumors, but also proved acutely radiotoxic. Animals treated with 1.0 microCi or more of the 225Ac radioconjugate died of a wasting syndrome within days with a dose dependent relationship. We conclude that the potential for 225Ac as a radioimmunotherapeutic agent is compromised not only by the slow release of 225Ac from the HEHA chelator, but most importantly by the radiotoxicity associated with decay daughter radioisotopes released from the target organ.     

A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing

Chimeric antigen receptor (CAR)-expressing T cells are a promising therapeutic option for patients with cancer. We developed a new CAR directed against the disialoganglioside GD2, a surface molecule expressed in neuroblastoma and in other neuroectoderm-derived neoplasms. The anti-GD2 single-chain variable fragment (scFv) derived from a murine antibody of IgM class was linked, via a human CD8α hinge-transmembrane domain, to the signaling domains of the costimulatory molecules 4-1BB (CD137) and CD3-ζ. The receptor was expressed in T lymphocytes by retroviral transduction and anti-tumor activities were assessed by targeting GD2-positive neuroblastoma cells using in vitro cytotoxicity assays and a xenograft model. Transduced T cells expressed high levels of anti-GD2 CAR and exerted a robust and specific anti-tumor activity in 4- and 48-hour cultures with neuroblastoma cells. Cytotoxicity was associated with the release of pro-apoptotic molecules such as TRAIL and IFN-γ. These results were confirmed in a xenograft model, where anti-GD2 CAR T cells infiltrating tumors and persisting into blood circulation induced massive apoptosis of neuroblastoma cells and completely abrogated tumor growth. This anti-GD2 CAR represents a powerful new tool to redirect T cells against GD2. The preclinical results of this study warrant clinical testing of this approach in neuroblastoma and other GD2-positive malignancies.     

靶向纳米造影剂C225-USPIO标记裸鼠鼻咽癌移植瘤MR成像研究

  目的  制备针对EGFR的靶向纳米造影剂C225-USPIO,并检测其标记裸鼠鼻咽癌移植瘤MR成像（MRI）能力。  方法  将西妥昔单抗（C225）与USPIO结合成C225-USPIO;检测造影剂粒径;选择EGFR高表达的鼻咽癌SUNE1 5-8F细胞株,接种于12只裸鼠的大腿腹侧皮下,待瘤直径增长至5 mm时,随机分实验组和对照组各6只,将C225-USPIO或USPIO通过鼠尾静脉注射,于注射后0、8、24、72 h经磁共振成像仪行T2WI扫描;检测肿瘤组织内造影剂分布。  结果  C225-USPIO粒径约45~50 nm;MRI示实验组8 h T2值较0 h略降低（P>0.05）,24 h显著降低（P < 0.05),72 h无明显改变（P>0.05）。对照组8~72 h各时间点T2值与0 h相比无明显改变（P>0.05）;注射造影剂72 h后肿瘤组织未见明显铁颗粒。  结论  C225-USPIO颗粒可通过毛细血管,并能运用于体内磁共振分子成像,可以降低裸鼠移植瘤磁共振T2信号强度,具有一定的特异性和靶向性。      

Localization of GD2-specific monoclonal antibody 3F8 in human osteosarcoma

3F8 is a murine IgG3 monoclonal antibody specific for the antigen disialoganglioside GD2. Immunofluorescence staining showed strong binding of 3F8 to 15 of 17 human osteosarcomas, including primary and metastatic tumors. The targeting potentials of the native monoclonal antibody (3F8) and the F(ab')2 fragment (p-3F8) were tested in BALB/c athymic nude mice xenografted with human osteosarcomas. After radiolabeling with iodine using the chloramine T method, both 3F8 and p-3F8 retained immunoreactivities. The irrelevant IgG3 antibody TIB114 and its F(ab')2 fragment were used as negative controls. A Ewing's sarcoma xenograft, which was low in GD2 antigen, was also studied for comparison. Mice were sacrificed 1 day and 4 days after i.v. antibody injection. 3F8 and p-3F8 showed preferential accumulation in osteosarcoma over normal tissues with tumor:nontumor ratios of 2.7-58:1 and 1.4-82:1, respectively, on Day 1. These ratios improved to 10-163:1 and 6.0-75:1 on Day 4. The intact antibody 3F8 showed selective tumor uptake with a much higher percentage of injected dose per g than the fragment p-3F8 and exhibited a longer tissue half-life than p-3F8. These data indicate that anti-GD2 monoclonal antibodies may be useful for imaging and targeted therapy of human osteogenic sarcoma. The F(ab')2 fragment has the advantage of achieving favorable tumor:nontumor ratios sooner after antibody injection while the intact antibody shows better retention by tumor tissues.     

