Which radionuclide, carrier molecule and clinical indication for alpha-immunotherapy?

Beta-emitting radionuclides are not able to kill isolated tumor cells disseminated in the body, even if a high density of radiolabeled molecules can be targeted at the surface of these cells because the vast majority of emitted electrons deliver their energy outside the targeted cells. Alpha-particle emitting radionuclides may overcome this limitation. It is thus of primary importance to test and validate the radionuclide of choice, the most appropriate carrier molecule and the most promising clinical indication. Four α-particle emitting radionuclides have been or are clinically tested in phase I studies namely 213Bi, 225Ac, 212Pb and 211At. Clinical safety has been documented and encouraging efficacy has been shown for some of them (213Bi and 211At). 211At has been the most studied and could be the most promising radionuclide but 225Ac and 212Pb are also of potential great interest. Any carrier molecule that has been labeled with ß-emitting radionuclides could be labeled with alpha particle-emitting radionuclide using, for some of them, the same chelating agents. However, the physical half-life of the radionuclide should match the biological half-life of the radioconjugate or its catabolites. Finally everybody agrees, based on the quite short range of alpha particles, on the fact that the clinical indications for alpha-immunotherapy should be limited to the situation of disseminated minimal residual diseases made of small clusters of malignant cells or isolated tumor cells.

Radiolabeling and targeting of lipidic nanocapsules for applications in radioimmunotherapy.

AIM: Radioimmunotherapy is limited in some cases by the low radioactive doses delivered to tumor cells by antibodies or pretargeted haptens. In order to increase this dose, lipidic nanocapsules (LNC) with a hydrophobic core are proposed as radionuclide vectors that could be targeted to cancer cells by a bispecific anti-tumor x anti-hapten antibody after incorporation of different haptens in the nanocapsule membrane. METHODS: To bind different radionuclides to the nanocapsules, several bifunctional chelating agents (BCA) were used to form stable complexes with the radionuclides. Some of them are hydrophilic for LNC shell while others are lipophilic to radiolabel the core. Poly(ethylene glycols) (PEG) were used to increase the residence time in blood. Since PEG can modify haptens recognition by the bispecific antibody and radiolabeling efficiency, haptens, BCA or Bolton-Hunter reagent (BH) were attached to the PEG extremity to optimize accessibility. Specific constructs (DSPE-PEG-haptens, DSPE-PEG-BCA, and DSPE-PEG-BH) were synthesized to develop these new radiolabeled vector formulations. Large amounts of PEG have been introduced by a postinsertion method without important change in nanocapsule size and properties. The nanocapsule core was radiolabeled with a lipophilic [(99m)Tc]SSS complex. RESULTS: Serum stability studies showed that this (99m)Tc-labeling method was efficient for at least 20 h. Concerning the nanocapsule surface, several methods have been performed for (111)In-labeling by using DSPE-PEG-DTPA and for (125)I-labeling with DSPE-PEG-BH. CONCLUSIONS: The nanocapsules labeling feasibility with a variety of radionuclides and their stability were demonstrated in this paper.

Comparison of positron emission tomography and lymphangiography in the diagnosis of infradiaphragmatic Hodgkin's disease.

PURPOSE: To evaluate the respective roles of positron emission tomography using 18F-fluorodeoxyglucose (FDG-PET) and lymphangiography (LAG) in staging Hodgkin's disease (HD) patients with negative contrast-enhanced infradiaphragmatic computed tomography (CT). MATERIAL AND METHODS: 28 patients underwent FDG-PET and LAG at initial staging. Concordant positive findings on both tests were regarded as actual HD locations and concordant negative findings as true negative. In case of discrepancy, the reference was biopsy or magnetic resonance imaging (MRI). RESULTS: Concordant results were obtained in 26 patients (24 negative, two positive). In two of the 24 negative patients, PET showed additional lesions in the spleen and one celiac lymph node (one patient), and in the right kidney and the right iliac crest (one patient). Discordant results were obtained in two patients. Both methods indicated infradiaphragmatic involvement in different locations in one patient. In the other, PET was falsely positive (PET done within 24 hours after a negative LAG), which was confirmed by biopsy (benign inflammatory, probably due to LAG medium). CONCLUSION: FDG-PET and LAG gave comparable results, making invasive LAG unnecessary. Furthermore, LAG, when performed before PET, can be responsible for false-positive PET results.

