RESUMO
Sticholysin I (StI), a potent cytolysin isolated from the sea anemone Stichodactyla helianthus, was linked to the monoclonal antibody (mAb) ior C5. StI acts by forming hydrophilic pores in the membrane of the attacked cells leading to osmotic lysis. ior C5 is a murine IgG1, which recognizes the tumor associated antigen (TAA) ior C2. The cytolysin and the mAb were coupled by using the heterobifunctional cross-linking reagent sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). Two hybrid molecules composed by one ior C5 and one or two StI molecules were obtained (named conjugated I and II, respectively). The purified conjugates were evaluated by a binding affinity assay against an ior C2-positive colon cancer cell line (SW948). Both molecules were able to recognize the antigen (Ag) in the same way that unconjugated ior C5 does. The activity of both conjugates against human erythrocytes and SW948 cells was assessed. They lost most of their hemolytic activity but their residual activity was very similar. Nevertheless, when their cytotoxicity was studied on the SW948 cell line, only conjugate II killed efficiently the cells, indicating a specific mAb-Ag interaction. In this chimeric molecule the ratio between the cytotoxic and the hemolytic activity was larger than that of the free cytolysin. This fact indicates an increase of the specificity of the toxic effect toward the SW948 cell line and consequently an increase of the difference between its hemolytic and cytotoxic doses. The results herein support the feasibility of directing StI to the surface of cancer cells expressing ior C2 Ag via the mAb ior C5.
Assuntos
Adenocarcinoma/tratamento farmacológico , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Neoplasias do Colo/tratamento farmacológico , Proteínas Hemolisinas/química , Proteínas Hemolisinas/farmacologia , Imunotoxinas/farmacologia , Porinas/química , Porinas/farmacologia , Adenocarcinoma/patologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Proteínas Hemolisinas/isolamento & purificação , Hemólise/efeitos dos fármacos , Humanos , Indicadores e Reagentes , Camundongos , Camundongos Endogâmicos BALB C , Compostos Orgânicos , Baço/citologia , Baço/efeitos dos fármacosRESUMO
Factors affecting the successful therapy of malignant diseases include the antibody dose used and the schedule of administration, the half-life and fast blood clearance of the antibodies, the presence of circulating antigen, poor tumor penetration of the high/molecular-weight monoclonal antibody (mAb) and the way in which these molecules are catabolized. To circumvent these limitations and achieve higher uptake, increased tumor penetration, faster blood clearance and longer retention in the tumors, there is a need to generate mAbs suitable for diagnosis as well as therapy and to develop novel strategies to increase the efficacy of immunotherapeutic treatments. There is a lack of knowledge about many aspects of the physiological function and metabolism of antibodies. This paper is intended to discuss factors that affect the pharmacokinetics of mAbs in human subjects with the purpose of forming possible strategies to optimize this approach for tumor diagnosis and therapy.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Animais , Previsões , Meia-Vida , Humanos , Neoplasias/diagnóstico , Neoplasias/terapiaRESUMO
Monoclonal Antibody (mAb) ior C5 is a murine IgG(1) that recognizes the tumor associated antigen (TAA) ior C2, a cell surface O-linked glycoprotein carbohydrate chain not present in most normal tissues and homogeneously expressed in the cytoplasm of normal colon epithelium and heterogeneously expressed in more than 83% of primary colorectal carcinomas. This study was designed to investigate the pharmacokinetics, biodistribution and the absorbed radiation doses of (99m)Tc-labeled mAb ior C5 antibody in colorectal tumor patients. Ten patients were administered 3 mg of anti-O-linked glycoprotein carbohydrate chain TAA ior C2 murine monoclonal antibody ior C5 radiolabeled with (99m)Tc activity of 1435.0 +/- 123 MBq by intravenous (i.v.) bolus infusion. Blood and urine samples were collected from 4 out of 10 patients at timed intervals from 10 min and up to 24 h after injection of the (99m)Tc-labeled mAb ior C5 for pharmacokinetic studies. Whole body images