RESUMEN
BACKGROUND: Paclitaxel has recently been reported by this laboratory to potentiate the high-LET radiation therapeutic (212)Pb-TCMC-trastuzumab, which targets HER2. To elucidate mechanisms associated with this therapy, targeted α-particle radiation therapeutic (212)Pb-TCMC-trastuzumab together with paclitaxel was investigated for the treatment of disseminated peritoneal cancers. METHODS: Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pre-treated with paclitaxel, followed by treatment with (212)Pb-TCMC-trastuzumab and compared with groups treated with paclitaxel alone, (212)Pb-TCMC-HuIgG, (212)Pb-TCMC-trastuzumab and (212)Pb-TCMC-HuIgG after paclitaxel pre-treatment. RESULTS: (212)Pb-TCMC-trastuzumab with paclitaxel given 24 h earlier induced increased mitotic catastrophe and apoptosis. The combined modality of paclitaxel and (212)Pb-TCMC-trastuzumab markedly reduced DNA content in the S-phase of the cell cycle with a concomitant increase observed in the G2/M-phase. This treatment regimen also diminished phosphorylation of histone H3, accompanied by an increase in multi-micronuclei, or mitotic catastrophe in nuclear profiles and positively stained γH2AX foci. The data suggests, possible effects on the mitotic spindle checkpoint by the paclitaxel and (212)Pb-TCMC-trastuzumab treatment. Consistent with this hypothesis, (212)Pb-TCMC-trastuzumab treatment in response to paclitaxel reduced expression and phosphorylation of BubR1, which is likely attributable to disruption of a functional Aurora B, leading to impairment of the mitotic spindle checkpoint. In addition, the reduction of BubR1 expression may be mediated by the association of a repressive transcription factor, E2F4, on the promoter region of BubR1 gene. CONCLUSION: These findings suggest that the sensitisation to therapy of (212)Pb-TCMC-trastuzumab by paclitaxel may be associated with perturbation of the mitotic spindle checkpoint, leading to increased mitotic catastrophe and cell death.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/radioterapia , Radioisótopos de Plomo/uso terapéutico , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Paclitaxel/uso terapéutico , Radioinmunoterapia , Animales , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Terapia Combinada , Femenino , Humanos , Ratones , Ratones Desnudos , Paclitaxel/farmacología , Radioinmunoterapia/métodos , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
INTRODUCTION: Tumors over-expressing the human epithelial receptor 2 (HER2) or exhibiting amplification or mutation of its proto-oncogene have a poorer prognosis. Using trastuzumab and/or other HER2 targeted therapies can increase overall survival in patients with HER2(+) tumors making it critical to accurately identify patients who may benefit. We report on a Phase 0 study of the imaging agent, 111In-CHX-A"-DTPA trastuzumab, in patients with known HER2 status to evaluate its safety and biodistribution and to obtain preliminary data regarding its ability to provide an accurate, whole-body, non-invasive means to determine HER2 status. METHODS: 111In-CHX-A"-DTPA trastuzumab was radiolabeled on-site and slowly infused into 11 patients who underwent single (n=5) or multiple (n=6) ɣ-camera (n=6) and/or SPECT (n=8) imaging sessions. RESULTS: No safety issues were identified. Visual and semi-quantitative imaging data were concordant with tissue HER2 expression profiling in all but 1 patient. The biodistribution showed intense peak liver activity at the initial imaging timepoint (3.3h) and a single-phase clearance fit of the average time-activity curve (TAC) estimated t1/2=46.9h (R2=0.97; 95%CI 41.8 to 53h). This was followed by high gastrointestinal (GI) tract activity peaking by 52h. Linear regression predicted GI clearance by 201.2h (R2 =0.96; 95%CI 188.5 to 216.9h). Blood pool had lower activity with its maximum on the initial images. Non-linear regression fit projected a t1/2=34.2h (R2 =0.96; 95%CI 25.3 to 46.3h). Assuming linear whole-body clearance, linear regression projected complete elimination (x-intercept) at 256.5hr (R2=0.96; 95%CI 186.1 to 489.2h). CONCLUSION: 111In-CHX-A"-DTPA trastuzumab can be safely imaged in humans. The biodistribution allowed for visual and semiquantitative analysis with results concordant with tissue expression profiling in 10 of 11 patients. Advances in Knowledge and Implications for Patient Care Using readily available components and on-site radiolabeling 111In-CHX-A"-DTPA trastuzumab SPECT imaging may provide an economical, non-invasive means to detect HER2 over-expression.
RESUMEN
We describe here the first in vivo targeting of tumors with a single-chain antigen-binding protein. The molecule, which was constructed and expressed in Escherichia coli, is a novel recombinant protein composed of a variable light-chain (VL), amino acid sequence of an immunoglobulin tethered to a variable heavy-chain (VH) sequence by a designed peptide. We show that this protein, derived from the DNA sequence of the variable regions of the antitumor monoclonal antibody B6.2, has the same in vitro antigen-binding properties as the B6.2 Fab' fragment. Comparative pharmacokinetic studies in athymic mice demonstrate much more rapid alpha and beta phases of plasma clearance for the single-chain antigen-binding protein than for the Fab' fragment, as well as an extremely rapid whole-body clearance. Half-life values for alpha and beta phases of single-chain antigen-binding protein clearance were 2.4 minutes and 2.8 hours, respectively, versus 14.8 minutes and 7.5 hours for Fab'. Furthermore, the single-chain antigen-binding protein molecule did not show accumulation in the kidney as did the Fab' molecule or, as previously shown, the F(ab')2 molecule. Despite its rapid clearance, the single-chain antigen-binding protein showed uptake in a human tumor xenograft comparable to that of the Fab' fragment, resulting in tumor to normal tissue ratios comparable to or greater than those obtained with the Fab' fragment. These studies thus demonstrate the in vivo stability of recombinant single-chain antigen-binding proteins and their potential in some diagnostic and therapeutic clinical applications in cancer and other diseases.