Current status and perspectives in alpha radioimmunotherapy.

Systemic administration of radiolabeled antibody directed against tumor antigens in radioimmunotherapy (RIT) enables to specifically target the cancer cells and to destroy them. So far, this strategy has proven its efficiency in the treatment of some hematological cancers with antibodies labeled with beta emitting radionuclides. In the last 2 decades, availability of short half life alpha emitters prompted to consider their use in RIT. Contrary to beta particles, alpha particles have a short path length and display a high lineic energy transfer. Those physical characteristics open new fields of clinical applications complementary to beta-RIT. To date, alpha-RIT is still at a preclinical stage of development: the radiolabeling methods need to be optimized to ensure in vivo stability of the radiopharmaceuticals. Some radionuclides have complex decay schemes with daughters emitting further alpha particles whose toxicity needs to be investigated. The modalities of administration of radiolabeled antibodies in animal models require also to be improved for delivering higher doses to tumor targets. A comprehensive analysis of the specific events occurring at cell or tissue level in response to alpha irradiation would be of great interest in order to define the best therapeutic association for residual disease or consolidation treatments. This approach has been proven to be efficient in increasing antitumor response either by using high doses with organ protection (kidney, bone marrow) or by a synergistic effect between alpha-RIT and associated treatments, such as chemotherapy.

Brown fat in breast cancer patients: analysis of serial (18)F-FDG PET/CT scans.

PURPOSE: It has recently been suggested that FDG accumulation in the brown adipose tissue varies as a function of age, sex and outdoor temperature. The aim of this study was to assess changes in FDG uptake in brown fat in patients based on serial PET/CT scans and to compare our results with previous findings. METHODS: Early response to neoadjuvant chemotherapy in 33 female breast cancer patients was assessed by FDG PET. Five PET/CT scans were performed for each patient. PET/CT images were analysed retrospectively. PET scans were considered positive when diffuse, symmetrical, abnormal "USA" (uptake in supraclavicular area) fat was detected. RESULTS: A total of 163 PET images were analysed. Seventy-four PET scans (45%) revealed abnormal FDG uptake in the supraclavicular area. These foci were present on uncorrected and attenuation-corrected images. FDG uptake was identical on all five scans in only five patients. No significant relationship was found between abnormal FDG uptake and outdoor temperature, age or time interval between chemotherapy and PET. Abnormal FDG uptake in the neck seemed to predominantly occur in patients with a low body mass index (p<0.05). Most significant changes in the PET/CT scan results were observed during chemotherapy with docetaxel (p<0.05). When observed, bilateral uptake in the neck was more intense than background uptake (p<0.00001). CONCLUSION: This study shows that FDG uptake in the neck varies as a function of time, that it is unrelated to age or outdoor temperature, and that bilateral uptake is generally intense.

Synthesis of new bivalent peptides for applications in the Affinity Enhancement System.