were taken in 5 out of 10 patients for quantitative normal organ biodistribution and dosimetry studies and planar anterior and posterior and SPECT images were taken in 5 out of 10 patients for tumor localization. Mean absorbed doses were estimated using the methods developed by the Medical Internal Radiation Dose (MIRD) committee. The effective dose equivalent (EDE) and effective dose (ED) were calculated as prescribed in International Commission on Radiological Protection (ICRP) publications 30 and 60. Plasma disappearance curves of (99m)Tc-labeled murine antibody ior C5 were best fit by a two-compartment model in all patients with (t(1/2alpha)) of 4.32 +/- 2.18 h and (t(1/2beta) of 32.6 +/- 3.82 h. Among the main target organs, accumulation of the radiolabeled antibody was found in liver (9.38 +/- 0.80%), heart (8.92 +/- 0.94%) and spleen (1.37 +/- 0.30%) at 5 min post-administration. These values were reduced at 24 h to (5.91 +/- 0.73%) and (0.62 +/- 0.22%), respectively, for the heart and spleen and increased to (9.78 +/- 1.99%) for liver. Estimates of radiation absorbed dose to normal organs in rad/mCi administered were: whole body, 0.0181 +/- 0.0017; heart wall, 0.0768 +/- 0.0090; kidneys, 0.0530 +/- 0.0260; liver, 0.0565 +/- 0.0109 and spleen, 0.0540 +/- 0.0128. The effective dose equivalent and effective dose estimates for adults were 0.0314 +/- 0.0031 and 0.0249 +/- 0.0027 rem/mCi administered. This feasibility study indicates that the O-linked glycoprotein carbohydrate chain TAA ior C2 is expressed in primary and metastatic colorectal carcinomas and shows very limited expression in normal adult tissues. The very good pattern of biodistribution of (99m)Tc-labeled mAb ior C5 in patients will allow imaging of colorectal carcinoma lesions.
Assuntos
Anticorpos Monoclonais/farmacocinética , Neoplasias Colorretais/diagnóstico , Complemento C5/farmacocinética , Dosagem Radioterapêutica , Tecnécio/farmacocinética , Distribuição Tecidual , Idoso , Animais , Anticorpos Monoclonais/administração & dosagem , Antígenos Glicosídicos Associados a Tumores/química , Antígenos Glicosídicos Associados a Tumores/genética , Complemento C5/administração & dosagem , Cuba , Estudos de Viabilidade , Feminino , Meia-Vida , Corpo Humano , Humanos , Injeções Intravenosas , Masculino , Camundongos , Pessoa de Meia-Idade , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/sangue , Compostos Radiofarmacêuticos/urina , Tecnécio/administração & dosagemRESUMO
The use of antibodies as targeting agents for the delivery of radioisotopes to tumors is an appealing concept that has received widespread attention since the advent of monoclonal antibody (mAb) technology. The present study describes the (188)Re-direct labeling of anti-epidermal growth factor receptor (EGF-R) humanized mAb h-R3; the analytical methods for quality control of radiopharmaceuticals such as instant thin layer chromatography-silica gel (ITLC-SG); the immunoreactivity and biological recognition of the target antigen assessment of the radiolabeled molecule using flow cytometry analysis; in vitro stability studies using saline 0.9% solution, cysteine, diethylenetriaminepentaacetic acid (DTPA), human serum and human serum albumin (HSA) 1% challenge; and the assessment of in vivo stability through biodistribution studies in normal Balb/c mice. No fragmentation of the reduced molecules was found using 2-ME as a reducing agent. Labeling efficiency was greater than 98.5 +/- 0.6% of rhenium-188 (188Re) bound to IgG1 after 5 h, as determined by paper chromatography in saline 0.9% solution. Radiocolloids determined by albumin impregnated ITLC was 1.04 +/- 0.07% in all cases. The biological activity measured by flow cytometry analysis showed an immunoreactivity fraction and the biological recognition of the target antigen overexpressed on H-125 human lung adenocarcinoma cell line greater than 87%. Challenge studies with cysteine, DTPA, human serum and HSA 1% demonstrated no evidence of transcomplexation of 188Re to DTPA or HSA and showed that 30% and 85% of the 188Re-radiolabeled was transcomplexed to human serum and to 100 mM cysteine after 24 h for human serum and 1 h incubation for cysteine at 37 masculine C, respectively. Biodistribution studies indicated no accumulation of the radiolabeled antibodies in normal organs.