Asunto(s)
Carcinoma/metabolismo , Proteínas Portadoras/farmacocinética , Neoplasias del Colon/metabolismo , Inmunotoxinas/farmacocinética , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/uso terapéutico , Femenino , Semivida , Humanos , Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Fragmentos Fab de Inmunoglobulinas/farmacocinética , Inmunotoxinas/genética , Inmunotoxinas/uso terapéutico , Riñón/metabolismo , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéutico , Distribución TisularRESUMEN
Single-chain antigen-binding proteins, or sFvs, represent potentially unique molecules for targeted delivery of drugs, toxins, or radionuclides to a tumor site. In previous studies (Cancer Res., 51:6363-6371, 1991) using a human colon carcinoma xenograft model, it was demonstrated that the sFv has an extremely rapid plasma and whole body clearance, as compared to intact IgG or Ig fragments. One potential consequence of the rapid sFv pharmacokinetic properties was the reduced percentage of injected dose/g of the radiolabeled sFv found in the tumor throughout a range of time points. The present study was designed to define the tumor penetration properties of a radiolabeled sFv in comparison with other Ig forms. 125I-labeled sFv, Fab', F(ab')2, and IgG forms of monoclonal antibody CC49, directed against the human pancarcinoma antigen TAG-72, were used to target the LS-174T human colon carcinoma xenograft in athymic mice. At various time points after systemic Ig administration, quantitative autoradiographic analyses of surgically removed tumors were used to define the rate and degree of penetration of the various Ig forms. These studies revealed that most of the intact IgG delivered to the tumor was concentrated in the region of or immediately adjacent to vessels, while the sFv was more evenly distributed throughout the tumor mass. The distributions of the Fab' and F(ab')2 fragments showed intermediate penetration in a size-related manner. The sFv demonstrated maximum tumor penetration at 0.5 h postinjection, while the intact IgG reached an equivalent degree of penetration at 48 to 96 h postinjection. These studies thus reveal a greater degree of uptake throughout the tumor for the sFv than would be expected by gross analyses of percentage injected dose/g and demonstrate an extremely rapid tumor penetration of the sFv. These studies should aid in the rational design of potential applications of drug-, toxin-, and radionuclide-conjugated sFvs in cancer therapy.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Antígenos/metabolismo , Neoplasias del Colon/metabolismo , Glicoproteínas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Radioisótopos de Yodo/metabolismo , Animales , Autorradiografía , Femenino , Inmunoglobulina G/inmunología , Ratones , Ratones Desnudos , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
In previous studies, we have compared the immunochemical properties, the in vivo pharmacokinetics, and the tumor penetrance of a radioiodinated single-chain Fv (sFv) in comparison with other immunoglobulin (Ig) forms (intact IgG, F(ab')2, and Fab') (Cancer Res., 51: 6363-6371, 1991). Biodistribution studies demonstrated a higher percent injected dose/g in the liver and spleen for the intact IgG and F(ab')2. Renal uptake was observed with the Fab' and F(ab')2, whereas the sFv demonstrated no specific localization in either of these organs. The 125I-labeled sFv also demonstrated a more even distribution throughout the tumor xenografts as compared to the other Ig forms (Cancer Res., 52: 3402-3408, 1992). Subsequent studies utilizing the sFv conjugated with a radiometal (177Lu) demonstrated that the sFv was being metabolized by the kidney, and a significantly higher percent injected dose/g was obtained with a 177Lu-labeled sFv as compared to a 125I-labeled sFv (Cancer Res., 52: 6413-6417, 1992). These previous studies indicated the potential utility of radioiodinated sFv and other Ig fragments for use in radioimmunoguided surgery with a hand-held probe, diagnostic imaging, and possibly therapy. The present study compares the distribution in normal tissues of the 4 Ig forms of monoclonal antibody (MAb) CC49, which is directed against a pancarcinoma antigen (tumor-associated glycoprotein-72). 125I-labeled sFv, Fab', F(ab')2, and IgG of MAb CC49 were administered to athymic mice either bearing or not bearing the tumor-associated glycoprotein-72 positive human colon carcinoma xenograft (LS-174T). At various intervals following the i.v. injection of the Ig forms, the liver, spleen, kidneys, and lungs were removed for autoradiographic analyses. Dramatic differences were observed in the kidney; the IgG was found only in the renal vasculature, whereas the Fab', F(ab')2, and sFv showed a high density of grains in the cortical tubules. In the liver, the IgG and F(ab')2 were found in association with hepatocytes, Kupffer cells, and in the sinusoids; the Fab' and sFv were primarily associated with the Kupffer cells. In the spleen, the Ig forms localized to the marginal zones surrounding the lymphoid follicles. No specific accumulation of grains for any of the Ig forms was observed in the lung. In each of the tissues, the clearance rates were related to the size of the Ig form. The localization in the liver and spleen was determined to be antigen-mediated.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Antígenos de Neoplasias/inmunología , Glicoproteínas/inmunología , Fragmentos de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Antígenos de Neoplasias/metabolismo , Autorradiografía , Neoplasias del Colon , Glicoproteínas/metabolismo , Humanos , Riñón/metabolismo , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Tejido Linfoide/metabolismo , Ratones , Ratones Desnudos , Bazo/metabolismo , Factores de Tiempo , Distribución Tisular , Trasplante HeterólogoRESUMEN