The feasibility of two-step radioimmunotherapy (RIT) of cancer by the Affinity Enhancement System (AES) has been demonstrated in experimental and clinical studies. This technique, associating a bispecific antibody and a bivalent peptide radiolabeled with iodine-131, has been developed to reduce toxicity and to improve therapeutic efficacy compared to one-step targeting methods. The use of AES with different beta-emitters such as rhenium-188, samarium-153, or lutetium-177 or alpha-emitters such as actinium-225 or bismuth-213 is now considered. Thus three new peptides, designed to allow for the coupling of a variety of bifunctional chelating agents BCA, were synthesized by associating two glycyl-succinyl-histamine (GSH) arms, which are recognized by the 679 monoclonal antibody (mAb-679), with different binding agents, such as p-nitrophenylalanine or N,N-bis(carboxymethyl)-4-N'-(9-fluorenylmethyloxycarbonyl)aminobenzylamine. Immunoreactivity and serum stability evaluation were performed for each synthesized peptide. One of the three peptides (LM218) proved to be more stable than the others, and three different BCAs were coupled to LM218 (CITC-DTPA, CITC-TTHA, and CITC-CHXA''DTPA). One of these products, LM218-BzTTHA was radiolabeled with indium-111 without loss of immunoreactivity toward the mAb-679. These new peptides will allow pretargeted RIT with a large variety of radionuclides, to adapt the choice of the radionuclide (LET, half-life, penetrating emission) to the nature and size of targeted tumors.

High frequency of bone/bone marrow involvement in advanced medullary thyroid cancer.

High hematological toxicity has been observed with anti-carcinoembryonic antigen radioimmunotherapy (RIT) in medullary thyroid carcinoma (MTC), suggesting metastatic bone involvement (BI). This retrospective study evaluated the rate of BI in MTC patients enrolled in two phase-I/II RIT trials using anti-carcinoembryonic antigen x anti-diethylenetriamine pentaacetic acid bispecific antibodies and [(131)I]di-diethylenetriamine pentaacetic acid hapten. Thirty-five patients underwent bone scintigraphy, bone magnetic resonance imaging (MRI), and post-RIT immunoscintigraphy (IS). IS performed in MTC patients was compared with IS conducted in 12 metastatic colorectal carcinoma (CRC) patients. Quantitative analysis of bone uptake was performed in three MTC and three CRC patients. In the MTC group, bone scintigraphy detected BI in 56.6% of patients, MRI in 75.8%, and IS in 88.6%. BI was confirmed by undirected (random) bone marrow biopsy, by bone surgery, or by two positive imaging methods in 74.3% of the patients. Sensitivity per patient of bone scintigraphy, MRI, and IS were 72.7, 100, and 100%, respectively. In contrast, IS visualized BI in only 33.3% of CRC patients; bone uptake was lower in CRC than in MTC patients. Bone MRI combined with post-RIT IS disclosed a much higher BI rate in advanced MTC than previously reported in the literature.

Ovarian cancer management. Practice guidelines for nuclear physicians.

Ovarian cancer is a frequent and severe malignancy. Over 75% of cases are diagnosed at an advanced stage with disease spread beyond the ovaries. Despite the high response rates of initial treatments (i.e.,70-80%), the median progression-free survival of advanced ovarian cancer is 16-22 months, and the 5-year overall survival, 20-30%. The majority of these patients relapse, with metastatic peritoneal spread, unresectable, or drug resistant disease. Our goal was to outline current knowledge about diagnosis, prognostic factors, and treatments, and to dwell on non-nuclear medicine and nuclear-medicine diagnostic procedures.

Lymphoscintigraphy in the sentinel lymph node technique for breast tumor: value of early and late images for the learning curve.

As the performance of early (H+1 to H+4) and late (D1) lymphoscintigraphic images raises organizational problems in outpatient surgery for breast cancer, only early images are generally obtained. The present study evaluated whether two series of images are better than one and defined the advantages of both methodologies. One hundred and eighteen patients with infiltrating breast carcinoma (T(0), T(1) and T(2)) were included in the study: 87 in group A (early and late images) and 31 in group B (only early images). All patients received two peritumoral injections of (99m)Tc-sulfur colloid, 15-18 MBq (group A) and <15 MBq (group B). During the operation, the patent blue bye technique was associated with radioactivity detection. The two groups were comparable for histological type and tumor size and localization. Successful localization of sentinel nodes on early lymphoscintigraphic images was significantly greater for group B. The identification of a sentinel node focus on early lymphoscintigraphy increased by 10% during the study. Sentinel node detection by the isotopic method alone, or the two methods combined, was comparable for both groups. In radioactivity detection, the count rate for sentinel nodes versus background (contralateral breast) was similar for the two groups. During the learning phase, two series of images gave a definite advantage. Subsequently, lymphoscintigraphy performed at +2 h was sufficient (the results for the two groups became indistinguishable).