Assuntos
Anticorpos Monoclonais/metabolismo , Receptores ErbB/metabolismo , Radioisótopos/metabolismo , Rênio/metabolismo , Coloração e Rotulagem/métodos , Animais , Sítios de Ligação de Anticorpos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Radioisótopos/análise , Radioisótopos/sangue , Rênio/análise , Rênio/sangueRESUMO
188Re is one of the radioisotopes expected to emerge as useful for therapy. Development of new radiopharmaceuticals based on 188Re depends on the radiolabeling methods used, which would give stable complexes having predefined radiochemical properties and in vitro and in vivo stability. This paper has attempted to provide a perspective of 188Re-labeled monoclonal antibodies, their radiolabeling characteristics, methods for quality control of radioimmunoconjugates and in vitro stability for radioimmunotherapy of solid tumors. The direct method of 188Re radiolabeling of antibodies by reductive attachment of 188Re in which free sulfhydryl groups have been generated by reduction of the intramolecular S-S disulfide bonds has been shown to be a promising approach in particular. Moreover, excellent methods have been developed to test the radionuclide, radiochemical purity and stability of 188Re-radioimmunoconjugates using high performance liquid chromatography (HPLC) and paper chromatography.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunoconjugados/uso terapêutico , Neoplasias/terapia , Radioimunoterapia/métodos , Radioisótopos/uso terapêutico , Rênio/uso terapêutico , Animais , Anticorpos Monoclonais/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Cromatografia em Papel , Estabilidade de Medicamentos , Humanos , Imunoconjugados/isolamento & purificação , Técnicas In Vitro , Camundongos , Controle de Qualidade , Radioimunoterapia/normas , Radioisótopos/isolamento & purificação , Radioisótopos/normas , Rênio/isolamento & purificação , Rênio/normasRESUMO
The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG(1)), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 microg/100 microCi of (99m)Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of (99m)Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significative accumulation was found in tumor (6.14 +/- 2.50 %ID/g, 5.06 +/- 2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.
Assuntos
Anticorpos Monoclonais , Receptores ErbB/imunologia , Compostos Radiofarmacêuticos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Ligação Competitiva , Carcinoma de Células Escamosas/diagnóstico por imagem , Ensaio de Imunoadsorção Enzimática , Feminino , Liofilização , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Epiteliais e Glandulares/diagnóstico por imagem , Radioimunoensaio , Ensaio Radioligante , Cintilografia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Compostos de Sulfidrila/química , Distribuição Tecidual , Transplante HeterólogoRESUMO
Brain tumors are often incurable despite current aggressive treatment modalities. Regional intracerebral administration of labeled monoclonal antibodies (Mabs) can maximize the radioisotope and Mab concentration to tumor sites while reducing systemic toxicity. h-R3 is a humanized antiepidermal growth factor receptor Mab that successfully targets the epidermal growth factor receptor, which is overexpressed in glioblastomas. We studied the acute local and systemic toxicity effects of intraventricular 188Re-h-R3 in rats. Forty rats were distributed into four groups with five animals of each sex in each group. A single 5 -microl dose (2.5 microl into the left and 2.5 microl into the right lateral ventricles) of neutral solution containing 50 microg of h- R3 labeled with 49.5 +/- 1.7,284 +/- 13.7 or 579 +/- 23.7 muCi of 188Re were stereotactically administered to each animal. Control animals received vehicle alone. Each animal was observed twice daily for detection of toxicity signs. Body weights were recorded on days 0, 7 and 14. Blood samples for analysis of hematological and clinical chemistry parameters were taken on days 0 and 14. Necropsy and histopathological studies were carried out after completion of the study. All animals, but one, remained clinically stable. Toxicities included local radionecrosis, discrete increase in ALAT and creatinine blood values at higher dose level. We concluded that a single intraventricular administration of relatively large doses of 188Re-h-R3 is tolerable and causes minimal local and systemic toxicity effects in rats. Nevertheless, further studies are necessary to discard learning and behavioral problems.