Previous studies have shown that (a) single-chain antibody binding proteins, or sFvs, localize experimental tumor xenografts (D.E. Milenic et al, Cancer Res., 51: 6363-6371, 1991) and (b) the administration of gamma-interferon (IFN-gamma) increases the expression of a high molecular weight glycoprotein, tumor-associated glycoprotein 72 (TAG-72), which improves mAb-based tumor targeting as well as radioimmunotherapy (J. W. Greiner et al., Cancer Res., 53: 600-608, 1993). The present experimental study was designed to determine whether exploiting those two observations in combination could augment tumor detection. Initial results revealed significant localization of a single-chain antibody binding protein of CC49 (i.e., CC49 sFv), a second generation anti-TAG-72 mAb, to human colon tumor xenografts (HT-29), which express low constitutive TAG-72 levels. IFN-gamma treatment of mice bearing HT-29 tumors significantly increased TAG-72 levels in the tumor xenografts. Increased TAG-72 expression was accompanied by a 2-4-fold augmentation of CC49 sFv localized to the HT-29 tumors, measured by direct quantitation of 125I-labeled CC49 sFv tumor deposition as well as tumor:normal tissue ratios. Enhanced CC49 sFv tumor localization improved HT-29 tumor visualization by external scintigraphy as well as when using a hand-held gamma-detecting probe to discriminate between normal (i.e., heart, hind leg) and tumor tissue. The gamma-detecting probe was the same as that used intraoperatively with 125I-labeled CC49 IgG to identify occult tumors in patients. The present experimental findings indicate that the efficiency by which 125I-labeled CC49 sFv localizes tumor in vivo can be enhanced with IFN-gamma. Results of the present study suggest that (a) the incorporation of an IFN-gamma treatment schema prior to radioimmunscintigraphy may increase the signal from the tumor site(s), thus providing a better discrimination between tumor and background, and (b) combining 125I-labeled CC49 sFv with IFN-gamma will not only reduce the time interval between antibody injection and surgery, but will also increase the efficiency of tumor localization using the intraoperative gamma-detecting probe.
Asunto(s)
Anticuerpos Antineoplásicos , Fragmentos de Inmunoglobulinas , Neoplasias Experimentales/diagnóstico por imagen , Animales , Antígenos de Neoplasias/análisis , Antígenos de Carbohidratos Asociados a Tumores/análisis , Neoplasias del Colon/diagnóstico por imagen , Rayos gamma , Glicoproteínas/análisis , Humanos , Fragmentos de Inmunoglobulinas/química , Interferón gamma/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Cintigrafía , Trasplante HeterólogoRESUMEN
177Lutetium (177Lu) is a member of the family of elements known as lanthanides or rare earths. Monoclonal antibody (MAb) CC49, a murine IgG1, which is reactive with the tumor-associated antigen, TAG-72, has been shown previously to react with a wide range of human carcinomas; CC49 reacts to a different epitope on the TAG-72 molecule than MAb B72.3 and has a higher binding affinity. We report here the first use of a 177Lu-labeled immunoconjugate, 177Lu-CC49, in an experimental therapy model for human carcinoma. 177Lu-CC49 was shown to delay the growth of established LS-174T human colon carcinomas in athymic mice at a single dose of 50 microCi. Overt toxicity was observed with the administration of approximately 500 microCi of 177Lu-CC49 in which 5 of 9 mice died of apparent marrow toxicity. A single administration of 200 or 350 microCi of 177Lu-CC49, however, was shown to eliminate established tumors through the 77-day observation period after MAb administration. Dose fractionation experiments revealed that at least 750 microCi of 177Lu-CC49 (250 microCi/week for 3 consecutive weeks) was well tolerated in that 9 of 10 mice survived. Moreover, this dose schedule was able to eliminate the growth of relatively large (300 mm3) human colon tumor xenografts in 90% of the animals treated. Single-dose and dose fractionation studies were also carried out with an isotype-matched control MAb, 177Lu-MOPC-21. In all dose schedules, a large differential was seen between the therapeutic effects of the 177Lu-CC49 versus that of the 177Lu-control MAb. The merits and limitations of the use of 177Lu-labeled immunoconjugates (in particular, 177Lu-CC49) are discussed in terms of potential novel therapeutics for human carcinoma.