Ganglioside GD2 specific monoclonal antibody 3F8: a phase I study in patients with neuroblastoma and malignant melanoma

The murine IgG3 monoclonal antibody (MoAb) 3F8, specific for the ganglioside GD2, activates human complement, is active in antibody-dependent cell-mediated cytotoxicity (ADCC), and can target specifically to human neuroblastoma in patients with metastatic disease. In a phase I study, 3F8 was administered intravenously (IV) to 17 patients with metastatic GD2 positive neuroblastoma or malignant melanoma at doses of 5, 20, 50, and 100 mg/m2. Serum 3F8 levels achieved were proportional to the dose of 3F8 infused. However, serum antimouse antibody levels did not increase with the amount of 3F8 administered. Toxicities included pain, hypertension, urticaria, and complement depletion. All acute side effects were controllable with symptomatic therapy. No long-term side effects were detected in patients observed for more than 14 months. None of the 17 patients received any antitumor therapy postantibody treatment. Antitumor responses occurred in seven of 17 patients. These ranged from complete clinical remissions to mixed responses. The murine monoclonal antibody (MoAb) 3F8 has clinical utility for the diagnosis and therapy of neuroblastoma and melanoma.     

Changes in the ganglioside composition of human neuroblastoma cells under different growth conditions

The ganglioside composition of human neuroblastoma cells (LA-N-1 and LA-N-5) was studied in samples obtained from (1) original cells in tissue cultures, (2) tumors grown in nude mice inoculated with original cells and (3) cells in tissue cultures re-established from the mouse tumors. The amounts of "a" pathway gangliosides (GM2, GM1 and GD1a) and those of the "b" pathway (GD3, GD2, GD1b and GT1b) differed according to the culture conditions. The "b" pathway gangliosides were markedly increased in the tumors grown in nude mice. In contrast, the "a" pathway gangliosides were abundant in cultures of both original and re-established cells. We also measured the enzymatic activities of UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyl transferase (EC 2.4.1.92) and of CMP-N-acetylneuraminic acid: GM3 sialyl transferase (EC 2.4.99.8) in neuroblastoma cells cultured under these conditions. These enzymes are thought to be the key enzymes involved in the synthesis of the "a" and "b" pathway gangliosides. Though there was no significant difference in the activity of N-acetylgalactosaminyl transferase between original cells and tumors in nude mice, re-established cells showed a definitely higher activity (3.5 times higher than in the original cells). On the other hand, tumors grown in nude mice had a markedly higher activity of sialyl transferase than that of original cells or re-established cells. These findings suggest that the culture conditions and/or the type of cell growth play some role in the synthesis and expression of gangliosides in neuroblastoma cells.     

Complete tumor ablation with iodine 131-radiolabeled disialoganglioside GD2-specific monoclonal antibody against human neuroblastoma xenografted in nude mice

The antibody 3F8, an IgG3 murine monoclonal antibody (MoAb) against disialoganglioside GD2, could target iodine-131 (131I) to established subcutaneous human neuroblastoma (NB) xenografts in BALB/c nude mice. 131I-radiolabeled MoAb (0.125-1 mCi) was injected iv. Tumor radioactivity over time was calculated from scintigraphy, and radiation dose to individual tumors was calculated. Tumor shrinkage occurred only with 131I-labeled 3F8, but not with nonradioactive 3F8 or radiolabeled irrelevant antibody. While the tumor of the control mice enlarged by tenfold, the treated tumor showed over 95% shrinkage by 12 days. Both the rate of shrinkage and duration of tumor response were dose dependent. Calculated doses of more than 10,000 rad could be achieved. Only those tumors that received more than 4,200 rad were completely ablated without recurrence. Recurrent tumors were not antigen negative or radioresistant. These results confirmed the prediction based on imaging studies that human NB xenografts could be effectively eradicated with the use of 131I-labeled MoAb 3F8 with tolerable toxicities.     