Assuntos
Anticorpos Monoclonais/toxicidade , Receptores ErbB/imunologia , Lesões Experimentais por Radiação , Compostos Radiofarmacêuticos/toxicidade , Adenocarcinoma , Animais , Anticorpos Monoclonais/administração & dosagem , Peso Corporal/efeitos da radiação , Encéfalo/patologia , Encéfalo/efeitos da radiação , Testes de Química Clínica , Feminino , Testes Hematológicos , Humanos , Injeções Intraventriculares , Neoplasias Pulmonares , Masculino , Necrose , Tamanho do Órgão/efeitos da radiação , Radioisótopos , Compostos Radiofarmacêuticos/administração & dosagem , Ratos , Ratos Sprague-Dawley , Rênio , Técnicas Estereotáxicas , Testes de Toxicidade , Células Tumorais CultivadasRESUMO
Monoclonal antibodies (MAbs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in nuclear medicine practice. The MAb ior egf/r3 developed at the Center of Molecular Immunology (Havana, Cuba) is a murine antibody that recognizes the human epidermal growth factor receptor (EGF-R) and has been used widely in the radioimmunodiagnosis of tumors of epithelial origin. Based on the direct Schwarz method, the present report describes the preparation of a freeze-dried formulation for radiolabeling the MAb ior egf/r3 with 99mTc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, biodistribution, pharmacokinetic, and stability of the formulation are reported. The study demonstrated that the freeze-dried formulation can be labeled with 99mTc at high yield. The resulting 99mTc-labeled ior egf/r3 MAb can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies. The kit does not need any other addition or purification at the time of tagging other than the requisite amount of pertechnetate (40-50 mCi). Because the contents of the kit are lyophilized, no special storage or transportation is required.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Tecnécio/farmacocinética , Animais , Estabilidade de Medicamentos , Feminino , Liofilização , Glucose , Humanos , Ratos , Ratos Wistar , Distribuição TecidualRESUMO
The anti-human epidermal growth factor receptor (EGF-R) humanized monoclonal antibody (MAb) h-R3 is an (IgG1), which binds to an extracellular domain of EGF-R. It was used to evaluate the biodistribution on nude mice xenografted with H-125 human lung adenocarcinoma cell line. Results were compared with its murine version of the MAb ior-egf/r3. Twenty-one athymic female 4NMRI nu/nu mice were injected intraperitoneally with 10 microg/100 muCi of 99mTc-labeled MAbs. Immunoreactivity of 99mTc-labeled MAbs were measured by enzyme-linked immunosorbent assay (ELISA) on H-125 cell line and the immunoreactive fractions was determined by the Lindmo method. Among all organs, significant accumulation was found in serum (27.05 +/- 2.08 %ID/g) and tumor (3.903 +/- 0.89 %ID/g) at 4 h after injection. These values decreased to 5.03 +/- 0.50 %ID/g and 2.19 +/- 0.56 %ID/g for serum and tumor, respectively. The immunoreactive fraction was found to be 0.70, with a correlation coefficient r = 0.9984. With the good biodistribution and tumor uptake of the 99mTc-labeled humanized antibody h-R3, a phase I diagnostic clinical trial of tumor with epithelial origin should be pursued.
Assuntos
Adenocarcinoma/metabolismo , Receptores ErbB/imunologia , Imunoconjugados/farmacocinética , Neoplasias Pulmonares/metabolismo , Compostos de Organotecnécio/farmacocinética , Tecnécio , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Feminino , Humanos , Imunoconjugados/química , Marcação por Isótopo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Compostos de Organotecnécio/síntese química , Distribuição Tecidual , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
UNLABELLED: Monoclonal antibody (moAb) ior egf/r3 is an IgG2a that recognizes the epidermal growth factor receptor (EGF-R). The aim of this study was to evaluate the diagnostic efficacy of the 99mTc-labeled moAb ior egf/r3 for the detection of epithelial-derived tumors, their metastases and recurrences. METHODS: One hundred forty-eight adult patients (51 women, 97 men; mean age 53 +/- 13 y) who were suspected of having cancer of epithelial origin were administered 3 mg/50 mCi (1.85 GBq) 99mTc-labeled moAb ior egf/r3 by intravenous bolus injection. Planar anterior and posterior images of the lesion sites and suspected metastases were acquired at 2, 4, 6 and 24 h after injection, and SPECT images were scanned at 5 h postinjection, using a 360 degrees circular orbit with 64 images. The backprojection method was used for image reconstruction with a Hamming-Hann filter. RESULTS: Labeling efficiency was always greater than 98.5% +/- 2.1 %. No adverse reactions or side effects were observed. Results of the biopsy specimens showed that 85.1% (126/148) of the patients had tumors of epithelial origin, 14.2% (21/148) were negative and 0.7% (1/148) had non-Hodgkin's lymphoma. The sensitivity rate by organ was as follows: brain (8/8, 100%), digestive tract (10/11, 90.9%), head and neck (17/23, 73.9%), lung (52/62, 83.9%) and breast (16/18, 88.9%). Overall sensitivity, specificity, accuracy, and positive and negative predictive values of the immunoscintigraphic imaging were 84.2% (106/126), 100.0% (22/22), 86.5% (128/148), 100% (106/106) and 52.4% (22/42), respectively. New metastases not identified previously by other diagnostic methods were detected in the 50% of the patients. CONCLUSION: Immunoscintigraphy with 99mTc-labeled moAb ior egf/r3 could be a useful procedure for the diagnosis and follow-up of the patients with tumors of epithelial origin.