Asunto(s)
Adenocarcinoma Mucinoso/radioterapia , Anticuerpos Monoclonales/uso terapéutico , Neoplasias del Colon/radioterapia , Inmunoglobulina G/uso terapéutico , Lutecio/uso terapéutico , Radioisótopos/uso terapéutico , Adenocarcinoma Mucinoso/patología , Animales , Anticuerpos Monoclonales/inmunología , Neoplasias del Colon/patología , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Inmunoglobulina G/inmunología , Lutecio/farmacocinética , Ratones , Ratones Desnudos , Radioisótopos/farmacocinética , Dosificación Radioterapéutica , Distribución Tisular , Células Tumorales CultivadasRESUMEN
CC49 is a "second generation" monoclonal antibody to B72.3, which reacts with the pancarcinoma antigen TAG-72. CC49 has been shown to efficiently target human colon carcinoma xenografts and is currently being evaluated in both diagnostic and therapeutic clinical trials. We describe here the construction and characterization of a recombinant single-chain Fv (sFv) of CC49. The sFv was shown to be a Mr 27,000 homogeneous entity which could be efficiently radiolabeled with 125I or 131I. Comparative direct binding studies and competition radioimmunoassays using CC49 intact IgG, F(ab')2, Fab', and sFv revealed that the monomeric CC49 Fab' and sFv had relative binding affinities 8-fold lower than the dimeric F(ab')2 and intact IgG. Nonetheless, the 131I-labeled sFv was shown to bind biopsies of TAG-72-expressing tumors. Metabolism studies in mice, using radiolabeled CC49 IgG, F(ab')2, Fab', and sFv, demonstrated an extremely rapid plasma and whole body clearance for the sFv. CC49 sFv plasma pharmacokinetic studies in rhesus monkeys also showed a very rapid plasma clearance (T1/2 alpha of 3.9 min and T1/2 beta of 4.2 h). Tumor targeting studies with all four radiolabeled Ig CC49 forms, using the LS-174T human colon carcinoma xenograft model, revealed a much lower percentage injected dose/g tumor binding for the CC49 monomeric sFv and Fab' as compared to the dimeric F(ab')2 and intact IgG. However, tumor:normal tissue ratios (radiolocalization indices) for the sFv were comparable to or greater than those of the other Ig forms. High kidney uptake with 125I-labeled Fab' and F(ab')2 was not seen with 125I-sFv. Gamma scanning studies also showed that 131I-CC49 sFv could efficiently localize tumors. The CC49 sFv may thus have utility in diagnostic and perhaps therapeutic applications for a range of human carcinomas.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Neoplasias del Colon/terapia , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Proteínas Recombinantes/farmacocinética , Animales , Neoplasias del Colon/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Técnicas para Inmunoenzimas , Radioisótopos de Yodo/metabolismo , Macaca mulatta , Ratones , Ratones Desnudos , Peso Molecular , Radioinmunoensayo , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Distribución TisularRESUMEN
Genetically engineered single-chain Fvs (sFv) are defined as recombinant proteins composed of a variable light chain amino acid sequence of an immunoglobulin tethered to a variable heavy chain sequence by a designed peptide. Previous studies using iodine-labeled sFv, derived from the anticarcinoma monoclonal antibody CC49, showed that the 125I-sFv could efficiently target antigen-positive tumors in a human tumor xenograft model while demonstrating rapid plasma clearance and minimal uptake in normal organs. One of the issues we raised in the analysis of the iodinated sFv metabolic studies was whether similar metabolic patterns would be observed if the sFv were labeled with a radiometal. In the studies reported here, 125I-CC49 sFv and 177Lu-CC49 sFv were co-injected in mice bearing antigen-positive carcinoma xenografts. Both sFv forms showed similar tumor targeting and plasma clearance pharmacokinetics. The 177Lu-sFv, however, showed a greater uptake in liver and spleen and a much higher uptake in kidney. These studies thus demonstrate that despite their small size (M(r) 27,000), the metal-chelated sFv shows a metabolic pattern very different than that of the iodinated sFv, which is most likely due to retention of the metal by organs metabolizing the sFv.
