RESUMEN
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.
Asunto(s)
3-Yodobencilguanidina/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Antígenos de Superficie/inmunología , Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/terapia , Glicoproteínas de Membrana/inmunología , Moléculas de Adhesión de Célula Nerviosa/inmunología , Neuroblastoma/diagnóstico por imagen , Neuroblastoma/terapia , Radiofármacos/uso terapéutico , Animales , Niño , Preescolar , Femenino , Humanos , Interpretación de Imagen Asistida por Computador , Lactante , Complejo de Antígeno L1 de Leucocito , Masculino , Ratones , Ratones Desnudos , Recurrencia Local de Neoplasia , Trasplante de Neoplasias , Cintigrafía , Trasplante Heterólogo , Células Tumorales Cultivadas , Recuento Corporal TotalRESUMEN
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.
RESUMEN
Radioimmunotherapy of cancer utilizes anti-tumor antibodies or antibody fragments conjugated to radionuclides to deliver radiation selectively to tumors. However, radiolabeled proteins deposit radioactivity in normal organs that metabolize or conserve proteins and peptides, primarily liver and kidneys. To accelerate the clearance of radioactivity from normal tissues, linkers between the antibody or antibody fragment and the radioactive moiety have been designed for cleavage in the liver and kidneys, to liberate low molecular weight radioactive species for rapid excretion. Modest success in improving the tumor-to-liver and tumor-to-kidney radiation dose ratios have been achieved in preclinical studies. Such changes when taken to clinical studies have suggested useful impact on therapeutic work. Recent advances in the development of cleavable linkers are described.
Asunto(s)
Inmunoconjugados/farmacocinética , Neoplasias/terapia , Radioinmunoterapia , Radiofármacos/farmacocinética , Animales , Reactivos de Enlaces Cruzados , Humanos , Inmunoconjugados/química , Riñón/metabolismo , Hígado/metabolismo , Tasa de Depuración Metabólica , Radiofármacos/químicaRESUMEN
In order to determine if tumor/nontarget tissue ratios of 67Cu-labeled antibody fragments can be improved, modifying the DO3A copper chelate with tripeptide linkers was investigated. The peptide-linked chelates 1,4,7,10-tetraazacyclodecane-N,N',N",N"'-tetraacetate (DOTA)-triglycyl-L-p-isothiocyanato-phenylalanine (DOTA-R1-NCS), DOTA-glycyl-phenylalanyl-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R2-NCS), DOTA-glycyl-prolyl-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R3-NCS) and DOTA-glycyl-L-p-isothiocyanato-phenylalanine (DOTA-R4-NCS) were synthesized and coupled to F(ab')2 fragments of anti-colon carcinoma mAb35. In vitro, the 67Cu-labeled antibody fragments were fully immunoreactive and stable in human serum. In vivo in nude mice bearing human colon carcinoma xenografts the conjugates R1 and R3 showed improved tumor uptake and lower levels of radioactivity in the liver compared with the other conjugates. Biodistributions of the DOTA-R2-F(ab')2 showed at early time points after injection higher levels of radioactivity in the liver, lower levels of activity persisting in the blood and lower accumulation of activity in the tumor. When liver homogenates were analyzed 30 min post injection by SDS-PAGE or FPLC gel chromatography, it was found that radioactivity was released more slowly from the triglycine (R1)-F(ab')2 than from the immunoconjugates with the R2 or the R4 linker. The main radioactive metabolites were protein bands at 66 kD, 31 kD and low molecular weight fragments. The results show that the rate of cleavage of the copper complex from F(ab')2 fragments in vivo can be influenced by the amino acid sequence close to the complex, with significant consequences on biodistributions.
