Spatial and temporal expression of the Dictyostelium discoideum G alpha protein subunit G alpha 2: expression of a dominant negative protein inhibits proper prestalk to stalk differentiation.

Previous results have shown that the G alpha protein subunit G alpha 2 is required for aggregation in Dictyostelium discoideum and is essential for coupling cell-surface cAMP receptors to downstream effectors in vivo during this stage of development. G alpha 2 expresses at least four distinct transcripts that are differentially regulated during development; two of the transcripts are expressed exclusively in the multicellular stages and their expression is restricted to prestalk cells. We partially dissected the G alpha 2 promoter and identified a component that is expressed exclusively during the multicellular stages using luciferase gene fusions. When this promoter region is coupled to lacZ, beta-gal expression is restricted to the multicellular stages and localized in prestalk cells with a pattern similar to that of the ecmA prestalk-specific promoter. We show that expression in wild-type cells of the G alpha 2 mutant protein [G alpha 2(G206T)] during the early stages of development blocks aggregation and cAMP-mediated activation of adenylyl cyclase and guanylyl cyclase, suggesting it functions as a dominant negatively active G alpha subunit. When this mutant G alpha protein is expressed from the ecmA prestalk-specific promoter, abnormal stalk differentiation during culmination is observed. Expression of the mutant G alpha 2 from the SP60 prespore promoter or wild-type G alpha 2 from either the ecmA or the SP60 promoter results in no detectable phenotype. The results suggest that G alpha 2 plays an essential role during the culmination stage in prestalk cells and may mediate cAMP receptor activation of these processes during multicellular development.

Tumor uptake and metabolism of copper-67-labeled monoclonal antibody chCE7 in nude mice bearing neuroblastoma xenografts.

METHODS: ChCE7, an internalizing, neuroblastoma-specific monoclonal antibody (MAb), and its F(ab')2 fragments were derived with the bifunctional ligand 4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl benzoic acid tetrahydrochloride (CPTA) and labeled with the potential therapeutic nuclide 67Cu. After internalization and degradation of these immunoconjugates in SKN-AS human neuroblastoma cells, the terminal degradation product was found to be the lysine adduct of the copper complex. In vivo distributions in nude mice bearing neuroblastoma xenografts were studied and extracts from tumor and tissue samples were analyzed. RESULTS: The intact MAb showed high tumor uptake, stable over 4 days postinjection (33.7% +/- 2.8% ID/g), with tumor/blood ratios increasing from 4.4 on Day 1 to 23.0 on Day 7 postinjection and low levels of radioactivity in other tissues. Analysis of tumor extracts by gel filtration chromatography and high-pressure liquid chromatography (HPLC) showed that over the period of 4 days radioactivity was present both in a high M(r) form, consisting of the MAb/antigen complex, as well as in a low M(r) form, consisting of the copper complex attached to short peptides, including the lys-CPTA complex. There was no evidence of aggregates or MAb/antigen complexes in the blood, radioactivity being exclusively in the form of intact MAb, and radioactivity in the liver was found to consist of intact MAb, MAb fragments and the lys-CPTA metabolite. In the case of the F(ab')2 fragments, high accumulation of radioactivity in the kidneys was observed and analysis of kidney extracts showed it to be due to rapid accumulation of the lys-CPTA complex. When kidney uptake and retention of the CPTA complex as well as of its lysine and glycine adducts was investigated, the lysine complex was taken up more strongly and retained longer in the kidneys than the other compounds. CONCLUSION: Copper-67-labeled MAb chCE7 F(ab')2 fragments were prepared using a novel bifunctional copper ligand 1-(p-aminobenzyl)-1,4,7,10-tetraazacyclodecane-4,7,10-triacetate (DO3A). Compared with MAb-chCE7 F(ab')2 fragments labeled by the CPTA ligand, labels using the DO3A ligand showed improved biodistributions resulting, 48 hr postinjection, in a 4-fold increase in tumor uptake and a 4-fold reduction of radioactivity in the kidneys.