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Radioisótopos de Yodo , Lutecio , Proteínas de Neoplasias/metabolismo , Radioisótopos , Animales , Anticuerpos Monoclonales/metabolismo , Quelantes/metabolismo , Quelantes/farmacocinética , Cromatografía Líquida de Alta Presión , Neoplasias del Colon/metabolismo , Femenino , Humanos , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/metabolismo , Región Variable de Inmunoglobulina/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Recombinantes/metabolismo , Distribución TisularRESUMEN
CC49 is a second-generation monoclonal antibody (MAb) that has high affinity for the tumor-associated pancarcinoma antigen tumor-associated glycoprotein-72. In clinical trials using gamma scanning, radiolabeled CC49 has facilitated the detection of more than 90% of carcinomas. We report here the development of a constant heavy-chain 2 (CH2) domain-deleted chimeric (c) CC49 MAb by transfecting an expression construct consisting of the CC49 murine variable region and a CH2 domain-deleted human IgG1 constant region into cCC49 kappa producing SP2/0 murine myeloma cells. As determined by SDS-PAGE, the intact cCC49 delta CH2 has a molecular weight of 153,000 and, under reducing conditions, molecular weights of 43,000 and 27,000. The plasma clearance and tumor-targeting properties of cCC49 delta CH2 were evaluated and compared with those of mouse/human chimeric forms cCC49 delta CH1 and intact cCC49. Previous studies have shown that the in vitro antigen-binding properties of cCC49 delta CH1 are similar to those of cCC49. Biodistribution studies reported here, using 131I-labeled cCC49 delta CH1 and 125I-labeled cCC49 in athymic mice bearing human colon carcinoma xenografts, demonstrated that both cMAbs localized to the tumor and cleared from the normal tissues similarly. However, in comparison with 125I-labeled cCC49, 131I-labeled cCC49 delta CH2 localized to tumors earlier and had a significantly lower percentage of the injected dose of cMAb/g (%ID/g) in normal tissues than cCC49. Immunoscintigraphy of 131I-labeled cCC49 delta CH2 and 125I-labeled cCC49 in athymic mice bearing human tumor xenografts demonstrated a clear image of the tumor by 24 h after i.v. administration of the delta CH2 cMAb versus the 72 h required for cCC49. Biodistribution studies using 177Lu-conjugated cCC49 delta CH1 and cCC49 showed no significant difference between the radiolocalization indices (% ID/g in tumor divided by % ID/g in normal tissue). 177Lu-conjugated cCC49 delta CH2, however, had lower % ID/g values in tumor xenografts and lower radiolocalization indices than either 177Lu-conjugated cCC49 delta CH1 or 177Lu-conjugated cCC49. Pharmacokinetic studies in non-tumor-bearing athymic mice using cCC49 delta CH1 and cCC49 revealed no significant difference between these cMAbs. However, the plasma clearance of cCC49 delta CH2 in non-tumor-bearing mice was significantly faster than that of cCC49. These results were similar when the cMAbs were labeled with either iodine or lutetium. In nonhuman primates, 131I-labeled cCC49 delta CH2 cleared significantly faster than 125I-labeled cCC49. The similar plasma clearance and tumor localization of cCC49 and cCC49 delta CH1 suggest that these two cMAbs may be used in similar clinical settings. However, because of the unique pharmacokinetics and tumor targeting of cCC49 delta CH2 versus cCC49 or cCC49 delta CH1, this chimeric immunoglobulin form may be useful in clinical settings that require efficient tumor targeting and rapid serum and whole-body clearance.
Asunto(s)
Anticuerpos Antineoplásicos/genética , Anticuerpos Antineoplásicos/inmunología , Animales , Anticuerpos Antineoplásicos/metabolismo , Carcinoma/diagnóstico por imagen , Neoplasias del Colon/diagnóstico por imagen , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Escherichia coli/genética , Eliminación de Gen , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Radioisótopos de Yodo , Macaca mulatta , Ratones , Ratones Desnudos , Radioinmunodetección , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacocinética , Transfección , Células Tumorales CultivadasRESUMEN
CC49, a murine monoclonal antibody that recognizes the tumor-associated glycoprotein 72, was conjugated to the chemical chelate 1,4,7,10-tetraaza-1-(1-carboxy-3-(4-aminophenyl) propyl)-tris-4,7,10- ((carboxy)methyl)cyclododecane that had been labeled with a beta emitter, Lu. Preclinical studies had shown that Lu-labeled CC49 caused regression of human colon adenocarcinoma xenografts in nude mice. Patients with advanced adenocarcinoma who had failed standard treatment and whose tumors expressed the tumor-associated glycoprotein 72 antigen were eligible for treatment to determine the maximum tolerated dose of Lu-labeled CC49. The starting dose of Lu was 10 mCi/m2 given i.v. with the dose of CC49 held constant at 20 mg. Pharmacokinetic sampling and immunoscintigraphy were performed over the ensuing 3 weeks. The dose of radioactive Lu was escalated by 15 mCi/m2 for each successive dose level. Unexpected bone marrow toxicity developed in patients treated at the second dose level with 25 mCi/m2 Lu; two patients developed grade 4 thrombocytopenia, while the third patient developed grade 3 thrombocytopenia. Pharmacokinetic studies showed that the plasma half-life of the immunoconjugate was 67 h; whole-body retention, however, was prolonged with a biological half-life of 258 h. Serial gamma camera imaging localized known tumor in all patients, and also demonstrated prolonged Lu retention in the reticuloendothelial system (RES). Bone marrow dosimetry estimates ranged from 4 to 5 REMS/mCi Lu based on imaging and biopsy data. Analysis of bone marrow biopsies demonstrated that most of the Lu was localized in the cellular compartment and not in the bone. No antitumor responses were observed. Intravenous administration of 15 mCi/m2 Lu-labeled CC49 to previously treated advanced cancer patients was associated with acceptable hematological toxicity and was the maximum tolerated dose. However, prolonged retention of Lu in the RES, including the bone marrow, was observed and limited the dose of Lu that could be given. Additional studies are indicated to reduce RES uptake and retention of this immunoconjugate.