Asunto(s)
Neoplasias del Colon/metabolismo , Radioisótopos de Cobre/farmacocinética , Inmunoconjugados/farmacocinética , Fragmentos Fab de Inmunoglobulinas/inmunología , Hígado/metabolismo , Oligopéptidos/farmacocinética , Animales , Anticuerpos Antineoplásicos/inmunología , Cromatografía Líquida de Alta Presión , Neoplasias del Colon/radioterapia , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Marcaje Isotópico , Ratones , Ratones Desnudos , Radioinmunoterapia , Trasplante HeterólogoRESUMEN
Immunopotentiating reconstituted influenza virosomes (IRIV) are 150-nm proteoliposomes composed of influenza surface glycoproteins and a mixture of natural and synthetic phospholipids. Due to size, structure and composition of the IRIVs, they serve as an antigen carrier system for efficacious vaccination, as was demonstrated for hepatitis A and influenza. This paper reviews the unique properties of IRIVs and describes the in vivo biodistribution of model antigens using 14C-labeled IRIVs and 125I-labeled streptavidin. IRIV formulated streptavidin induced a strong depot effect after intra muscular (i.m.) vaccination of mice, whereas soluble streptavidin was soon eliminated via the kidney of the animals. A mixture of antigen and IRIVs yielded higher antibody titers after i.m. inoculation than streptavidin alone. The highest immunostimulation was achieved by the binding of the antigen to the investigated adjuvant. The potential penetration of inactivated hepatitis A virions into lipid membranes was assessed by measuring the area increase of a lipid monolayer kept at a constant surface pressure corresponding to that of lipid bilayer vesicles. The monolayers were composed of phosphatidylcholine (POPC) and phosphatidylethanolamine (POPE) (75/25 mol/mol), thus resembling the lipid composition of the IRIV. The results suggested that the hepatitis A antigen may spontaneously bind to the reconstituted IRIV membranes.
Asunto(s)
Adyuvantes Inmunológicos/farmacocinética , Antígenos Virales/metabolismo , Hepatitis A/inmunología , Vacunas contra la Influenza/farmacocinética , Estreptavidina/farmacocinética , Vacunas contra Hepatitis Viral/farmacocinética , Absorción , Animales , Formación de Anticuerpos , Radioisótopos de Carbono , Femenino , Inyecciones Intramusculares , Radioisótopos de Yodo , Riñón/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Músculo Esquelético/metabolismo , Bazo/metabolismoRESUMEN
5-Bromo-2'-deoxyuridine (BrUdR) labeled with 77Br and 76Br was compared with 5-iodo-2'-deoxyuridine (IUdR) labeled with 125I or 131I, first in vitro then in in vivo experiments in mice. The results showed a significantly higher incorporation of BrUdR into DNA than IUdR, which can be explained by the greater similarity (size and surface hydrophilicity of the molecules) of BrUdR to thymidine. Both tracers are dehalogenated quickly in vivo but not in vitro. Free bromide is excreted more slowly than iodide, resulting in a higher background activity level after the application of [76Br]BrUdR and compensates for the favorable DNA incorporation. 76Br has more favorable properties than 124I for imaging purposes with positron emission tomography (PET) because of a very convenient half-life (16 h vs. 4.15 days) and about double the positron yield per decay. However, the more favorable physical properties are balanced by the slower excretion and thus the estimated radiation dose is higher in the case of 76Br than 124I. Thus, both tracers, [124I]IUdR and [76Br]BrUdR are potentially suitable but not optimal to measure cell proliferation in vivo. The difference between the two tracers is small and the extrapolation from mice to human difficult, and thus it cannot be concluded if one of the tracers would be better than the other for imaging of cancer patients.
