Intraperitoneal radioimmunotherapy for ovarian cancer: pharmacokinetics, toxicity, and efficacy of I-131 labeled monoclonal antibodies

Thirty-six patients with ovarian cancer were treated with intraperitoneal I-131 labeled monoclonal antibodies to tumor associated antigens. The activity of I-131 administered was increased from 20 mCi to 158 mCi and the pharmacokinetics and toxicity evaluated. Five patients who had developed HAMA (Human Antimouse Antibodies) were retreated, and the pharmacokinetics and toxicity of the first and second treatment compared. Patients receiving their first therapy (HAMA negative), had a maximum of 25% (range 19.8-39.8%) of the injected activity in their circulation. This was accompanied by severe marrow suppression at I-131 activities over 120 mCi. The 5 HAMA positive patients had only 5% injected activity in the systemic circulation (range 3.8-6%), with rapid urinary excretion and neglible marrow suppression. In 31 patients with assessable disease there were no responses in 8 patients with gross disease (nodules greater than 2 cms), partial responses in 2 out of 15 patients with nodules less than 2 cms, and complete responses in 3 out of 6 patients with microscopic disease. The non specific radiation dose to the peritoneal cavity was estimated to be less than 500 cGy by lithium fluoride TLD, and could not be expected to account for the responses seen.     

The nucleolin targeting aptamer AS1411 destabilizes Bcl-2 messenger RNA in human breast cancer cells

We sought to determine whether nucleolin, a bcl-2 mRNA-binding protein, has a role in the regulation of bcl-2 mRNA stability in MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, we examined the efficacy of the aptamer AS1411 in targeting nucleolin and inducing bcl-2 mRNA instability and cytotoxicity in these cells. AS1411 at 5 micromol/L inhibited the growth of MCF-7 and MDA-MB-231 cells, whereas 20 micromol/L AS1411 had no effect on the growth rate or viability of normal MCF-10A mammary epithelial cells. This selectivity of AS1411 was related to a greater uptake of AS1411 into the cytoplasm of MCF-7 cells compared with MCF-10A cells and to a 4-fold higher level of cytoplasmic nucleolin in MCF-7 cells. Stable siRNA knockdown of nucleolin in MCF-7 cells reduced nucleolin and bcl-2 protein levels and decreased the half-life of bcl-2 mRNA from 11 to 5 hours. Similarly, AS1411 (10 micromol/L) decreased the half-life of bcl-2 mRNA in MCF-7 and MDA-MB-231 cells to 1.0 and 1.2 hours, respectively. In contrast, AS1411 had no effect on the stability of bcl-2 mRNA in normal MCF-10A cells. AS1411 also inhibited the binding of nucleolin to the instability element AU-rich element 1 of bcl-2 mRNA in a cell-free system and in MCF-7 cells. Together, the results suggest that AS1411 acts as a molecular decoy by competing with bcl-2 mRNA for binding to cytoplasmic nucleolin in these breast cancer cell lines. This interferes with the stabilization of bcl-2 mRNA by nucleolin and may be one mechanism by which AS1411 induces tumor cell death.     

Intraperitoneal administration of an I125 labelled monoclonal antibody for localization of human colorectal cancer in the nude mouse

The monoclonal antibody (MoAb) HMGF-1 was evaluated in the radioimmunodetection of human colonic cancer transplanted intraperitoneally (i.p.) into athymic nude mice. This antibody reacts with a component of the human milkfat globule, as well as a wide range of epithelial cells and adenocarcinomas of various origins. Purified MoAb was iodinated with 125I and administered i.p. into nu/nu mice bearing (i.p.) xenografts of human colonic adenocarcinoma (X56). Differential tissue counts of radioactivity demonstrated preferential localization of the antibody in i.p. and subcutaneous (s.c.) tumor tissue as compared to normal tissues. Maximum per cent dose per g of tumor (25.17 +/- 1.37), maximum tumor: blood ratio (4.45 +/- 0.14) and maximum tumor: tissue ratios (34.2 +/- 0.12) were obtained at the optimal labelling time of 5 days after antibody injection. Selective localization to tumor was confirmed with a control anti-hepatitis virus MoAb of the same isotype and by localization studies in non-tumor bearing athymic mice. Half lives of the persistence of the iodine 125 in the tumor bearing and non tumor bearing mice were 5 and 7 days, respectively, indicating approximate antibody half lives. Whole body scans showed distinct tumor images without the use of subtraction techniques. This pilot experimental study demonstrates the feasibility of i.p. administration of labelled antitumor MoAb in the imaging of i.p. tumors in an athymic mouse system. Whether or not these observations are applicable to the human situation remains to be carefully established.     