Asunto(s)
Adenocarcinoma/radioterapia , Anticuerpos Antineoplásicos/administración & dosificación , Antineoplásicos/administración & dosificación , Lutecio/administración & dosificación , Neoplasias/radioterapia , Radioisótopos/administración & dosificación , Adenocarcinoma/patología , Adulto , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Antineoplásicos/efectos adversos , Antineoplásicos/efectos adversos , Neoplasias de la Mama/patología , Neoplasias de la Mama/radioterapia , Neoplasias del Colon/patología , Neoplasias del Colon/radioterapia , Femenino , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/radioterapia , Lutecio/efectos adversos , Masculino , Persona de Mediana Edad , Neoplasias/patología , Radioinmunoterapia/métodos , Radioisótopos/efectos adversos , Dosificación RadioterapéuticaRESUMEN
Novel strategies to increase the therapeutic ratio in clinical radioimmunotherapy studies are needed. Limitations to radioimmunotherapy include bone marrow suppression due to the long circulating half-life of radiolabeled monoclonal antibodies (mAbs) and heterogeneous tumor penetration of the high-molecular-weight mAb. An approach to overcome these problems is the use of genetically engineered mAbs. The engineered mAb discussed in this paper contains a deletion in the constant region of the mAb that increases its tumor penetration and blood clearance compared with the intact mAb. Radiolabeling of this mAb should lead to a similar radiation-absorbed dose to tumor compared with the intact mAb, but reduce the radiation absorbed dose to bone marrow. In addition, low or variable expression of tumor-associated target antigens or receptors may lead to low or heterogeneous tumor uptake of radiolabeled mAbs. This report also discusses a novel approach toward systemic radiotherapy that combines gene transfer techniques (to increase tumor receptor expression) with radiolabeled peptides that target the induced receptor. The radiolabeled peptides achieve good tumor uptake, rapid tumor penetration, and rapid blood clearance. A humanized construct of the CC49 (HuCC49) high-affinity anti-TAG-72 mAb, as well as a construct with the CH2 region deleted (HuCC49deltaCH2), were labeled with 131I and 177Lu. Biodistribution of the radiolabeled constructs was evaluated 24 h after regional i.p. injection in athymic nude mice bearing i.p. LS174T human colon cancer xenografts. The 131I-HuCC49deltaCH2 showed a median tumor uptake of 5.5% ID/g which was similar to that of 131I-HuCC49 at 5.2% ID/g. However, the median blood concentration of 131I-HuCC49deltaCH2 was 0.2% ID/g which was significantly lower than 0.8% ID/g for 1311-HuCC49. The uptake of the constructs in other normal tissues were similar. The 177Lu-HuCC49deltaCH2 showed a median tumor uptake of 9.4% ID/g, which was slightly higher than that of 177Lu-HuCC49 at 7.9% ID/g. The median blood concentration of 177Lu-HuCC49deltaCH2 was 0.2% ID/g, which was significantly lower than 0.4% ID/g for 177Lu-HuCC49. The uptake of the antibody constructs in other normal tissues were similar except for the kidney. The tumor:blood ratios of 177Lu-HuCC49 and 177Lu-HuCC49deltaCH2 were 19.4 and 60.2, respectively, at 24 h after injection. The purpose of the second aspect of the study was to determine the biodistribution of 64Cu-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA)-octreotide in a human ovarian cancer model induced to express human somatostatin receptor subtype 2 (SSTr2) using gene transfer techniques as a prelude to future therapy studies. Mice bearing i.p. SKOV3.ip1 tumors transduced with an adenoviral vector encoding the cDNA for SSTr2 (AdSSTr2) and injected i.p. with 64Cu-TETA-octreotide showed a median uptake of 24.3% ID/g in tumor at 4 h postinjection compared with 4.9% ID/g at 18 h after injection. Also, tumor uptake of 64Cu-TETA-octreotide at 4 h was not significantly different when administered either 2 or 4 days after injection of AdSSTr2 (P = 0.076). 64Cu-TETA-octreotide should be useful for targeted radiotherapy against tumors that are genetically induced to express high levels of SSTr. These two novel targeting strategies show promise for improved cancer radioimmunotherapy.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Neoplasias Experimentales/radioterapia , Radioinmunoterapia , Animales , Anticuerpos Monoclonales/farmacocinética , Radioisótopos de Cobre/farmacocinética , Humanos , Marcaje Isotópico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Octreótido/farmacocinética , Distribución Tisular , Células Tumorales CultivadasRESUMEN
One of the major obstacles in the successful clinical application of monoclonal antibodies has been the development of host immune responses to murine Ig constant and variable regions. While the CDR grafting of MAbs may alleviate many of these problems, the potential remains that one or more murine CDRs on the human Ig backbone of a "humanized" MAb may still be immunogenic. Studies were undertaken employing a MAb of potential clinical utility, CC49, to define those CDRs that are essential for antigen binding and those that may be immunogenic in humans. We previously developed a humanized CC49 (HuCC49) by grafting the MAb CC49 hypervariable regions onto frameworks of human MAbs. To identify those CDRs essential for binding, a panel of variant HuCC49 MAbs was generated here by systematically replacing each of the murine CDRs with their human counterparts. The relative affinity constant of each variant was determined. Serum from a patient who received murine CC49 was used to determine the potential immunogenicity of each CDR in humans. The serum was shown to react with the anti-CC49 variable region. Results showed that patients' anti-idiotypic responses are directed mainly against LCDR3 and moderately against LCDR1 and HCDR2. These studies demonstrate for the first time that variants containing individual CDR substitutions of a humanized MAb can be constructed, and each CDR can be defined for the two most important properties for potential clinical utility: antigen binding and immunogenicity.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Anticuerpos Antiidiotipos/biosíntesis , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Baculoviridae/genética , Sitios de Unión/genética , Unión Competitiva , Línea Celular , Humanos , Región Variable de Inmunoglobulina/inmunología , Cinética , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , SpodopteraRESUMEN
Monoclonal antibody (MAb) CC49 reacts with a pancarcinoma antigen, tumor associated glycoprotein (TAG)-72. To circumvent human anti-murine antibody (HAMA) responses in patients, we earlier developed a humanized CC49 (HuCC49) by grafting the complementarity-determining regions (CDRs) of MAb CC49 onto variable light (VL) and variable heavy (VH) frameworks of the human MAbs LEN and 21/28'CL, respectively. With the aim of minimizing its immunogenicity further, we have now generated a variant HuCC49 MAb by grafting the specificity-determining residues (SDRs) of MAb CC49 onto the frameworks of the human MAbs. Based on the evaluation of its binding affinity for TAG-72 and its reactivity with anti-idiotypic antibodies present in sera from patients who have been treated with murine CC49, this variant retains its antigen-binding activity and shows minimal reactivity with anti-idiotypic antibodies in patients' sera. Development of this variant, which is a potentially useful clinical reagent for diagnosis and therapy of human carcinomas, demonstrates that for humanization of a xenogeneic antibody grafting of the potential SDRs should be sufficient to retain its antigen-binding properties.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Neoplasias/tratamiento farmacológico , Animales , Anticuerpos Heterófilos/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Anticuerpos Antineoplásicos/uso terapéutico , Antineoplásicos/inmunología , Antineoplásicos/uso terapéutico , Regiones Determinantes de Complementariedad/análisis , Regiones Determinantes de Complementariedad/genética , Diseño de Fármacos , Ingeniería Genética , Variación Genética , HumanosRESUMEN
While in vivo gene inoculation is being increasingly exploited to express genes of choice and elicit specific immune responses in animal models, the utility of this method has not been explored extensively for the expression of antibody genes. The primary constraint of this method is the need to deliver to, and express in, a single cell two functional genes, i.e., those encoding heavy and light chains of an antibody molecule. Several single-gene constructs encoding variants of the monoclonal antibody (MAb) CC49 have been developed, MAb CC49 recognizes a tumor-associated glycoprotein, TAG-72. SP2/O myeloma cells, transfected with the CC49 single gene, express a single-chain protein which is secreted by the transfectoma as a homodimer. Following intramuscular injection of mice with the expression plasmids of the single-gene constructs, the encoded CC49 antibody (AB1) was detected in the plasma of the host. In addition, cellular and humoral immune responses to AB1 have been demonstrated. Antibodies (AB2) to the in vivo-produced variable region of AB1 have been detected and persisted for at least 70 days post-inoculation of the recombinant plasmid. Thus, in vivo gene inoculation of single-chain immunoglobulins may be an alternative or complimentary approach to the induction of anti-idiotypic responses in immunotherapy protocols.
Asunto(s)
Anticuerpos Antineoplásicos/genética , Técnicas de Transferencia de Gen , Terapia Genética , Inmunoglobulinas/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Expresión Génica , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
The evolution of monoclonal antibody forms for radioimmunotherapy and other antibody-based applications has been driven by a series of problems that each new form has introduced. Ehrlich was the first to present the concept that antibodies could be exploited in such a manner. Four decades were required before technological advances allowed the exploration of the potential of antibodies for radioimaging and radioimmunotherapeutic applications. Advances in DNA technology have led to the ability to tailor and manipulate the immunoglobulin molecule for specific functions and in vivo properties. This article discusses the use of monoclonal antibodies for radiotherapy with an emphasis on the problems that have been encountered and the subsequent solutions.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Neoplasias/radioterapia , Radioinmunoterapia , Animales , Anticuerpos Monoclonales/química , Diseño de Fármacos , Humanos , Péptidos/uso terapéuticoRESUMEN
Immunoglobulin fragments, whether of polyclonal or monoclonal antibodies, offer a number of advantages over the intact immunoglobulin. The generation of immunoreactive fragments from monoclonal antibodies (MAb) is not always a straightforward task. Both pepsin and papain can be used to digest MAb to a bivalent molecule with a Mr of 100,000. However, pepsin pepsin digestions does not always result in immunoreactive fragments and a stable consistent product by papain digestion is often difficult to obtain. MAb B72.3 is an example of both situations. MAb B72.3 reacts with a glycoprotein (TAG-72) with a molecular weight greater than 10(6). MAb B72.3 has been shown to exhibit a high degree of selective reactivity with colon, breast and ovarian carcinomas and has been used for radioimmunodiagnosis in model systems and in clinical trials. A third enzyme, bromelain, in the same family of sulfhydryl proteases as papain, has been used to generate a fragment of MAb B72.3, with a Mr of approximately 100,000. The bromelain-generated fragment of MAb B72.3 retained 100% immunoreactivity as measured in competitive solid-phase radioimmunoassays and could be generated with consistent results from one preparation to another. Both the bromelain- and papain-generated fragments were radiolabelled with 125I without significant loss of the MAb's reactivity to tumor extracts. Differences were observed between the bromelain- and papain-generated fragment when compared in vivo. Fragmentation of MAb B72.3 with bromelain has yielded a superior bivalent fragment for radioimmunolocalization.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antineoplásicos/farmacocinética , Bromelaínas , Línea Celular , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Inmunoterapia , Ratones , Ratones Desnudos , Peso Molecular , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/terapia , Papaína , Pepsina A , CintigrafíaRESUMEN
Radiolabeled B72.3 (anti-TAG-72) has been shown to selectively localize metastatic lesions in 70%-80% of the cases. Serum samples from 27 colorectal carcinoma patients who received iodine-131-(131I) B72.3 by i.v. administration were analyzed. Circulating immunoreactive antibody followed a biphasic clearance pattern. High-performance liquid chromatography (HPLC) and SDS-polyacrylamide gel electrophoresis demonstrated that 131I-B72.3 retained its integrity in the patients' sera. HPLC analysis also demonstrated the presence of immune complexes in the sera of 12 patients; this correlated with elevated levels of circulating TAG-72. Several different HAMA response patterns were detected in the 25 patients' sera that were analyzed; some patients developed HAMA as early as 5-7 days post-MAb injection. Higher doses of administered MAb B72.3 correlated with the development of HAMA (p = 0.007). The presence of elevated levels of TAG-72 in the patients' pre-inoculum serum was shown to correlate with the detection of lesions by gamma scanning. Serum TAG-72 may serve as a criteria for patient selection for immunodiagnostic or immunotherapeutic procedures using MAb B72.3.