Assuntos
Receptores ErbB/imunologia , Neoplasias Epiteliais e Glandulares/diagnóstico por imagem , Radioimunodetecção , Pertecnetato Tc 99m de Sódio , Animais , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Compostos Radiofarmacêuticos , Segurança , Sensibilidade e Especificidade , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
UNLABELLED: Radiolabeled antitumor antibodies hold promise for diagnostic imaging and therapy in oncology. The purpose of this study was to investigate the pharmacokinetics, clearances and possible differences of two dosage administrations of the 99mTc-labeled antiepidermal growth factor (EGF)-receptor antibody and to predict the best dose and schedule for future clinical evaluations of this radiopharmaceutical. METHODS: Nine patients (4 women, 5 men; mean age 46.4 +/- 14.0 yr) were administered 1-3 mg 99mTc-labeled anti-EGF-receptor antibody (a murine IgG2a isotype) by intravenous bolus infusion. After administration, blood samples were collected from 7 patients from an antecubital vein opposite to the injection side at intervals from 2 min to 24 hr after injection, and plasma samples were obtained for pharmacokinetic analysis. Appropriate plasma samples were examined for isotope clearance (i.e., microCi/ml at various intervals) and 99mTc complexation to plasma proteins by fast protein liquid chromatography (FPLC) analysis. Urine was collected from each patient at 3 hr intervals up to 24 hr after monoclonal antibody administration to monitor 99mTc clearance. Plasma time-activity curves were fitted to a two-compartment model using nonlinear least-squares regression analysis by the method of flexible polyhedrals. RESULTS: Plasma disappearance curves of 99mTc-labeled anti-EGF-receptor antibody were best fit by biexponential equation with a distribution half-life (t(1/2alpha)) of 0.137 +/- 0.076 hr (n = 7) and elimination half-life (t(1/2beta)) of 20.3 +/- 8.0 hr. Analysis of urine showed that activity clearance by this route amounted to 4.9% +/- 0.6% of the injected dose in 24 hr, and FPLC analysis showed no evidence of decomposition, only 6%-7% of 99mTc was in a low molecular weight species. CONCLUSION: Plasma pharmacokinetics and urine clearance indicate comparability in both doses. The pharmacokinetic properties of the 99mTc-labeled anti-EGF-receptor antibody were found to be dose-independent. These findings provide an initial characterization of the radiopharmaceutical disposition in patients and may be used as the basis for calculating a better estimate of biodistribution and dosimetry for patients who will receive 188Re-labeled anti-EGF-receptor antibody (MAb ior egf/r3) injection for radioimmunotherapy and warrants further controlled clinical trials to define the efficacy of the radiopharmaceutical.