Radiation-induced apoptosis in human myeloma cell line increases BCL-2/BAX dimer formation and does not result in BAX/BAX homodimerization.

A popular model of BCL-2 and BAX involvement in apoptosis suggests that upon apoptosis induction cytosolic BAX translocates to the mitochondria, where it displays the pro-apoptotic function, which involves its homodimerization. BCL-2 exerts anti-apoptotic function by forming heterodimers with BAX, thus neutralizing the pro-apoptotic activity of the latter. We have shown that irradiation of the human myeloma cell line RPMI-8226 induced apoptosis as determined by DNA degradation, cytochrome c release into cytoplasm and BCL-2 caspase-mediated cleavage. BCL-2 protein was present only in the membrane fraction, whereas BAX was found both in cytosol and membranes isolated from non-irradiated cells. Radiation induced moderate redistribution of BAX from cytosol to membranes with a concomitant increase in BCL-2/BAX heterodimer formation. Rapid and transient BCL-2 phosphorylation in membrane fractions of irradiated cells did not affect BCL-2/BAX heterodimerization. We failed to detect any BAX/BAX homodimers in apoptotic cells. Our findings show that in irradiated RPMI-8226 cells the formation of BCL-2/BAX heterodimers correlates with apoptosis. We conclude that BCL-2/BAX heterodimers are negative regulators of death protection, and our data agree with those who propose that BCL-2 does not require BAX to exert its survival function.

Two-step targeting of xenografted colon carcinoma using a bispecific antibody and 188Re-labeled bivalent hapten: biodistribution and dosimetry studies.

UNLABELLED: Radioimmunotherapy (RIT) is currently being considered for the treatment of solid tumors. Although results have been encouraging for pretargeted 131I RIT with the affinity enhancement system (AES), the radionuclide used is not optimal because of its long half-life, strong gamma emission, poor specific activity, and low beta particle energy. 188Re, though unsuitable for direct antibody labeling, could be used with the AES two-step targeting technique. The purpose of this study was to compare the distribution and dosimetry of a bivalent hapten labeled with 188Re or 125I. For dosimetry calculations and biodistribution data, 125I was substituted for 131I. METHODS: After preliminary injection of a bispecific anticarcinoembryonic antigen (CEA) or antihapten antibody (Bs-mAb F6-679), AG 8.1 or AG 8.0 hapten radiolabeled with 188Re or 125I was injected into a nude mouse model grafted subcutaneously with a human colon carcinoma cell line (LS-174-T) expressing CEA. A dosimetry study was performed for each animal from the concentration of radioactivity in tumor and different tissues. RESULTS: Radiolabeling of AG 8.1 with 125I afforded a 40% yield with a specific activity of 11.1 MBq/nmol after purification. Radiolabeling of AG 8.0 with 188Re afforded a 72% yield with a specific activity of 31.82 MBq/nmol. In all experiments, the percentage of tumor uptake of 125I-AG 8.1 was always significantly greater than that of 188Re-AG 8.0. The corresponding tumor-to-tissue ratios reflected uptake values. The least favorable tumor-to-normal tissue ratios in the dosimetry study were 8.1 and 8.5 for 131I (tumor-to-blood ratio and tumor-to-kidney ratio, respectively) and 2.3 for 188Re (tumor-to-intestine ratio). CONCLUSION: This study indicates that 188Re can be used for radiolabeling of hapten in two-step radioimmunotherapy protocols with the AES technique. 188Re has a greater range than 131I, which should allow the treatment of solid tumors around 1 cm in diameter. Although the method used for hapten radiolabeling did not provide optimal tumor uptake, the use of a bifunctional chelating agent associated with AG 8.1 should solve this problem.