Asunto(s)
Adenocarcinoma/metabolismo , Anticuerpos Monoclonales/farmacocinética , Neoplasias Colorrectales/metabolismo , Adenocarcinoma/sangre , Adenocarcinoma/inmunología , Adolescente , Adulto , Anciano , Animales , Anticuerpos Antiidiotipos/sangre , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/inmunología , Complejo Antígeno-Anticuerpo/sangre , Antígenos de Neoplasias/sangre , Antígenos de Neoplasias/inmunología , Cromatografía Líquida de Alta Presión , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/inmunología , Electroforesis en Gel de Poliacrilamida , Femenino , Glicoproteínas/sangre , Glicoproteínas/inmunología , Humanos , Inmunoglobulina G/inmunología , Radioisótopos de Yodo , Masculino , Ratones , Persona de Mediana Edad , RadioinmunoensayoRESUMEN
Radioisotopes of Pb(II) have been of some interest in radioimmunotherapy and radioimmunoimaging (RII). However, the absence of a kinetically stable bifunctional chelating agent for Pb(II) has hampered its use for these applications. 203Pb (T(1/2) = 52.02 h) has application potential in RII, with a gamma-emission that is ideal for single photon emission computerized tomography, whereas 212Pb (T(1/2) = 10 h) is a source of highly cytotoxic alpha-particles via its decay to its 212Bi (T(1/2) = 60 min) daughter. The synthesis of the novel bifunctional chelating agent 2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra- (2-carbamoyl methyl)-cyclododecane (4-NCS-Bz-TCMC) is reported herein. The Pb[TCMC]2+ complex was less labile to metal ion release than Pb[DOTA]2- at pH 3.5 and below in isotopic exchange experiments. In addition to increased stability to Pb2+ ion release at low pH, the bifunctional TCMC ligand was found to have many other advantages over the bifunctional 1,4,7,10-tetraazacyclodocane-1,4,7,10-tetraacetic acid (DOTA) ligand. These include a shorter and more straightforward synthetic route, a more efficient conjugation reaction to a monoclonal antibody (mAb), with a higher chelate to protein ratio, a higher percent immuroreactivity, and a more efficient radiolabeling reaction of the mAb-ligand conjugate with 203Pb.
Asunto(s)
Quelantes/síntesis química , Compuestos Heterocíclicos/síntesis química , Isotiocianatos/síntesis química , Radioisótopos de Plomo , Quelantes/química , Electroforesis en Gel de Poliacrilamida , Compuestos Heterocíclicos/sangre , Compuestos Heterocíclicos/química , Humanos , Concentración de Iones de Hidrógeno , Isotiocianatos/sangre , Isotiocianatos/química , Estructura MolecularRESUMEN
Detailed synthesis of the bifunctional chelating agents 2-methyl-6-(p-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (1B4M-DOTA) and 2-(p-isothiocyanatobenzyl)-5, 6-cyclohexano-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetate (CHX-DOTA) are reported. These chelating agents were compared to 2-(p-isothiocyanatobenzyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (C-DOTA) and 1, 4, 7, 10-Tetraaza-N-(1-carboxy-3-(4-nitrophenyl)propyl)-N', N", N"'-tris(acetic acid) cyclododecane (PA-DOTA) as their (177)Lu radiolabeled conjugates with Herceptin. In vitro stability of the immunoconjugates radiolabeled with (177)Lu was assessed by serum stability studies. The in vivo stability of the radiolabeled immunoconjugates and their targeting characteristics were determined by biodistribution studies in LS-174T xenograft tumor-bearing mice. Relative radiolabeling rates and efficiencies were determined for all four immunoconjugates. Insertion of the 1B4M moiety into the DOTA backbone increases radiometal chelation rate and provides complex stability comparable to C-DOTA and PA-DOTA while the CHX-DOTA appears to not form as stable a (177)Lu complex while exhibiting a substantial increase in formation rate. The 1B4M-DOTAmay have potential for radioimmunotherapy applications.