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The L1-cell adhesion molecule (L1-CAM) is highly expressed in various cancer types including ovarian carcinoma but is absent from most normal tissue. A chimeric monoclonal antibody, chCE7, specifically binds to human L1-CAM and exhibits anti-proliferative effects on L1-CAM-expressing tumor cells. The goal of this study was to evaluate the efficacy of a novel (177)Lu-chCE7 radioimmunotherapeutic agent and to compare it to a treatment protocol with unlabeled, growth-inhibiting chCE7 in a mouse xenograft model of disseminated ovarian cancer. chCE7agl, an aglycosylated IgG1 variant with improved pharmacokinetics, was conjugated with 1,4,7,10-tetraazacyclododecane-N-N'-N'-N‴-tetraacetic acid (DOTA) and labeled with the low-energy β-emitter (177)Lu. Tumor growth and survival were assessed after a single i.v. dose of 8 MBq (60 μg) radioimmunoconjugate in nude mice bearing either subcutaneous or intraperitoneal SKOV3.ip1 human ovarian cancer tumors. Therapeutic efficacy was compared with three times weekly i.p. administration of 10 mg/kg unconjugated chCE7. In vivo analysis of (177)Lu-chCE7agl biodistribution demonstrated high and specific accumulation of radioactivity at the tumor site with maximal tumor uptake of up to 48.0 ± 8.1% ID/g at 168 h postinjection. A single treatment with (177)Lu-DOTA-chCE7agl caused significant retardation of tumor growth and prolonged median survival from 33 to 71 days, while administration of a nontargeted (177)Lu-immunoconjugate had no beneficial effect. Three times weekly i.p. application of unlabeled chCE7 10 mg/kg similarly increased survival from 44 to 72 days. We conclude that a single dose of (177)Lu-DOTA-chCE7agl is as effective as repeated administration of nonradioactive chCE7 for treatment of small intraperitoneal tumors expressing L1-CAM.

Immunoprecipitation after cell surface labeling of human neuroblastoma cells showed that the anti-neuroblastoma monoclonal antibody (mAb) chCE7 binds to a 200,000 M(r) cell surface protein. The protein was partially purified by immuno-affinity chromatography from a human renal carcinoma and a human neuroblastoma cell line, which both showed high levels of binding of MAb chCE7. NH(2)-terminal sequences of 18 and 15 amino acid residues were determined. Both sequences isolated from the renal carcinoma and the neuroblastoma cells showed strong homology to human cell adhesion molecule L1 (L1-CAM), and both were characterized by the NH(2)-terminal deletion of 5 amino acids, comprising exon 2 of L1-CAM. Reverse trancription-polymerase chain reaction (RT-PCR) analysis of the regions spanning exon 2 and exon 27 of L1-CAM indicated that in neuroblastoma cells both transcripts for the full-length and exon-deleted forms are present, whereas in the renal carcinoma cell lines only the exon-deleted L1-CAM isoform were detected. Western blot analysis showed that 6 of 7 tested renal carcinoma cell lines and 5 of 15 renal carcinoma tissues expressed L1-CAM. In normal adult kidney tissue, very low levels of protein expression were found. Northern blot analysis confirmed that in renal carcinoma and neuroblastoma cell lines L1-CAM mRNA levels are correlated with protein expression.

Internalization and cellular degradation of a chimeric monoclonal anti-neuroblastoma antibody (MAb chCE7) is described. Immunofluorescence localization showed temperature-dependent redistribution of MAb chCE7 from the cell surface at 0 degrees C to vesicular structures after heating to 37 degrees C. SKN-AS cells which were pulse-labelled at 0 degrees C with radioiodinated chCE7 released 50% of the initial cell-bound radioactivity into the medium after 20 hr at 37 degrees C. Low-molecular-weight radioactivity in the medium was identified as iodotyrosine and the major intracellular degradation product was found to be an antibody fragment of 43 kDa. Degradation was blocked by chloroquine, an inhibitor of lysosomal function. MAb chCE7 binds to a carbohydrate epitope, as treatment with tunicamycin abolishes cell binding. In adherent cells in culture, inhibition of protein synthesis by cycloheximide affected cell-surface binding sites for chCE7 in a biphasic manner, with an initial increase followed by long-term decrease. Expression of binding sites was also found to be inhibited by sodium azide, by the lysosomotropic drug chloroquine and by Brefeldin A, a drug which prevents export of newly synthetized proteins to the cell surface. When cells were treated with high doses of chCE7 up to 24 hr and cell-surface binding sites were then measured after an acidic buffer wash, no loss of surface binding sites was found, indicating that pretreatment with MAb chCE7 does not induce down-regulation of its binding sites.