Asunto(s)
Radioisótopos de Bromo/farmacocinética , Bromodesoxiuridina/farmacocinética , Tomografía Computarizada de Emisión , Animales , División Celular , Ciclotrones , Semivida , Humanos , Idoxuridina/farmacocinética , Radioisótopos de Yodo/farmacocinética , Ratones , Distribución TisularRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Neoplasias Renales/química , Glicoproteínas de Membrana/análisis , Proteínas de Neoplasias/análisis , Moléculas de Adhesión de Célula Nerviosa/análisis , Neuroblastoma/química , Animales , Células CHO , Cricetinae , Humanos , Riñón/química , Complejo de Antígeno L1 de Leucocito , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/inmunología , Pruebas de Precipitina , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
The peptide-linked copper chelators CPTA-triglycyl-L-p-isothiocyanato-phenylalanine (CPTA-R1-NCS) as well as DOTA-triglycyl-L-p-isocyanato-phenylalanine (DOTA-R1-NCS) were synthesized and coupled to F(ab')2 fragments of the anti-neuroblastoma monoclonal antibody (MAb) chCE7. 67Cu-labeled conjugates were compared with the original CPTA- and DO3A-F(ab')2 in vitro and in vivo in mice bearing neuroblastoma xenografts. With the CPTA-R1-F(ab')2, biodistributions were improved, because radioactivity present in the kidney was reduced. With the DOTA-R1-F(ab')2, clearance from the blood was slower and tumor uptake was higher compared with the other conjugates. DOTA-R1-F(ab')2 achieved the best tumor/tissue ratios.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Neoplasias Encefálicas/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Neuroblastoma/metabolismo , Oligopéptidos/química , Radiofármacos/farmacocinética , Animales , Anticuerpos Monoclonales/química , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Radioisótopos de Cobre , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Ligandos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Radiofármacos/química , Espectrofotometría Ultravioleta , Distribución TisularRESUMEN
The pentadentate H3bhci [1,3,5-trideoxy-1, 3-bis((2-hydroxybenzyl)amino)-cis-inositol] and its bifunctionalized analogue H3bhci-glu-H [1,3,5-trideoxy-1, 3-bis((2-hydroxybenzyl)amino)-5-glutaramido-cis-inositol] were synthesized, and their coordination chemistry was investigated with inactive rhenium, with no carrier added Re-188 and with carrier added Re-186. The neutral Re(V) complexes [ReO(bhci)] and [ReO(bhci-glu-H)] are formed in good yields starting from [ReOCl3(P(C6H5)3)2] or in quantitative yield directly from [186/188ReO4]- in aqueous solution by reduction with Sn(II) or Sn(0). The X-ray structures of [ReO(bhci)] and [ReO(bhci-glu-H)] were elucidated revealing pentadentate "side on" coordination of the ligands to the "Re=O" core. The basic cyclohexane frame adopts a chair form in the case of [ReO(bhci)] and a twisted boat form in the case of [ReO(bhci-glu-H)]. [ReO(bhci)] crystallizes in the monoclinic space group C2/c with a = 27.425(3), b = 14.185(1), c = 19.047(2) A, and beta = 103.64(2) degrees and [ReO(bhci-glu-H)] in the monoclinic space group P21/c with a = 13.056(3), b = 10.180(1), c = 22.378(5) A, and beta = 98.205(9) degrees. Both 188Re complexes are stable in human serum for at least 3 days without decomposition. After injection into mice, [ReO(bhci-glu)]- is readily excreted through the intestines, while [ReO(bhci)] is excreted by intestines, liver, and the kidneys. TLC investigations of the urine showed exclusively the complexes [ReO(bhci-glu-H)] and [ReO(bhci)], respectively, and no decomposition products. For derivatization of antibodies, the carboxylic group of [ReO(bhci-glu-H)] was activated with N-hydroxysuccinimide, which required unusually vigorous reaction conditions (heating). The anti colon cancer antibody mAb-35 [IgG and F(ab')2 fragment] was labeled with [186/188ReO(bhci-glu)] to a specific activity of up to 1.5 mCi/mg (55 MBq/mg) with full retention of immunoreactivity. Labeling yields followed pseudo-first-order kinetics in antibody concentration with the ratio of rates between aminolysis and hydrolysis being about 2. Biodistributions of 186Re-labeled intact mAb-35 as well as of its F(ab')2 fragment in tumor-bearing nude mice revealed good uptake by the tumor with only low accumulation of radioactivity in normal tissue.