9-O-acetyl GD3 protects tumor cells from apoptosis

The ganglioside GD3 (Neu5Ac alpha8Neu5Ac alpha3Gal beta4GlcCer) is an intracellular lipid messenger that induces apoptosis by targeting mitochondria in various cell types. GD3 can also promote apoptosis when externally added to cells. Previous studies showed that the proapoptotic effects of GD3 can be counteracted by 9-O-acetylation. To determine whether 9-O-acetyl GD3 (acGD3) has a general antiapoptotic potential, the apoptosis-sensitive Jurkat cell line and an apoptosis-sensitive variant of the cell line Molt-4 were preincubated with micromolar concentrations of acGD3 and then treated with inducers of apoptosis. A reduced apoptotic index and an increased cell viability were observed. On the other hand, when the Jurkat cells were treated with GD3 for extended periods of time, a population was selected that was resistant to apoptosis induction by N-acetyl sphingosine as well as by the anti-leukemic drug daunorubicin. Comparative analysis of gangliosides revealed the formation of acGD3 in the resistant Jurkat cells that was not found in the apoptosis-sensitive cells. Conversely, exposing the acGD3 positive and apoptosis-resistant cell line Molt-4 to the O-deacetylating activity of salicylate resulted in a complete disappearance of acGD3 and an enhanced sensitivity to N-acetyl sphingosine-mediated apoptosis. Formation of acGD3 might thus represent a new mechanism how tumor cells can escape apoptosis.     

High-linear energy transfer (LET) alpha versus low-LET beta emitters in radioimmunotherapy of solid tumors: therapeutic efficacy and dose-limiting toxicity of 213Bi- versus 90Y-labeled CO17-1A Fab' fragments in a human colonic cancer model.

Recent studies suggest that radioimmunotherapy (RIT) with high-linear energy transfer (LET) radiation may have therapeutic advantages over conventional low-LET (e.g., beta-) emissions. Furthermore, fragments may be more effective in controlling tumor growth than complete IgG. However, to the best of our knowledge, no investigators have attempted a direct comparison of the therapeutic efficacy and toxicity of a systemic targeted therapeutic strategy, using high-LET alpha versus low-LET beta emitters in vivo. The aim of this study was, therefore, to assess the toxicity and antitumor efficacy of RIT with the alpha emitter 213Bi/213Po, as compared to the beta emitter 90Y, linked to a monovalent Fab' fragment in a human colonic cancer xenograft model in nude mice. Biodistribution studies of 213Bi- or 88Y-labeled benzyl-diethylene-triamine-pentaacetate-conjugated Fab' fragments of the murine monoclonal antibody CO17-1A were performed in nude mice bearing s.c. human colon cancer xenografts. 213Bi was readily obtained from an "in-house" 225Ac/213Bi generator. It decays by beta- and 440-keV gamma emission, with a t(1/2) of 45.6 min, as compared to the ultra-short-lived alpha emitter, 213Po (t(1/2) = 4.2 micros). For therapy, the mice were injected either with 213Bi- or 90Y-labeled CO17-1A Fab', whereas control groups were left untreated or were given a radiolabeled irrelevant control antibody. The maximum tolerated dose (MTD) of each agent was determined. The mice were treated with or without inhibition of the renal accretion of antibody fragments by D-lysine (T. M. Behr et al., Cancer Res., 55: 3825-3834, 1995), bone marrow transplantation, or combinations thereof. Myelotoxicity and potential second-organ toxicities, as well as tumor growth, were monitored at weekly intervals. Additionally, the therapeutic efficacy of both 213Bi- and 90Y-labeled CO17-1A Fab' was compared in a GW-39 model metastatic to the liver of nude mice. In accordance with kidney uptake values of as high as > or = 80% of the injected dose per gram, the kidney was the first dose-limiting organ using both 90Y- and 213Bi-labeled Fab' fragments. Application of D-lysine decreased the renal dose by >3-fold. Accordingly, myelotoxicity became dose limiting with both conjugates. By using lysine protection, the MTD of 90Y-Fab' was 250 microCi and the MTD of 213Bi-Fab' was 700 microCi, corresponding to blood doses of 5-8 Gy. Additional bone marrow transplantation allowed for an increase of the MTD of 90Y-Fab' to 400 microCi and for 213Bi-Fab' to 1100 microCi, respectively. At these very dose levels, no biochemical or histological evidence of renal damage was observed (kidney doses of <35 Gy). At equitoxic dosing, 213Bi-labeled Fab' fragments were significantly more effective than the respective 90Y-labeled conjugates. In the metastatic model, all untreated controls died from rapidly progressing hepatic metastases at 6-8 weeks after tumor inoculation, whereas a histologically confirmed cure was observed in 95% of those animals treated with 700 microCi of 213Bi-Fab' 10 days after model induction, which is in contrast to an only 20% cure rate in mice treated with 250 microCi of 90Y-Fab'. These data show that RIT with alpha emitters may be therapeutically more effective than conventional beta emitters. Surprisingly, maximum tolerated blood doses were, at 5-8 Gy, very similar between high-LET alpha and low-LET beta emitters. Due to its short physical half-life, 213Bi appears to be especially suitable for use in conjunction with fast-clearing fragments.     