Assuntos
Anticorpos Monoclonais/farmacocinética , Receptores ErbB/imunologia , Neoplasias Pulmonares/diagnóstico por imagem , Compostos Radiofarmacêuticos/farmacocinética , Pertecnetato Tc 99m de Sódio/farmacocinética , Anticorpos Monoclonais/administração & dosagem , Cromatografia Líquida , Feminino , Meia-Vida , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Radioimunodetecção , Radioimunoterapia , Radiometria , Compostos Radiofarmacêuticos/administração & dosagem , Pertecnetato Tc 99m de Sódio/administração & dosagem , Distribuição TecidualRESUMO
The increased interest in the availability of radionuclides for therapy has resulted from the recent success and potential importance of radiolabeled antibodies for both diagnosis and therapy. There is a widespread interest in the availability of 188Re for various therapeutic applications, particularly for attachment to tumor-specific monoclonal antibodies for radioimmunotherapy. This review provides a perspective of 188Re-direct labeled MAbs for radioimmunotherapy of solid tumors, normal organ biodistribution, absorbed radiation doses to normal organs and tumors, and the toxicity to bone marrow and normal tissues. Methods for calculation of mean absorbed radiation doses to the whole body, various normal organs, and tumors have been developed using source-organ residence times and the methods developed by the Medical Internal Radiation Dose (MIRD) committee. The toxicity for 188Re-labeled antibodies is predominantly hematopoietic, with platelets and white blood cells being most sensitive to the effects of radiation. Rhenium-188 would be the isotope of choice for radioimmunotherapeutic applications because of cost, availability, and favorable radiation characteristics. Rhenium-188 has a half-life of 16.9 h and maximum beta energy of 2.118 MeV. This isotope is particularly attractive because it can be supplied conveniently from 188W/188Re-radionuclide generator system.
Assuntos
Anticorpos Monoclonais/farmacocinética , Neoplasias/terapia , Radioimunoterapia/métodos , Rênio , Animais , Anticorpos Monoclonais/efeitos adversos , Humanos , Marcação por Isótopo , Radioisótopos , Radiometria , Distribuição TecidualRESUMO
Monoclonal antibodies (MAbs) are being widely used for imaging studies, coupled mainly with 99mTc. The antibody ior egf/r3 is a MAb against human epidermal growth factor receptor (hEGF-r), and we have developed a method for optimum labeling of this MAb with 99mTc. The reduction was performed with 2-mercaptoethanol (2-ME) at a molar ratio of 2000:1 (2-ME:MAb) and methylene diphosphonate as transchelant. The integrity of reduced MAb was checked by mean of native polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography on Superose 12 (purity >99%). Radio colloids remained lower than 2%, and the labeling efficiency was 98.5%. The number of sulfhydryl groups generated was quantified using Ellman's reagent and was found to be 6.65+/-0.69 per antibody molecule. In vitro stability studies in several challenging conditions (DTPA, human serum albumin and human serum) were performed, and no significant loss in binding percentage was seen. Radio receptor assay was used to test immunoreactivity of the reduced MAb. Both labeled and unlabeled MAbs were able to compete for binding to the hEGF-r with radioiodinated EGF. Biodistribution studies in BALB/c mice are reported.
Assuntos
Anticorpos Monoclonais/química , Receptores ErbB/imunologia , Imunoconjugados/química , Marcação por Isótopo/métodos , Tecnécio/química , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Estabilidade de Medicamentos , Feminino , Humanos , Imunoconjugados/imunologia , Imunoconjugados/farmacocinética , Mercaptoetanol/química , Camundongos , Camundongos Endogâmicos BALB C , Oxirredução , Compostos de Sulfidrila/química , Distribuição TecidualRESUMO
The pharmacokinetics, biodistribution and dosimetry of 99mTc-labeled anti-human epidermal growth factor receptor (anti-hEGF-r) humanized monoclonal antibody (MAb) R3 was investigated following intravenous injection in normal Wistar rats. Serum disappearance curves were best fit by a two-compartment model having a mean distribution half-life (t 1/2alpha) of 0.250 h and a mean elimination (t 1/2beta) of 13.89 h. Among the various organs, a little accumulation of the radiolabeled antibody was found only in kidneys. Biodistribution and dosimetry studies in humans were performed by extrapolation of the animal data to humans. Absorbed dose to normal organs and the remainder of the whole body were estimated using the medical internal radiation dose formula, and dose contributions from radioactivity in transit through the gastrointestinal tract were estimated using a compartment model. Extrapolated values of radiation absorbed dose to normal organs in rads per millicurie administered were whole body, 0.0085; lower large intestine wall, 0.0898; small intestine, 0.0530; upper large intestine wall, 0.0731; and kidneys, 0.0455. The effective dose equivalent predicted was 0.0162 rem/mCi and the effective dose was found to be 0.015 rem/mCi. On the basis of the pharmacokinetics, biodistribution and internal radiation dosimetry information obtained in this study, a diagnostic phase I clinical trial with 99mTc-labeled humanized MAb R3 conjugate in patients should be supported.