[Immunotargeting of tumors: state of the art and prospects in 2000]. / Immunociblage des tumeurs: situation et perspectives en 2000.

Following 15 years of experimental studies, tumor immunotargeting using monoclonal antibodies directed against tumor associated antigens shows now important monoclonal antibodies directed against tumor associated antigens shows now important clinical developments. This is mainly due to encouraging therapeutic results which have obtained using humanized antibodies such as the anti-CD20 rituximab in follicular B lymphomas and the anti-DrbB2 herceptin in breast carcinomas. Thanks to genetic engineering it is possible to graft variable or hypervariable regions from murine antibodies to human IgG, and even to obtain fully human antibodies by using either transgenic mice containing a large part of the human repertoire of human IgG, or selection of human antibody fragments expressed by phages. Radiolabeling of antibodies played a major role to demonstrate the tumor immunotargeting specificity and remains attractive for the diagnosis by immunoscintigraphy as well as for the treatment by radioimmunotherapy of some cancers. In this review, the current results and the prospects of diagnostic and therapeutic uses of anti-tumor antibodies and their fragments will be described. Concerning diagnosis, 123-iodine or 99m-technetium labeled Fab fragments allowed very demonstrative tumor images but this technique has a limited effect upon the therapeutic attitude. Immuno-PET (positron emission tomography) could enhance the sensitivity of this imaging method. Radio-immunoguided surgery and immunophotodetection are attractive techniques still under evaluation. Concerning therapy, 131-iodine labeled anti-CD20 antibodies gave spectacular results in non-Hodgkin's B lymphomas. In solid tumors which as less radiosensitive, radioimmunotherapy could concern small tumors and need the use of two-steps targeting and/or alpha emitters radioisotopes. Some other strategies will be described such as bispecific antibodies directed against tumors and immune effector cells, some antibody fragments expressed on T cells called T-bodies or some biological studies using intrabodies. Published data and works in progress demonstrate that immunotargeting of tumors will have a growing place in the treatments of cancer patients.

Treatment of bone metastases of prostate cancer with strontium-89 chloride: efficacy in relation to the degree of bone involvement.

This retrospective study evaluated the toxicity and efficacy of strontium-89 chloride (Metastron, Amersham) in 94 patients with painful bone metastases of prostate cancer (117 injections of 150 MBq) and compared the efficacy of treatment in patients with moderate and extensive bone involvement. The predictive value of flare response with regard to analgesic response was also studied. High-grade leukothrombopenias were observed after only 5% of injections. An improvement in quality of life was obtained in 65% of cases, a decrease in pain in 78% (31% complete response) and a reduction of analgesics in 60%. Efficacy was significantly better for pain decrease (P=0.005) and reduction of analgesics (P=0.018), and response was significantly longer (P<0.0035) in patients with moderate than in patients with extensive bone involvement. The flare response observed in 23% of cases was not predictive of pain response (P=0.919) or reduction of analgesics (P=0.353). A second dose prolonged analgesia in three-quarters of cases without any apparent increase in toxicity. These results confirm the benefit of 89Sr chloride for the treatment of metastatic bone pain and suggest that internal radiotherapy should be started earlier. A bone scan could be proposed at the time of hormonal escape resulting in bone pain, and internal radiotherapy could be initiated when several metastatic foci exist, even if only one is painful. In this way, pain-free follow-up could be prolonged, and the transition to other therapeutic approaches, particularly opioids, delayed.

Nuclear medicine applications for neuroendocrine tumors.