Shedding of GD2 ganglioside by human neuroblastoma

Substantial concentrations of the cell-surface glycosphingolipid, the disialoganglioside GD2, are uniformly present in human neuroblastoma tumors. This ganglioside can also be detected in the plasma of patients with neuroblastoma by direct thin-layer chromatographic analysis. Among 32 neuroblastoma patients in all clinical stages studied prior to the initiation of treatment, 27 (84%) showed measurably elevated plasma concentrations of GD2 (greater than or equal to 50 pmol/ml). The mean level (545 +/- 108 pmol/ml) was more than 50 times the normal plasma GD2 concentration of less than or equal to 10 pmol/ml. Circulating GD2 was not detected in the plasma of patients with the related, more differentiated tumors, ganglioneuroblastoma and ganglioneuroma, indicating an association of the shedding of this ganglioside with the undifferentiated phenotype. Circulating GD2 diminished in patients in response to therapy, and reappeared in patients whose disease recurred. The results suggest that the sequential determination of circulating GD2 will be of value in monitoring individual patients with neuroblastoma.     

Compartmental intrathecal radioimmunotherapy: results for treatment for metastatic CNS neuroblastoma

Innovation in the management of brain metastases is needed. We evaluated the addition of compartmental intrathecal antibody-based radioimmunotherapy (cRIT) in patients with recurrent metastatic central nervous system (CNS) neuroblastoma following surgery, craniospinal irradiation, and chemotherapy. Twenty one patients treated for recurrent neuroblastoma metastatic to the CNS, received a cRIT-containing salvage regimen incorporating intrathecal ¹³¹I-monoclonal antibodies (MoAbs) targeting GD2 or B7H3 following surgery and radiation. Most patients also received outpatient craniospinal irradiation, 3F8/GMCSF immunotherapy, 13-cis-retinoic acid and oral temozolomide for systemic control. Seventeen of 21 cRIT-salvage patients are alive 7–74 months (median 33 months) since CNS relapse, with all 17 remaining free of CNS neuroblastoma. One patient died of infection at 22 months with no evidence of disease at autopsy, and one of lung and bone marrow metastases at 15 months, and one of progressive bone marrow disease at 30 months. The cRIT-salvage regimen was well tolerated, notable for myelosuppression minimized by stem cell support (n = 5), and biochemical hypothyroidism (n = 5). One patient with a 7-year history of metastatic neuroblastoma is in remission from MLL-associated secondary leukemia. This is significantly improved to published results with non-cRIT based where relapsed CNS NB has a median time to death of approximately 6 months. The cRIT-salvage regimen for CNS metastases was well tolerated by young patients, despite their prior history of intensive cytotoxic therapies. It has the potential to increase survival with better than expected quality of life.     