Assuntos
Anticorpos Monoclonais/farmacocinética , Receptores ErbB/imunologia , Imunoconjugados/farmacocinética , Compostos de Tecnécio/farmacocinética , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Feminino , Humanos , Imunoconjugados/sangue , Imunoconjugados/imunologia , Marcação por Isótopo , Planejamento da Radioterapia Assistida por Computador , Ratos , Ratos Wistar , Compostos de Tecnécio/sangue , Distribuição TecidualRESUMO
UNLABELLED: Accurate estimation of biodistribution and absorbed dose to normal organs and tumors is important for immunoscintigraphic studies and radioimmunotherapy treatment planning. METHODS: Four patients (3 men, 1 woman; mean age 54.8 +/- 9.2 yr; range 42-64 yr) were administered 3 mg of anti-human epidermal growth factor receptor (anti-hEGF-r) antibody (ior egf/r3), radiolabeled with 99mTc activity of 39.5 +/- 1.1 mCi (range 38.5 mCi-40.7 mCi) by intravenous bolus infusion. After administration, blood and urine samples were collected from three patients up to 24 hr after injection. Whole-body anterior and posterior scans were obtained at 5 min and 1, 3, 5 and 24 hr after injection. Using a computer program, regions of interest were drawn over the heart, liver, spleen, bladder and tumor to measure the activity in the source organs at each scanning time. Time-activity curves for each source organ were then fitted to monoexponential or biexponential functions by nonlinear least squares regression using the flexible polyhedrals method, which adequately fit our data with the correlation coefficient of 0.985 +/- 0.013, and were integrated to determine organ residence times. The mean absorbed doses to the whole body and various normal organs were then estimated from residence times and from blood and urine samples using the methods developed by the Medical Internal Radiation Dose Committee. The effective dose equivalent and effective dose were calculated as prescribed in ICRP Publication Nos. 30 and 60. RESULTS: Plasma disappearance curves of 99mTc-labeled anti-hEGF-r antibody were best-fit by a two-compartment model in all patients with a distribution half-life (t(1/2alpha)) of 0.207 hr +/- 0.059 hr (mean +/- s.d., n = 3) and an elimination half-life (t(1/2beta)) of 13.9 hr +/- 2.2 hr. Among the various organs, significant accumulation of the radiolabeled antibody was found in the liver (48.5% +/- 4.4%, mean +/- s.d.), heart (3.50% +/- 0.17%) and spleen (3.1% +/- 1.8%) at 5 min postadministration. These values were reduced to 3.2% +/- 0.4%, 0.1% +/- 0.01% and 0.1% +/- 0.1%, respectively, at 24 hr. Mean cumulative urinary excretion of 99mTc-labeled anti-hEGF-r antibody was 4.6% +/- 0.6% at 24 hr postinjection. Estimates of radiation absorbed dose to normal organs in rad/mCi administered (mean +/- s.d., n = 4) were: whole body 0.017 +/- 0.002; gallbladder wall 0.074 +/- 0.007; spleen 0.136 +/- 0.076; and liver 0.267 +/- 0.036. The effective dose equivalent and effective dose estimates for adults were 0.041 +/- 0.008 rem/mCi and 0.027 +/- 0.004 rem/mCi administered. CONCLUSION: This feasibility study indicates that 99mTc-labeled anti-hEGF-r antibody (ior egf/r3) can be used safely; this analysis provides a dosimetric framework for future studies. This monoclonal antibody, labeled with 188Re, could possibly permit a successful regional radioimmunotherapy of tumors of epithelial origin.