Evaluation of radioiodinated and radiocopper labeled monovalent fragments of monoclonal antibody chCE7 for targeting of neuroblastoma.

Monovalent fragments of antineuroblastoma antibody mAb chCE7 were evaluated for their in vitro and in vivo tumor cell binding properties. Single chain fragments were constructed from the variable region genes cloned from hybridoma cells, expressed in E.coli and purified by metal chelate affinity chromatography. Radioiodinated CE7-scFv fragments were found to bind with high affinity (Kd approximately 10(-9) M) to target cells in vitro but formed aggregates at 37 degrees C, and bound to serum proteins in vitro and in vivo. Circular Dichroism spectra revealed the protein to be in a conformationally altered form and no permanent "refolding" could be achieved. In contrast, chCE7- Fab fragments were found to bind to target tumor cells with similar affinity than the parent mAb chCE7 (Kd approximately 10(-10) M), showed no tendency to aggregate and were stable in serum both in vitro and in vivo. Kinetics of association and dissociation of radioiodinated scFv and Fab fragments were found to be rapid. Radioiodination with the Iodogen method led to impaired immunoreactivity which was found to further increase the off- rates of radioiodinated fragments from tumor cells. Radioiodination with the Bolton-Hunter reagent as well as labeling of chCE7-Fab fragments with 67Cu via the macrocyclic CPTA ligand led to fully immunoreactive Fab fragments. Radioiodinated and radiocopper labeled monovalent CE7 fragments did not internalize into target tumor cells as the parent mAb and its F(ab')2 fragment. A comparison of the biodistribution in tumor bearing nude mice of the radiocopper labeled monovalent, non internalizing Fab fragments with the internalizing divalent F(ab')2 fragments showed in both cases high levels of radioactivity in the kidneys. Concerning tumor uptake, radioactivity from both internalizing and non internalizing fragments remained associated with tumor tissue for longer times than in case of the corresponding radioiodinated fragments. When compared with the radioiodinated forms, tumor uptake of radiocopper-labeled 67Cu-chCE7 and its F(ab')2 fragments was found to be higher. However in the case of the non internalizing 67Cu-chCE7-Fab fragment no increase in the absolute amount of radioactivity in tumor tissue compared with the radioiodinated Fab was observed, indicating an advantage of using radiocopper labeling in conjunction with internalizing antibody fragments for delivering high doses of radioactivity to neuroblastoma.

Anti-neuroblastoma antibody chCE7 binds to an isoform of L1-CAM present in renal carcinoma cells.

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.

A comparison of targetting of neuroblastoma with mIBG and anti L1-CAM antibody mAb chCE7: therapeutic efficacy in a neuroblastoma xenograft model and imaging of neuroblastoma patients.

Iodine-131 labelled anti L1-CAM antibody mAb chCE7 was compared with the effective neuroblastoma-seeking agent (131)I-labelled metaiodobenzylguanidine (MIBG) with regard to (a) its therapeutic efficacy in treating nude mice with neuroblastoma xenografts and (b) its tumour targetting ability in neuroblastoma patients. The SK-N-SH tumour cells used in the mouse experiments show good MIBG uptake and provide a relatively low number of 6,300 binding sites/cell for mAb chCE7. Tumours were treated with single injections of (131)I-MIBG (110 MBq) and with (131)I-labelled mAb chCE7 (17 MBq) and both agents showed antitumour activity. After therapy with (131)I-chCE7, the subcutaneous tumours nearly disappeared; treatment with (131)I-MIBG was somewhat less effective, resulting in a 70% reduction in tumour volume. A calculated tumour regrowth delay of 9 days occurred with a radioactivity dose of 17 MBq of an irrelevant control antibody mAb 35, which does not bind to SK-N-SH cells, compared with a regrowth delay of 34 days with (131)I-mAb chCE7 and of 24 days with (131)I-MIBG. General toxicity appeared to be mild, as assessed by a transient, approximate 10% maximum decrease in body weight during the treatments. The superior growth inhibition achieved by (131)I-chCE7 compared with (131)I-MIBG can be explained by its prolonged retention in the tumours, due to slower normal tissue and plasma clearance. Cross-reaction of mAb chCE7 with L1-CAM present in normal human tissues was investigated by direct binding of radioiodinated mAb to frozen tissue sections. Results showed a strong reaction with normal human brain tissue and weak but detectable binding to normal adult kidney sections. Seven patients with recurrent neuroblastoma were sequentially imaged with (131)I-MIBG and (131)I-chCE7. The results underlined the heterogeneity of neuroblastoma and showed the two imaging modalities to be complementary. (131)I-chCE7 scintigraphy may have clinical utility in detecting metastases which do not accumulate (131)I-MIBG, and the antibody may hold potential for radioimmunotherapy, either by itself or in combination with (131)I-MIBG.