Asunto(s)
Inositol/análogos & derivados , Proteínas/química , Renio/química , Animales , Anticuerpos Monoclonales/química , Antígeno Carcinoembrionario/química , Antígeno Carcinoembrionario/inmunología , Cristalografía por Rayos X , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/metabolismo , Inositol/sangre , Inositol/química , Ligandos , Ratones , Ratones Desnudos , Conformación Proteica , Radioisótopos , Distribución Tisular , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales , Radioisótopos de Cobre , Fragmentos Fab de Inmunoglobulinas , Radioisótopos de Yodo , Neuroblastoma/diagnóstico por imagen , Radioinmunodetección , Animales , Humanos , Fragmentos de Inmunoglobulinas , RatonesRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Compuestos Aza , Benzoatos , Radioisótopos de Cobre/farmacocinética , Neuroblastoma/metabolismo , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Humanos , Ratones , Ratones Desnudos , Proteínas de Neoplasias/inmunología , Trasplante de Neoplasias , Neuroblastoma/inmunología , Distribución TisularRESUMEN
Four different methods of radiolabelling the anti-granulocyte monoclonal antibody MAb47 were compared and their influence on diagnostic value studied. The best clinical images were obtained following labelling with iodine-123 by the Iodogen method and direct labelling with technetium-99m after tris-(carboxyethyl)-phosphine treatment of MAb47 to achieve disulphide bridge reduction. 99mTc labelling using a specific ligand (MAb47-mtp), or a second method involving direct reduction with mercaptoethanol, led to an increased background activity in clinical studies, thus impeding the diagnosis of chronic disease. Fresh infections were clearly localized by all four preparations. The elimination of the activity from the blood was slower in the case of the iodinated MAb47, while the collected urine samples showed an excretion of about 10% of the injected activity per day independent of the labelling method. The results in terms of sensitivity and specificity were rather similar for all labelling methods and ranged from 90% to 99%.
Asunto(s)
Anticuerpos Monoclonales , Granulocitos/inmunología , Inflamación/diagnóstico por imagen , Radioisótopos de Yodo , Tecnecio , Absceso/diagnóstico por imagen , Adulto , Anciano , Anticuerpos Monoclonales/farmacocinética , Aracnoiditis/diagnóstico por imagen , Humanos , Marcaje Isotópico/métodos , Masculino , Persona de Mediana Edad , Infecciones Relacionadas con Prótesis/diagnóstico por imagen , Cintigrafía , Juego de Reactivos para Diagnóstico , Sensibilidad y EspecificidadRESUMEN
Monoclonal antibody chCE7, an internalizing neuroblastoma-specific chimeric antibody, was derivatized with the macrocyclic amine ligand 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid tetrahydrochloride and labeled with the potential therapeutic nuclide 67Cu. Using pulse labeling and an acid elution endocytosis assay, 67Cu-chCE7 was found to be internalized into human neuroblastoma (SKN-AS) cells at a similar rate and to a similar extent as 125I-labeled chCE7. Uptake of 67Cu-chCE7 and 125I-chCE7 into the acid stable (intracellular) pool proceeded with similar kinetics during the first 2 h of internalization. However, in contrast to 125I-chCE7-loaded cells, at later times intracellular radioactivity kept increasing in the case of 67Cu-chCE7-loaded cells. It was shown that this effect is due to the intracellular accumulation of a low M(r) degradation product consisting of the 67Cu-4[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid complex, possibly with a short peptide attached to it. Degradation of both 125I-chCE7 and 67Cu-chCE7 was inhibited by chloroquine, indicating endosomal or lysosomal degradation, and a 43,000 M(r) fragment was found to be the major high M(r) degradation product in both cases. Although at times between 4 and 6 h of internalization intracellular breakdown of 67Cu-chCE7 was found to proceed more slowly, the major difference between the two immunoconjugates resides in the prolonged cellular retention of the 67Cu-chCE7 metabolite.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Radioisótopos de Cobre/metabolismo , Inmunoconjugados/metabolismo , Neuroblastoma/inmunología , Anticuerpos Monoclonales/administración & dosificación , Radioisótopos de Cobre/administración & dosificación , Endocitosis , Humanos , Inmunoconjugados/uso terapéutico , Neuroblastoma/metabolismo , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Neuroblastoma/metabolismo , Antígenos de Neoplasias/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Radioisótopos de Yodo/metabolismo , Neuroblastoma/inmunología , TemperaturaRESUMEN