Assuntos
Receptores ErbB/imunologia , Radioimunoterapia , Compostos Radiofarmacêuticos/uso terapêutico , Pertecnetato Tc 99m de Sódio/uso terapêutico , Adulto , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/uso terapêutico , Estudos de Viabilidade , Feminino , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/radioterapia , Masculino , Pessoa de Meia-Idade , Radioisótopos/farmacocinética , Radioisótopos/uso terapêutico , Radiometria , Cintilografia , Compostos Radiofarmacêuticos/farmacocinética , Dosagem Radioterapêutica , Rênio/farmacocinética , Rênio/uso terapêutico , Pertecnetato Tc 99m de Sódio/farmacocinética , Distribuição TecidualRESUMO
A microcomputer software package for determining the concentration of either the antibody or antigen from ELISA data for IBM PC compatible is presented. In the program optical densities (OD) and fluorescence obtained from 96-well ELISA plate can be input either directly, by interfacing with different brands of microplate reader such as Multiskan II Plus and Organon Teknika to the computer or manually. This software utilizes some mathematical and statistical models to fit the standard curve of each assay and interpolate analyte concentration using data from OD or fluorescence measurements. Cubic spline (Guardabasso et al., 1988), bezier and polynomial (Rodbard, 1979; Baud et al., 1991) interpolation formulas can be used to fit the data over the entire range for estimating the antibody or antigen concentration of the unknown samples whose OD or fluorescence is beyond the entire range. This software package, based on the concentration values of the analyte determined in different fluids (Núnez et al., 1994; Morales et al., 1994) and with some rules and algorithms, is used to calculate the parameters of screening and diagnostic tests such as sensitivity, specificity and predictive values (Coughlin et al., 1992). With the construction of the Receiver Operating Characteristic (ROC) curve it is possible to analyse different values of the sensitivity and specificity of the screening and diagnostic tests. A comparative statistical test for two populations that are non-normally distributed using a non-parametric Mann-Whitney test is provided. This software is an expandable tool designed for general use in clinical and experimental applications, including diagnostic and screening tests.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Programas de Rastreamento/métodos , Software , Anticorpos/análise , Antígenos/análise , Diagnóstico Diferencial , Humanos , Interface Usuário-ComputadorRESUMO
Reduction of disulfide bonds to sulfhydryl (SH) groups for direct radiolabeling of antibodies for immunoscintigraphic studies of colorectal and other cancers continues to be of considerable research interest. We have developed a general strategy and a versatile computer program for the quantification of the number of SH per molecule of antibody (Ab) generated after the treatment of monoclonal antibodies (MAbs) with reducing agents such as 2-mercaptoethanol (2-ME), stannous chloride (SnCl2), dithiothreitol (DTT), dithioerythritol (DTE), ascorbic acid (AA), and the like. The program we describe here performs an unweighted least-squares regression analysis of the cysteine standard curve and interpolates the cysteine concentration of the samples. The number of SH groups per molecule of antibody in the 2-mercaptoethanol and in the other reducing agents was calculated from the cysteine standard curve using Ellman's reagent to develop the yellow color. The linear least-squares method fit the standard data with a high degree of accuracy and with the correlation coefficient r of 0.999. A program has been written for the IBM PC compatible computer utilizing a friendly menu to interact with the users. The package allows the user to change parameters of the assay, to calculate regression coefficients slope, intercept and its standard errors, to perform statistical analysis, together with detailed analysis of variance, and to produce an output of the results in a printed format.
Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Compostos de Sulfidrila/análise , Reagentes de Sulfidrila , Animais , Ácido Ascórbico , Antígeno Carcinoembrionário/imunologia , Cisteína/análise , Ditioeritritol , Ditiotreitol , Receptores ErbB/imunologia , Mucosa Gástrica/imunologia , Humanos , Mucosa Intestinal/imunologia , Análise dos Mínimos Quadrados , Mercaptoetanol , Camundongos , Oxirredução , Análise de Regressão , Software , Compostos de EstanhoRESUMO
A simple, rapid, and reproducible micromethod for quantification of sulfhydryl (SH) groups generated after reduction of monoclonal antibody (MAb) disulfide bonds with 2-mercaptoethanol (2-ME) is described. The number of SH groups per molecule of antibody in the 2-ME and in the other reducing agents was calculated from the cysteine standard curve using Ellman's reagent to develop the yellow color. Results were plotted as absorbance at 405 nm vs. cysteine concentration (microgram/mL). After subtraction of the background due to Ellman's reagent, a straight-line relationship passing through the origin was obtained. Absorption spectrum of the yellow products was controlled, and no significative differences were found between optical density at 412 nm and 405 nm. Using a small quantity of antibody in the order of 37 micrograms, the lowest detection limit for cysteine quantification was 0.03 microgram. An excellent linear correlation was found between both cysteine concentration and absorbance (r = 0.999), and the mean value of the relative error in the quantification of cysteine from samples was 2.8%. A statistical Student t-test showed an excellent linearity and parallelism between cysteine standard and samples.