UNLABELLED Biodistribution studies demonstrate that intralesional administration of radiolabeled IgM results in high retention of radioactivity with little normal tissue uptake. This study examines the therapeutic potential of this modality. MATERIALS AND METHODS Nude mice bearing subcutaneous human head and neck squamous cell carcinoma xenografts were treated with single intralesional (IL) injections of tumor-reactive human monoclonal IgM (CR4E8) labeled with 25-394 microCi of yttrium-90 (90Y). Untreated mice, mice treated with IL unlabeled immunoconjugate or IL 90Y-aggregate, 160-400 microCi, served as controls. Mice were monitored for tumor growth and toxicity. RESULTS Mice received 80 Gy per 100 microCi of 90Y-labeled IgM and 110 Gy per 100 microCi of 90Y-aggregate. All tumors treated with 90Y-labeled IgM responded. In mice that received > or = 100 microCi, tumors macroscopically disappeared within three weeks from treatment with seventy-six percent tumor-free at 216 days. Acute toxicities associated with high activity 90Y-labeled IgM and 90Y-aggregate were moist skin desquamation and reduced blood counts. Late radiation damage to connective tissue was observed in mice treated with > 100 microCi of 90Y-labeled IgM. CONCLUSIONS Intralesional administration of 90Y-labeled IgM can ablate tumors in nude mice with modest acute or late toxicity. Further development of this modality for loco-regional therapy is warranted.

Purpose: The humanized monoclonal antibody, trastuzumab (Herceptin), directed against HER2/neu, has been effective in the treatment of breast cancer malignancies. However, clinical activity has depended on HER2/neu expression. Radiolabeled trastuzumab has been considered previously as a potential agent for radioimmunotherapy. The objective of this study was to investigate the efficacy of trastuzumab labeled with the α-particle emitting atomic generator, actinium-225 (225Ac), against breast cancer spheroids with different HER2/neu expression levels. 225Ac has a 10-day half-life and a decay scheme yielding four α-particles. Experimental Design: The breast carcinoma cell lines MCF7, MDA-MB-361 (MDA), and BT-474 (BT) with relative HER2/neu expression (by flow cytometry) of 1:4:18 were used. Spheroids of these cell lines were incubated with different concentrations of 225Ac-trastuzumab, and spheroid growth was measured by light microscopy over a 50-day period. Results: The activity concentration required to yield a 50% reduction in spheroid volume at day 35 was 18.1, 1.9, and 0.6 kBq/ml (490, 52, 14 nCi/ml) for MCF7, MDA, and BT spheroids, respectively. MCF7 spheroids continued growing but with a 20–30 day growth delay at 18.5 kBq/ml. MDA spheroid growth was delayed by 30–40 days at 3.7 kBq/ml; at 18.5 kBq/ml, 12 of 12 spheroids disaggregated after 70, days and cells remaining from each spheroid failed to form colonies within 2 weeks of being transferred to adherent dishes. Eight of 10 BT spheroids failed to regrow at 1.85 kBq/ml. All of the BT spheroids at activity concentrations 3.7 kBq/ml failed to regrow and to form colonies. The radiosensitivity of these three lines as spheroids was evaluated as the activity concentration required to reduce the treated to untreated spheroid volume ratio to 0.37, denoted DVR37. An external beam radiosensitivity of 2 Gy was found for spheroids of all three of the cell lines. After α-particle irradiation a DVR37 of 1.5, 3.0, and 2.0 kBq/ml was determined for MCF7, MDA, and BT, respectively. Conclusion: These studies suggest that 225Ac-labeled trastuzumab may be a potent therapeutic agent against metastatic breast cancer cells exhibiting intermediate to high HER2/neu expression.

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.

UNLABELLED:In an effort to improve loco-regional control of ovarian cancer, intraperitoneal (i.p.) administration of an yttrium-90 (90Y) labeled human IgM was studied in a nude mouse model of the disease.METHODS:Athymic nude mice bearing i.p. nodules of SKOV3 NMP2, a human ovarian carcinoma cell line, received single (50-400 microCi) or fractionated (150-510 microCi) administrations of 90Y-labeled 2B12. Untreated mice and mice treated with unlabeled immunoconjugate served as controls. Mice were monitored for weight loss, blood counts and survival.RESULTS:Mice that received at least 300 microCi of 90Y-labeled 2B12 in a single administration lost more than 10% of their body weight with some early deaths, both of which were prevented with fractionated administration. Granulocytes and lymphocytes declined with treatment while red blood cell counts were relatively stable. Untreated mice and mice treated with unlabeled immunoconjugate had a median survival time of 20 and 17 days respectively. Treatment with 90Y-labeled 2B12 increased median survival by 11-12 days per 100 microCi for single (50-300 microCi) and fractionated administrations (150-510 microCi). The highest fractionated activity produced over three logs of tumor cell kill without significant toxicity.CONCLUSION:Intraperitoneal RIT with 90Y-labeled 2B12 appears to be an attractive modality to treat peritoneal carcinomatosis and warrants further development.