Hybridoma CE7 produces a murine antibody (gamma 1/kappa) which binds to a 190-kDa cell-surface glycoprotein of human neuroblastoma. Because of its tumor specificity, it has been used routinely in clinical pathology to confirm diagnosis of neuroblastoma. We have isolated the gene segments coding for the variable regions of the immunoglobulin H and L chain of this hybridoma. These V genes were used to construct mouse/human chimeric H and L chain genes (gamma 1/kappa) which were then expressed in SP2/0 cells. A cell-binding inhibition assay showed that the specificity of the chimeric CE7 antibody (chCE7) is identical to that of the original CE7. Radioiodinated chCE7 binds to approximately 43,000 sites per neuroblastoma cell with an affinity of 10(10) M-1. In neuroblastoma-bearing nude mice, biodistribution studies with [125I]chCE7 were performed and tumor accumulations of up to 32% of injected dose/g tissue together with low blood and organ uptake were found.

One of the major limitations of using intact immunoglobulins for targeting tumors is the poor penetration into tissues. Although small single chain Fv (scFv) antibody fragments have been used because of their improved kinetics, some exhibit high renal accumulation. In this study we show a significant reduction of renal accumulation of His-tagged scFv's directly labelled with 99mTc-tricarbonyl by use of succinic anhydride. Further decrease of renal uptake will make this approach hopefully suitable for therapeutic application, using 188/186Re(I)-tricarbonyl.

Because of its suitable decay characteristics and favourable chemical behaviour, 67Cu is considered to have potential for application in radionuclide therapy. This has led to increasing interest in biochemical, preclinical and clinical studies with 67Cu-labelled radiopharmaceuticals. 67Cu can be produced in accelerators by bombarding a natZn- (1) or 68Zn target with protons (2) or by bombarding 67Zn with neutrons in a high flux reactor (3). At the present time we produce up to 3.7 Gbq of 67Cu at the 72 MeV accelerator of the Paul Scherrer Institute by irradiating natZn. In order to scale up the 67Cu production, we evaluated the use of 68Zn and 70Zn. Cross sections were measured using the reactions 65Cu(p, n)65Zn of monitor foils (4), natZn(p, 2pxn)67Cu in the p-energy-range 67 to 12 MeV, 68Zn(p, 2p)67Cu in the p-energy range 67 to 45MeV and 70Zn(p, 2p2n)67Cu (5) in the p-energy range 45 to 12 MeV. Results indicate that the use of 68Zn as target instead of natZn leads to a 4.5 — fold increase in 67Cu production and that the use of 70Zn leads to a 10–20-fold increase in the 20–30 MeV energy range.

Growth of the human mammary tumor cell line ZR-75-1 is stimulated by epidermal growth factor (EGF) and alpha-type transforming growth factor (alpha TGF), as well as by estradiol (E2). The role of activation of S6 kinase and S6 phosphorylation in the EGF(alpha TGF)-induced and E2-induced growth was investigated. Maximal effects on growth are observed at 10 nM EGF or alpha TGF. EGF as well as alpha TGF treatment of serum-starved cells leads to rapid activation of S6 kinase; the activity is increased about tenfold after 30 min of EGF treatment and declines with the time reaching about 25% of the maximal activity after 2 h of EGF treatment. Similar to the growth response, S6 kinase is activated at lower doses of EGF than alpha TGF and shows a maximal response at 10 nM for both growth factors. In contrast to this finding the incubation of serum-starved cells with E2 over a concentration range between 1 pM and 10 nM and times from 30 min to 4 h does not lead to increased S6 kinase activity. On investigating whether this lack of response to E2 is due to desensitization of the system by induction of alpha TGF it was found that preincubation of cells with alpha TGF for 2-6 h desensitizes them to reactivation of S6 kinase by alpha TGF, whereas preincubation with E2 does not. When S6 phosphorylation is monitored over times from 1 h to 6 h, it is observed that EGF leads to increased S6 phosphorylation, whereas E2 does not. The rate of onset of protein synthesis in the first 2 h of stimulation, when EGF-induced S6 phosphorylation is maximal, is more rapid with EGF than with E2. The results suggest that different pathway are involved in E2-induced and EGF(alpha TGF)-induced proliferation.