huBC1-IL12, an immunocytokine which targets EDB-containing oncofetal fibronectin in tumors and tumor vasculature, shows potent anti-tumor activity in human tumor models

IL-12 is a cytokine which showed anti-tumor effects in clinical trials, but also produced serious toxicity. We describe a fusion protein, huBCI-IL12, designed to achieve an improved therapeutic index by specifically targeting IL-12 to tumor and tumor vasculature. huBC-1 is a humanized antibody that targets a cryptic sequence of the human ED-B-containing fibronectin isoform, B-FN, present in the subendothelial extracellular matrix of most aggressive tumors. B-FN is oncofetal and angiogenesis-associated, and is undetectable in most normal adult tissues. The original murine BC-1 antibody has been used successfully for immunoscintigraphy to image brain tumor mass in glioblastoma patients. In huBCI-IL12, each of the lgG heavy chains is genetically fused to the N-terminus of the IL-12 p35 subunit, which in turn is disulfide-bonded to the p40 subunit,     

Immunolocalisation of testicular tumours using radiolabelled monoclonal antibody to placental alkaline phosphatase

Tumour associated monoclonal antibody against placental alkaline phosphatase (H17E2) was radiolabelled with Indium-111 and Iodine-123 and administered intravenously in 33 patients with primary and/or metastatic testicular tumour, as well as in 8 patients who were in complete remission after surgical excision of the tumour. The presence of a tumour was confirmed and correlated well with conventional diagnostic techniques and, in addition, the antibody scan revealed the presence of active disease in 2 patients with negative conventional imaging and with elevated serum markers. In addition, in one patient the CT produced a false positive result where the antibody scan was negative. Finally, the absence of tumour was confirmed in all 8 cases of patients in complete remission. All patients studied with Indium-labelled antibody had observable concentrations of the radiolabel in the liver (estimated to be approximately 30% of the administered dose), as well as in the kidneys and spleen. The patients studied with the Iodine-123 labelled antibody had observable concentrations in the thyroid gland and the stomach. The best images were seen at 48 and 24 hrs after the Indium and Iodine radiolabelled antibody respectively. No human anti-mouse antibody was detected in any of our patients, even in those who received 2 and 3 administrations, with the highest amount of administered protein being 800 micrograms. No toxicity was encountered in any of our patients in 4 months of follow-up. This method may be of clinical value in patients with testicular neoplasma and represents a new addition to current imaging techniques. A positive scan indicates the definite presence of a tumor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP: a mitogenic signal for Swiss 3T3 cells.

Addition of cholera toxin (100 ng/ml) to quiescent cultures of Swiss 3T3 cells acts synergistically with serum (2-4%), insulin, phorbol esters, epidermal growth factor, and fibroblast-derived growth factor to stimulate DNA synthesis. In the presence of insulin, cholera toxin caused a dose-dependent increase in cumulative [3H]thymidine incorporation into acid-insoluble material and in the intracellular cyclic AMP (cAMP) level. The dose--response curves for the two processes were similar. Furthermore, addition of 1-methyl-3-isobutylxanthine (15--500 microM) or of 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (5--100 microM), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase which are potent inhibitors of cyclic nucleotide phosphodiesterase activity, stimulated DNA synthesis and increased cAMP levels in Swiss 3T3 cells. These compounds strikingly potentiated the effect of cholera toxin on DNA synthesis and on cAMP levels. When quiescent Swiss 3T3 cells were exposed to cholera toxin (100 ng/ml) and insulin at 10 micrograms/ml (4- to 7-fold increase in cAMP level) or to these agents and 1-methyl-3-isobutyl xanthine at 50 microM (35-fold increase in cAMP level), DNA synthesis began after a lag of 16 hr. These results indicate that cAMP acts as a mitogenic signal for Swiss 3T3 cells and differ from the widely held view that cyclic AMP inhibits the proliferation of fibroblast cells.     