Sensitive, specific radiopharmaceuticals are available for scintigraphic diagnosis and internal radiotherapy of neuroendocrine tumors. (123)I-MIBG (metaiodobenzylguanidine) scintigraphy is the examination of choice for visualizing tumor sites of pheochromocytoma. In the event of malignant pheochromocytoma or carcinoid tumor, this examination allows assessment of the presence or absence of tumor uptake and can guide radiotherapy with (131)I-MIBG. The peptides secreted by neuroendocrine tumors can be radiolabeled for targeting of their specific receptors. Scintigraphy using a (111)In-labeled somatostatin analog (octreotide) is the examination of choice for diagnosis of the spread of gastroenteropancreatic and carcinoid tumors, as it is more sensitive than morphologic imaging techniques. It can also guide radiotherapy performed with the same pharmaceutical vector. These same two agents (MIBG and octreotide) can be used therapeutically by replacing (123)I with (131)I and (111)In by (90)Y. A transient palliative effect is obtained for a variable number of tumors (most often large ones) that take up the radiopharmaceutic agent well. There is general consensus that, for relatively radioresistant solid tumors, this type of radiotherapy is efficient only in the event of small tumor targets (a few millimeters in diameter) whose uptake is maximal, allowing more homogeneous distribution than that achieved with large tumors. Thus for optimal control of the disease it is recommended first to use scintigraphic imaging to confirm that the tumor takes up the radiopharmaceutical agent in question ((123)I-MIBG or (111)In-octreotide) and then reduce the tumor burden surgically before injecting high therapeutic activity (possibly with reinjection of peripheral stem cells). This treatment can be repeated three times every 3 months before evaluating the response. In these conditions, internal radiotherapy can be beneficial or even determinant for controlling disease progression.

Rhenium-188-labeled anti-neural cell adhesion molecule antibodies with 2-iminothiolane modification for targeting small-cell lung cancer.

We have evaluated the potential of 188Re-labeled monoclonal antibodies (MAbs) modified with 2-iminothiolane (2IT) for targeting small-cell lung cancer (SCLC). Radiolabeled MAbs NK1NBL1 and C218 recognizing neural cell adhesion molecule were injected i.v. into athymic mice inoculated with human SCLC tumors, and the biodistribution was examined. NK1NBL1 localized in the tumors better than C218. 188Re-labeled MAbs cleared from the blood faster than 125I-labeled counterparts, resulting in higher tumor-to-blood ratios. In conclusion, the 188Re-labeled MAbs are attractive candidates for imaging and therapy of SCLC.

Validation of 213Bi-alpha radioimmunotherapy for multiple myeloma.

The efficacy of radioimmunotherapy (RIT) with beta emitters has been clinically demonstrated in the treatment of refractory forms of lymphoma. Alpha-emitting radionuclides with a short half-life are also good potential candidates for RIT directed at tumor targets easily accessible to radioimmunoconjugate molecules and small enough to benefit from the short range of alpha particles (<100 microm). The purpose of this study was to demonstrate the feasibility of ex vivo purging of multiple myeloma-invaded bone marrow. Tumor cells were targeted by a specific monoclonal antibody (B-B4) coupled to 213Bi by a chelating agent (pentaacetic triamine diethylene p-aminobenzyl acid). The efficacy of alpha-RIT was assessed in vitro by analysis of thymidine incorporation, cell mortality, apoptosis of myeloma cells, and the study of nonspecific irradiation of hematopoietic cell lines not recognized by B-B4-pentaacetic triamine diethylene p-aminobenzyl acid immunoconjugate. High dose-dependent cell mortality of myeloma cells was found with radiolabeled B-B4, and this mortality was total at 30 kBq/10(5) cells. Cells were found in apoptotic state at rates of up to 40% for a dose of 7.4 kBq/10(5) cells. Nonspecific mortality was low compared with specific mortality (up to 1%).

Immunohistology of carcinoembryonic antigen (CEA)-expressing tumors grafted in nude mice after radioimmunotherapy with 131I-labeled bivalent hapten and anti-CEA x antihapten bispecific antibody.