A comparison of targeting of neuroblastoma with mIBG and anti L1-CAM antibody mAb chCE7: therapeutic efficacy in a neuroblastoma xenograft model and imaging of neuroblastoma patients.

Iodine-131 labelled anti L1-CAM antibody mAb chCE7 was compared with the effective neuroblastoma-seeking agent 131I-labelled metaiodobenzylguanidine (MIBG) with regard to (a) its therapeutic efficacy in treating nude mice with neuroblastoma xenografts and (b) its tumour targeting ability in neuroblastoma patients. The SK-N-SH tumour cells used in the mouse experiments show good MIBG uptake and provide a relatively low number of 6,300 binding sites/cell for mAb chCE7. Tumours were treated with single injections of 131I-MIBG (110 MBq) and with 131I-labelled mAb chCE7 (17 MBq) and both agents showed antitumour activity. After therapy with 131I-chCE7, the subcutaneous tumours nearly disappeared; treatment with 131I-MIBG was somewhat less effective, resulting in a 70% reduction in tumour volume. A calculated tumour regrowth delay of 9 days occurred with a radioactivity dose of 17 MBq of an irrelevant control antibody mAb 35, which does not bind to SK-N-SH cells, compared with a regrowth delay of 34 days with 131I-mAb chCE7 and of 24 days with 131I-MIBG. General toxicity appeared to be mild, as assessed by a transient, approximate 10% maximum decrease in body weight during the treatments. The superior growth inhibition achieved by 131I-chCE7 compared with 131I-MIBG can be explained by its prolonged retention in the tumours, due to slower normal tissue and plasma clearance. Cross-reaction of mAb chCE7 with L1-CAM present in normal human tissues was investigated by direct binding of radioiodinated mAb to frozen tissue sections. Results showed a strong reaction with normal human brain tissue and weak but detectable binding to normal adult kidney sections. Seven patients with recurrent neuroblastoma were sequentially imaged with 131I-MIBG and 131I-chCE7. The results underlined the heterogeneity of neuroblastoma and showed the two imaging modalities to be complementary. 131I-chCE7 scintigraphy may have clinical utility in detecting metastases which do not accumulate 131I-MIBG, and the antibody may hold potential for radioimmunotherapy, either by itself or in combination with 131I-MIBG.

The mass of 22Mg.

Mass measurements with a relative precision of better than 1.5 x 10(-8) were performed on 22Mg and its reaction partners 21Na and 22Na with the ISOLTRAP Penning trap mass spectrometer at CERN, yielding the mass excesses D(22Mg)=-399.92(27) keV, D(21Na)=-2184.71(21) keV, and D(22Na)=-5181.56(16) keV. The importance of these results is twofold. First, a comparative half-life (Ft value) has been obtained for the superallowed beta decay of 22Mg to further test the conserved-vector-current hypothesis. Second, the resonance energy for the 21Na proton capture reaction has been independently determined, allowing direct comparisons of observable gamma radiation in nova explosions with the yield expected from models.