Characterisation and internalisation of recombinant humanised HMFG-1 antibodies against MUC1

The humanised HMFG-1 immunoglobulin has been extensively developed as a clinical immunotherapeutic agent for MUC1 expressing tumours. We have constructed a single-chain Fv (scFv) and Fab fragment from this antibody and shown that both these species retain their specificity for MUC1. The scFv was less stable and less soluble than the Fab. Detailed analyses of the binding kinetics of the whole IgG and Fab fragment show that the affinity for MUC1 synthetic peptides is low (approximately 100 nM for the IgG and 10 muM for the Fab), with particularly low but similar dissociation rate constants (0.031-0.095 s(-1)). Binding to native antigen on the cell surface is over two orders of magnitude better. Confocal immunofluorescence microscopy shows that both the IgG and Fab are internalised rapidly (the IgG is internalised within 15 min) and colocalise to early endosomes. This work provides an appreciation of the binding, internalising and trafficking kinetics, important for the development of future therapeutics based on this antibody.     

Patients receiving murine monoclonal antibody therapy for malignancy develop T cells that proliferate in vitro in response to these antibodies as antigens

Peripheral blood mononuclear cells (PBMCs) were obtained from patients receiving radioactive murine monoclonal antibody (MAb) therapy for malignant epithelial tumours, as well as normal controls, and were tested for the ability of T cells to proliferate in vitro in the presence of the MAb administered for therapy (HMFG1), and another isotypically matched antibody of irrelevant specificity (11.4.1). We studied 13 patients who had one (ten patients) or two (three patients) courses of MAB treatment, 11 age matched patients with the same histologic types of tumours, that had not received MAbs, and four normal controls. There was a consistent dose dependent in vitro T cell proliferation in 11 of the 13 patients after MAb therapy. This was not observed in the pre-therapy group of patients or normal controls, where the T cell proliferative responses remained baseline. The mean stimulation index (S.I.) in the post-therapy group was significantly higher than that of the pre-therapy patients and that of normal controls. When the in vitro T cell proliferative responses of these patients were measured in the presence of HMGF1 MAb (IgG1) and an isotypically identical, but idiotypically unrelated 11.4.1 MAb (IgG1), there was no statistically significant difference in the mean S.I. For HMFG1 vs 11.4.1 for the whole group of treated patients. When patients were separated into those who received one and those who received two MAb treatments, a significant increase in the mean S.I. was observed in the presence of HMFG1, in the group of patients receiving two treatment courses, suggesting the generation of T cells with specificity for the idiotypic component of the administered murine immunoglobulin. In order to further characterise these in vitro cellular responses we incubated PBMCs with and without an optimal concentration of the MAb (100-300 micrograms ml-1), as defined by the proliferation assay, and compared the differences in cell subpopulations. A significant increase in the percentage of cells expressing interleukin-2 receptors (IL-2R) was observed after MAb stimulation. The percentage of CD4+ lymphocytes and the CD4/CD8 ratio increased in all the cases studied, after MAb stimulation, where the percentages of B cells and NK cells remained relatively constant at less than 2-3% of the total population. We therefore conclude that murine MAbs administered to patients with cancer can lead to the generation of T cells which can recognise these MAbs as antigens when presented appropriately in vitro. The main proliferating population appears to be T helper CD4+ lymphocytes which following stimulation can release interleukin-2 leading to the expression of high levels of IL-2R.