We have developed a pretargeting strategy, called the Affinity Enhancement System (AES), which uses bispecific antibodies (BsF(ab')2) to target radiolabeled bivalent haptens to tumor cells. We performed several radioimmunotherapy (RIT) experiments in nude mice grafted with LS174T colon carcinoma or TT medullary thyroid cancer. Mice were treated with 131I-labeled di-DTPA-indium-tyrosyl-lysine bivalent hapten (75-112 MBq) administered 15-48 h after anti-CEA x anti-DTPA-indium BsF(ab')2. Immunohistological studies were performed on tumors at their minimal relative volume (TT), on stabilized tumor nodules (LS174T), and on regrowing tumors (TT and LS174T). Untreated tumors were used as controls. On microscopic examination, regrowing tumors (2 months posttherapy) were similar to untreated tumors with cells showing their respective typical morphology (large cells with a high nucleocytoplasmic ratio for TT, small and very undifferentiated cells for LS174T). However, regrowing tumors showed larger necrotic areas and a higher mitotic index correlated with Ki-67 antigen staining. Immunostaining for CEA was as strong as for controls. By contrast, the immunohistology of TT tumors at their minimal relative volume (1 month posttherapy) or of LS174T residual nodules (8 months posttherapy) showed decreased mitotic indices correlated with poor Ki-67 antigen staining. Some clusters of LS174T presented with features of glandular lumen, which suggested a more differentiated and less aggressive status. In TT tumors, CEA expression remained unchanged (80-100% membrane and cytoplasmic staining), whereas only 70% of the LS174T tumors were stained, with 58% loss of the membrane expression. Repeated treatment early after the tumor has reached its minimal relative volume should thus be efficient and improve the overall efficacy of AES RIT.

Toxicity and efficacy of radioimmunotherapy in carcinoembryonic antigen-producing medullary thyroid cancer xenograft: comparison of iodine 131-labeled F(ab')2 and pretargeted bivalent hapten and evaluation of repeated injections.

This study compared the toxicity and efficacy of 131I-labeled bivalent hapten pretargeted by anti-carcinoembryonic antigen (CEA)/anti-N alpha-(diethylenetriamine-N,N,N',N''-tetraacetic acid-indium(F6-734) bispecific antibody [affinity enhancement system (AES) reagents] with 131I-labeled anti-CEA F(ab')2 (131I-F6) in mice grafted with a human medullary thyroid carcinoma. Repeated injections of AES reagents were also evaluated. Mice bearing TT tumor xenografts were treated with 37, 74, or 92.5 MBq of AES reagents, two injections of 74 MBq of AES reagents 45 days apart, or 37 or 92.5 MBq of 131I-F6. Control groups were treated with nonspecific 131I-labeled F(ab')2, nonspecific AES reagents, nonradiolabeled F6, F6-734 bispecific antibody, and nonradiolabeled bivalent hapten or received no injection. For AES treatments, bispecific antibody was injected 48 h before the hapten. Animal weight, hematological toxicity, tumor volume, and serum thyrocalcitonin were monitored during 5 months. At 92.5 MBq, weight loss was significantly lower after AES than F6 treatment (P = 0.004). The percentages of leukocyte count changes were significantly lower after AES than F6 at 37 and 92.5 MBq (P = 0.01 and 0.04, respectively). The percentage of platelet count changes was significantly lower with AES at the 92.5-MBq dose level (P = 0.04). In the group injected twice with AES reagents, toxicity was not significantly increased after the second treatment. Tumor response was observed in all cases but was significantly longer with repeated treatments of 74 MBq AES reagents than with a single treatment (P = 0.004). Two complete responses were observed with repeated treatments. Changes in thyrocacitonin level paralleled those in tumor volume. These results indicate that pretargeted radioimmunotherapy was at least as efficient as one-step radioimmunotherapy and markedly less toxic. Repeated treatments with AES reagents increased efficacy without increasing toxicity.