Five new epitope-defined monoclonal antibodies reactive with GM2 and human glioma and medulloblastoma cell lines

In order to investigate GM2 expression in gliomas, the GM2-positive human glioma cell line (HGL) D-54 MG, which contains 0.6 nmol GM2/mg protein, representing 77% of the total monosialoganglioside fraction, was used as an immunogen for the production of anti-GM2 monoclonal antibodies. For ganglioside designations, see IUPAC-IUB (Eur. J. Biochem., 79: 11-21, 1977) and Svennerholm (J. Neurochem., 10: 613-623, 1963). Five IgM monoclonal antibodies (DMAb-1 through DMAb-5) specifically recognizing the GalNAc beta1-4(NeuAc alpha 2-3)Gal-terminal epitope common to GM2 and GalNAC-GD1a are reported. The antibodies did not react with GM1, GM3, GD2, GD3, GD1a, GD1b, and GQ1b. Purified anti-GM2 MAbs were used to define the expression of the "GM2" terminal epitope by cultured human malignant and normal cells by radioimmunoassay and membrane immunofluorescence. Among neuroectodermal tissue-derived cell lines, DMAb-3, at an optimal concentration of 5 micrograms/ml, showed high reactivity (radioimmunoassay binding ratios greater than 20) with 9 of 19 HGLs, 3 of 5 medulloblastoma, 4 of 5 neuroblastoma, and 1 of 3 melanoma lines. Moderate reactivity (binding ratio, 10-20) was exhibited by 3 HGL, 2 medulloblastoma, and 1 neuroblastoma lines and low reactivity (binding ratio, 3-10) by 5 HGL lines; no reactivity was detected with 2 HGL and 2 melanoma lines. Densitometric evaluation of monosialoganglioside extracts from human glioma and medulloblastoma cell lines in conjunction with immunostaining on thin-layer chromatograms showed that GM2 represents the major monosialoganglioside in 8 of 10 HGL and in 3 of 4 Med lines. In these lines the amount of GM2 ranged from less than 0.1 to 0.6 nmol/mg protein. These results indicate that GM2 represents a proportionally increased ganglioside of most glioma, medulloblastoma, and neuroblastoma cells in vitro.     

Primary central nervous system involvement of the so called ‘peripheral T-cell lymphoma’. Report of a case and review of the literature

Epidural neuroblastoma xenografts in nude rats causing paraparesis were treated with intravenous injection of an anti-GD₂ monoclonal antibody 3F8. Metastatic or primary epidural tumors in humans cause rapid neurologic compromise. Treatment is often unsatisfactory. An animal model was established to study antibody targeted therapy of epidural tumor. Human neuroblastoma was xenotransplanted into the thoracic epidural space of nude rats. When paraparesis developed, animals were treated intravenously with an anti-GD2 monoclonal antibody, 3F8, either alone or radiolabeled with¹³¹Iodine. Improvement in neurologic function occurred in 2 of 20 (10%) animals receiving no treatment or control antibody, 14 of 17 (82%) animals receiving 3F8 alone and all 9 animals receiving¹³¹I-3F8 (p<0.0001 for 3F8 or¹³¹I-3F8 vs. control). Six animals treated with 3F8 alone recovered normal neurologic function and remained well until sacrifice 10 days later. Four animals treated with 3F8 alone had no tumor evident on pathologic examination. The percent injected dose of¹³¹I-3F8/g tumor in 5 samples ranged from 0.73% to 3.8%. These observations demonstrate that neoplastic epidural compression of the spinal cord in the rat can be treated successfully with intravenous unmodified monoclonal antibody and that signs of neurologic dysfunction can be reversed. The potential of this approach in treating patients with epidural tumors and other neoplasms, especially those that are not sensitive to chemotherapy or radiotherapy, deserves to be explored.     