The anti-carcinoembryonic antigen murine monoclonal antibody MAb35 and its F(ab')2 fragment were labeled with 131I or the potential therapeutic nuclide 67Cu. In vivo distribution patterns were compared in nude mice bearing human tumor xenografts by coinjection of the 131I- and 67Cu-labeled materials, thereby minimizing variations due to xenograft and host animal. The results showed that the 67Cu-labeled intact MAb35 achieved twice the percentage of injected dose/g tumor when compared to its 131I-labeled counterpart, without significant impairment of the wholebody distribution pattern. However, this effect was not evident in the case of F(ab')2, where high uptake of 67Cu was found in the kidney without any enhancement of accumulation in the target xenografts. To investigate the underlying causes of the different distribution patterns observed, iodine labeling was also performed using a more stable linkage, and the results indicated that the observed differences cannot be explained by simple deiodination of conventionally labeled preparations. We conclude that the intact form of the 67Cu-labeled antibody may be superior to the F(ab')2 fragment for use in our intended clinical studies. Our continuing work on the processing of radiometal-labeled F(ab')2 fragments, at the systemic and cellular level, will hopefully lead to a strategy to circumvent the problem of high kidney accumulation.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Neoplasias del Colon/inmunología , Radioisótopos de Cobre/metabolismo , Radioinmunoterapia/métodos , Animales , Anticuerpos Monoclonales/uso terapéutico , Neoplasias del Colon/radioterapia , Radioisótopos de Cobre/uso terapéutico , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Radioisótopos de Yodo/metabolismo , Ratones , Trasplante de Neoplasias , Distribución Tisular , Trasplante HeterólogoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos , Secuencia de Bases , Sitios de Unión , Complemento C3/metabolismo , Complemento C4/metabolismo , Sondas de ADN , Biblioteca Genómica , Humanos , Cadenas J de Inmunoglobulina/genética , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Neuroblastoma/inmunología , Transfección , Células Tumorales CultivadasRESUMEN
The labelling of the D1 antagonist SCH 23982 with 123I was studied in detail by following the nucleophilic and electrophilic approaches and the reaction conditions were optimized. The product was purified by reversed phase HPLC with a phosphoric acid/EtOH mixture which simply has to be neutralized and diluted before injection. Its binding was tested in vitro with rat striatal membranes proving the high affinity to D1 and very low affinity to D2 receptors.
Asunto(s)
Benzazepinas/análogos & derivados , Antagonistas de Dopamina , Radioisótopos de Yodo , Marcaje Isotópico/métodosRESUMEN
This study was performed to evaluate the tumor targeting ability of chCE7 with a view to clinical applications in neuroblastoma imaging and therapy. A chimeric (mouse/human) monoclonal antibody (chCE7) of gamma 1/kappa isotype directed against a neuroblastoma-associated cell-surface glycoprotein is described. In vitro chCE7 binds with high affinity (KD approximately 1 x 10(-10) M) to SKN-AS human neuroblastoma cells. Binding studies with 125I-labeled chCE7 show temperature-dependent modulation of antigen binding and indicate that a proportion of the bound antibody is internalized due to rapid antigen turnover. In vivo biodistribution of radioiodinated chCE7 in nude mice bearing SKN-AS tumors shows optimal tumor uptake after 24 hr with about 30% of the injected dose per g. Optimal tumor/blood ratios (3.4:1) are reached after 4-5 days. Uptake in other organs including the reticuloendothelial system is low with tumor/organ ratios of 10 and more. Tumor uptake of chCE7 and the parent murine CE7 are found to be similar. Stability of chCE7 during and after radiolabeling is good with no loss of immunoreactivity in preparations labeled with 123I up to 100 mCi/mg and 80% immunoreactivity after labeling with 13 mCi/mg of 131I. Neuroblastoma xenografts can be imaged by radioimmunoscintigraphy with 123I- and and 131I-labeled chCE7.