The promise of radiolabelled immunoglobulin therapy (RIT) for selective, patient friendly, cancer treatment has not yet been fulfilled. Only patients with haematological malignancies show sizable response rates after RIT. With solid tumours, intravenous administration of radiolabelled antibodies does not provide sufficient tumour targeting. However, intracompartmental administration may solve this problem, particularly if tumour reactive IgM is used. Clinical progress in RIT depends on understanding the important RIT variables: antigen, antibody, radioisotope, conjugation chemistry, activity escalation, fractionation and protein dose. These are reviewed and a new translational decision tree/flow diagram is presented that can limit analysis to the most important RIT variables for a particular disease. These variables may differ depending on the type and stage of cancer, but the guiding principles in RIT development remain the same: selectivity and accountability. The proper application of these principles leads to the definition of a new series of phase I, II, III studies. These studies are more appropriate for the clinical exploration of RIT and place an emphasis on therapeutic ratio rather than toxicity.

UNLABELLED:Most patients with ovarian cancer have disease in the peritoneal cavity. Treatment of this region is inadequate because recurrences are frequent. Increased radiation doses to tumor and, hence, greater tumor control may be possible with intraperitoneal (i.p.) administration of radiolabeled human monoclonal immunoglobulin M (IgM), which is reactive with tumor-associated antigens.METHODS:Biodistribution studies were performed in nude mice bearing i.p. nodules of human ovarian cancer after administration of human monoclonal IgMlambda (AC6C3-2B12), labeled with 111In or 90Y. Irrelevant 111In-labeled human IgMlambda (CH-1B9) and 90Y-aggregate served as specificity controls.RESULTS:Intravenous administration of 111In-labeled AC6C3-2B12 produced low tumor and high liver and spleen uptake. Intraperitoneal administration of AC6C3-2B12 labeled with 111In or 90Y resulted in rapid, high tumor uptake (>45% of injected dose per gram of tumor at 3 hr) that was at least three-fold higher than any normal organ. Biodistribution results were similar for 111In- and 90Y-labeled IgM. Tumor uptake of 111In-labeled AC6C3-2B12 was two-fold greater than that of 111In-labeled CH-1B9. Normal organ uptakes were similar for tumor-reactive and irrelevant IgM. Radioimmunoconjugates were retained in the peritoneal cavity for a prolonged period of time. Yttrium-90 aggregate demonstrated high tumor and bone uptake.CONCLUSION:Higher tumor uptake was observed after i.p. administration of tumor-reactive IgM than after irrelevant IgM. The in vivo behavior of tumor-reactive IgM was similar when it was radiolabeled with either 111In or 90Y. Therefore, 111In-based imaging studies can be used to predict the biodistribution of subsequently administered 90Y-labeled IgM. Further development of radiolabeled AC6C3-2B12 as a diagnostic and therapeutic agent for patients with advanced ovarian carcinoma is warranted.

BACKGROUND AND PURPOSE:Intralesional (i.l.) administration of radiolabeled human monoclonal IgM could provide a new method for increasing the radiation dose delivered to a tumor without exceeding normal tissue tolerance. MATERIALS AND METHODS:Nude mice with subcutaneous human head and neck squamous cell carcinoma nodules were injected either intralesionally or intravenously with a tumor-reactive human monoclonal IgM (CR4E8) labeled with indium-111 (111In) or yttrium-90 (90Y). Groups of mice were sacrificed at different time points and their tumors and major organs were excised and counted for radioactivity. Additional mice that were treated with i.l. 90Y-labeled CR4E8 were sacrificed at the same time points for tumor autoradiography. Serial whole-body gamma camera images were obtained from additional mice treated with i.l. 111In-labeled CR4E8. Intralesionally administered 111In-labeled irrelevant IgM (CH-1B9) and 90Y-aggregate served as specificity controls. RESULTS:Intralesional administration of radiolabeled IgM resulted in prolonged high tumor radioactivity with little normal tissue uptake, with kidney and liver having the highest values. The biodistribution of i.l. CR4E8 was similar whether labeled with 111In or 90Y. Tumor uptake of i.l. irrelevant IgM appeared to be lower and tumor retention appeared to be shorter. Intravenous administration of tumor-reactive IgM resulted in very low tumor radioactivity with high liver and moderate spleen uptake. The i.l. administration of 90Y-aggregate produced prolonged high tumor radioactivity with little normal tissue uptake, with bone having the highest value. Tumor autoradiographs demonstrated that the radiolabeled IgM diffused through the tumor over time while the 90Y-aggregate remained localized at the injection site. Gamma camera scintigraphy corroborated the results of the biodistribution studies. CONCLUSIONS:Intralesional but not intravenous administration of 111In- or 90Y-labeled human IgM results in high tumor radioactivity with low normal tissue exposure. Myelotoxicity is not anticipated to be the dose-limiting normal tissue toxicity of this treatment. Further development of human IgM for the i.l. treatment of human malignancies appears to be warranted.

An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.(ABSTRACT TRUNCATED AT 250 WORDS)