Efforts to control the errant products of a targeted in vivo generator

Alpha-particle immunotherapy by targeted alpha-emitters or alpha-emitting isotope generators is a novel form of extraordinarily potent cancer therapy. A major impediment to the clinical use of targeted actinium-225 (225Ac) in vivo generators may be the radiotoxicity of the systemically released daughter radionuclides. The daughters, especially bismuth-213 (213Bi), tend to accumulate in the kidneys. We tested the efficacy of various pharmacologic agents and the effect of tumor burden in altering the pharmacokinetics of the 225Ac daughters to modify their renal uptake. Pharmacologic treatments in animals were started before i.v. administration of the HuM195-225Ac generator. 225Ac, francium-221 (221Fr), and 213Bi biodistributions were calculated in each animal at different time points after 225Ac generator injection. Oral metal chelation with 2,3-dimercapto-1-propanesulfonic acid (DMPS) or meso-2,3-dimercaptosuccinic acid (DMSA) caused a significant reduction (P < 0.0001) in the renal 213Bi uptake; however, DMPS was more effective than DMSA (P < 0.001). The results with DMPS were also confirmed in a monkey model. The renal 213Bi and 221Fr activities were significantly reduced by furosemide and chlorothiazide treatment (P < 0.0001). The effect on renal 213Bi activity was further enhanced by the combination of DMPS with either chlorothiazide or furosemide (P < 0.0001). Competitive antagonism by bismuth subnitrate moderately reduced the renal uptake of 213Bi. The presence of a higher target-tumor burden significantly prevented the renal 213Bi accumulation (P = 0.003), which was further reduced by DMPS treatment (P < 0.0001). Metal chelation, diuresis with furosemide or chlorothiazide, and competitive metal blockade may be used as adjuvant therapies to modify the renal accumulation of 225Ac daughters.     

Occurrence of tumor-associated ganglioside antigens with Hanganutziu-Deicher antigenic activity on human melanomas

The specificity of antibody to NeuGc alpha 2-3Gal beta 1-4Glc-cer (GM3(NeuGc] was carefully reexamined by the method of enzyme-immunostaining on a thin layer plate. The affinity-purified antibody was found to react with NeuGc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuGc-NeuGc] and NeuGc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuGc-NeuAc], but not with NeuAc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuAc-NeuGc)) or NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuAc-NeuAc]. From this result together with the previous results, it (GD3(NeuAc-NeuAc], From this result together with the previous results, it could be concluded that the antibody recognizes the outer portion of molecular species of sialic acids in the gangliosides. By using this antibody, the expression of Hanganutziu-Deicher (HD) gangliosides could be demonstrated in human malignant melanoma. The molecular species were different among individuals examined. Among HD-antigenic gangliosides, GM3(NeuGc) was commonly found in melanoma tissues. One of the patients examined expressed GD3(NeuGc-NeuGc) and GD 3(NeuGc-NeuAc), which may be characteristic gangliosides in human melanomas, since these gangliosides could not be detected in human colon cancer or human fetal tissues.     

Clinically effective monoclonal antibody 3F8 mediates nonoxidative lysis of human neuroectodermal tumor cells by polymorphonuclear leukocytes.

Most studies of antibody-dependent cellular cytotoxicity (ADCC) by polymorphonuclear leukocytes (PMN) have supported oxidative lytic processes. This may be because the studies used nonhuman or nonneoplastic cells that were highly sensitive to reactive oxygen species or were small enough to be phagocytosed by PMN. We therefore investigated whether oxygen radicals participate in PMN cytotoxicity toward human neuroectodermal solid tumor cells sensitized by 3F8, which is an anti-ganglioside GD2 murine IgG3 monoclonal antibody with documented anticancer activity in humans. A 4-h 51Cr release assay was used to assess tumor cell lysis by hydrogen peroxide, superoxide, and hypochlorite. Nine of 11 GD2(+) human melanoma and neuroblastoma cell lines had equal or greater resistance to these oxidants as compared to a GD2(-) human carcinoma line (SKBr1-III) found by others (and confirmed by us) to be significantly more resistant to oxidative lysis than a murine cell line (P388D1) representative of those commonly used in cytotoxicity assays. To facilitate detection of oxidant-mediated lysis, subsequent studies of 3F8-mediated ADCC used GD2(+) targets that were relatively sensitive and others that were relatively resistant to oxygen radicals. Normal PMN and PMN obtained from children with chronic granulomatous disease, which do not generate reactive oxygen species, were equally effective in ADCC. Granulocyte-macrophage colony-stimulating factor, which primes oxidative responses of normal but not of chronic granulomatous disease PMN, enhanced ADCC by both kinds of PMN. During ADCC of 3F8-sensitized targets, with or without granulocyte-macrophage colony-stimulating factor, GD2(-) "innocent bystander" tumor cells (including P388D1) were not lysed, a finding consistent with unimportant extracellular release of cytotoxic mediators. Finally, antioxidant and antimyeloperoxidase moieties did not block ADCC. We conclude that oxidants are not key factors in 3F8-mediated lysis by PMN of human neuroectodermal tumor cells.