Asunto(s)
Anticuerpos Monoclonales , Neuroblastoma/diagnóstico por imagen , Radioinmunodetección/métodos , Animales , Anticuerpos Monoclonales/metabolismo , Femenino , Humanos , Inmunotoxinas/metabolismo , Radioisótopos de Yodo , Ratones , Trasplante de Neoplasias , Neuroblastoma/metabolismo , Temperatura , Distribución Tisular , Trasplante Heterólogo , Células Tumorales Cultivadas/diagnóstico por imagen , Células Tumorales Cultivadas/metabolismoRESUMEN
The high kinetic stability of the Cu2+ complex of the chelator 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl]benzoic acid was demonstrated at physiological pH as well as under acidic conditions. The chelating agent was conjugated to AB35, a monoclonal antibody directed against CEA, without a significant loss of immunoreactivity. The conjugate could, under optimal labeling conditions, be labeled with 67Cu in acetate buffer with a full occupancy of ligands within 20 min. This radiolabeled conjugate showed no transfer of radiocopper to serum proteins in human serum over 7 days. The biodistribution in tumor-bearing mice was measured and compared to that of iodinated AB35. Tumor uptake was high with 15 +/- 3% ID (injected dose)/g after 24 h and 32 +/- 7% ID/g after 96 h for the 67Cu-labeled antibody and 13 +/- 4% ID/g after 24 h and 14 +/- 2% ID/g after 96 h for the 125I-labeled antibody. Whereas radioactivity in normal organs decreased with time after 24 h, increased residence time was shown up to 4 days with the 67Cu-labeled AB35.
Asunto(s)
Anticuerpos Monoclonales , Antígeno Carcinoembrionario/inmunología , Quelantes , Radioisótopos de Cobre , Marcaje Isotópico , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Benzoatos/química , Sitios de Unión , Sangre , Proteínas Sanguíneas/metabolismo , Quelantes/química , Estabilidad de Medicamentos , Compuestos Heterocíclicos/química , Ratones , Ratones Desnudos , Neoplasias Experimentales/metabolismo , Soluciones , Distribución TisularRESUMEN
The expression of a nonspecific cross-reacting antigen (NCA) species on the cell surface of the human promyelocytic leukemia cell line HL-60 was investigated via binding of 125I-labeled carcinoembryonic antigen (CEA) and NCA-specific monoclonal antibodies (Mabs). Very low specific binding of the CEA-specific Mab35 was found, whereas the CEA- and NCA-recognizing Mab47 showed 20-fold higher binding. The number of binding sites for Mab47 on HL-60 cells is lower than on normal granulocytes and is modulated by inducers of cellular differentiation and growth. Dimethylsulfoxide (DMSO), an inducer of neutrophilic differentiation, increased Mab47 binding in a time-dependent manner up to 4-fold after 7 days. In contrast, phorbol-12-myristate-13-acetate which induces differentiation into monocyte/macrophages led to a loss of binding sites. Mab47 binding was also decreased by granulocyte-macrophage colony-stimulating factor and this effect was enhanced in the presence of DMSO during the first 3 days of DMSO treatment. It is concluded that agents affecting neutrophilic differentiation or cell growth act in an opposite manner on NCA expression of HL-60 cells. NCA expression is not crucial for neutrophilic differentiation because it can be suppressed by granulocyte-macrophage colony-stimulating factor early in the differentiation program without affecting cell maturation.