Pharmacokinetics and biodistribution of samarium-153-labelled OC125 antibody coupled to CITCDTPA in a xenograft model of ovarian cancer

The use of samarium-153 in the context of radioimmunotherapy of cancers has been limited by the instability of antibody labelling, which produces high uptake concentrations in liver and bone. This study compares the pharmacokinetics and biodistribution of¹⁵³Sm-labelled OC125 monoclonal antibody, in whole or F(ab)₂ fragment form and with diethylene triamine penta-acetic acid (DTPA) or 6-p-isothiocyanatobenzyl diethylene triamine penta-acetic acid (CITCDTPA) coupling, in nude mice grafted subcutaneously with an ovarian adenocarcinoma line (SHIN-3) expressing CA125 antigen. The specific activity of the immunoconjugates was 18.5–55.5 MBq/mg, and their immunoreactivity exceeded 65%. With¹⁵³Sm-DTPA-OC125F(ab)₂, the stability study in serum indicated that 50% of the metal remained bound to the antibody. The pharmacokinetic study showed a retention half-life of 25.1 h and blood clearance of 0.72 ml/h. The biodistribution study indicated tumour uptake of 4.53%±9% of injected activity per gram (%ID/g) at 24 h and tumour-to-liver and tumour-to-bone ratios of 0.23±0.02 and 1.54±0.49 respectively at 24 h. With¹⁵³Sm-CITCDTPA-OC125F(ab)₂, serum stability was greater (87% of the metal remaining bound to the antibody), retention half-life was 22.25 h and blood clearance was 2.23 ml/h. Tumour was better targeted (8.30%±3.56%ID/g at 24 h), and tumour-to-liver and tumour-to-bone ratios were 1.17±0.36 and 7.08±3.09 respectively at 24 h. However, renal retention remained elevated (29.76%±9.41%ID/g at 24 h). With intact IgG, renal uptake decreased (1.41%±0.49%ID/g at 24 h), but tumour uptake was lower than with fragments (1.46%±0.58%ID/g at 24 h). Liver uptake was higher (tumour-to-liver ratio 0.10±0.05), and blood clearance was slower. The stability and distribution of¹⁵³Sm-CITCDTPA were more favourable than those of¹⁵³Sm-DTPA for application in radioimmunotherapy. Quantitative analysis performed using digitized images obtained by conventional autoradiography and the imaging plate system indicated that the latter system is suitable for bio-distribution studies of immunoconjugates.     

In vivo evaluation of a lead-labeled monoclonal antibody using the DOTA ligand

The aim of this study was to assess the utility of a radioimmunoconjugate containing a lead radionuclide for therapy and scintigraphy applications. The radioimmunoconjugate evaluated consisted of a bifunctional DOTA ligand and monoclonal antibody (MAb) B72.3 using athymic mice bearing LS-174T tumors, human colon carcinoma xenografts. In the studies reported here, the lead-203-DOTA complex itself was first demonstrated to have in vivo stability. MAb B72.3 was then conjugated with the DOTA ligand and labeled with ²⁰³Pb, and the immunoreactivity of B72.3 was maintained. The localization of the radioimmunoconjugate to tumor tissue and other select organs paralleled that of DOTA-¹²⁵I-B72.3, suggesting a similar metabolic pattern of the two radioimmunoconjugates. Thus, the DOTA-metal complex does not alter the behavior of the radioimmunoconjugate. Tumor localization of the ²⁰³Pb-DOTA-B72.3 conjugate was demonstrated with biodistribution studies as well as immunoscintigraphy studies. Such data highlight the stability of a lead radionuclide in the DOTA ligand. The suitability of this chelation chemistry for labeling radioimmunoconjugates with a lead radionuclide now makes its application in nuclear medicine a feasible proposition. Received 25 October 1997 and in revised form 23 January 1998     

Pilot study with the monoclonal antibody IOR-C5 as a potential agent of radioimmunoscintigraphy in colorectal cancer

IOR C-5 is a G1 immunoglobulin type intact murine monoclonal antibody (MAb) that was developed in the Center of Molecular Immunology in Havana City, Cuba. In immunohistochemical studies, this demonstrated a significant affinity for the epithelial tissues so that it was used in a pilot clinical study to perform a radioimmunoscintigraphy of the colorectal primary tumors and their locoregional recurrences. It was labeled with 99mTc using the Schwarz method, with a > 95% performance. Planar images of the chest, abdomen and pelvis were performed at 10 minutes, 4-6 hours and 18-24 hours post-injection in the anterior and posterior projections and the SPECT was performed 4-6 hours and 18-24 hours post-injection of 1.85 GBq 99mTC. This study has aimed to verify in vivo the capacity of ior-C5 MAb to accumulate in the malignant colorectal lesions. ior-C5 accumulated in 5 out of the 7 patients who were studied and who were suffering from colorectal cancer or in whom there was suspicion of recurrence. There was a negative case of primary tumors, which was an adenocarcinoma in situ in a tubular-papillary adenoma. The second case with a negative radioimmunoscintigraphy was a true negative case.Conclusions: It can be concluded that even though the number of patients is quite low, ior-C5 fulfilled the expectations of recognizing the epitope expressed in colorectal tumors in an in vivo human environment.     

Radioiodinated B6.2 monoclonal antibody: further characterization of a potential radiopharmaceutical for the identification of breast tumors

A monoclonal antibody (B6.2) directed against human breast carcinomas and its F(ab')2 and Fab' fragments were labeled with 125I using Iodogen. Solid phase radioimmunoassay was used to evaluate the in vitro binding of the labeled antibodies to a human breast tumor metastasis to the liver, the MCF-7 breast tumor cell line and normal liver tissue. Scatchard analysis revealed that 125I-labeled B6.2 IgG and F(ab')2 bound to both breast tumors with affinity constants in the range 1.2-4.4 X 10(9) M-1, while the affinity constant for the binding of the 125I-labeled Fab' fragment to the metastasis and the MCF-7 line was 5.9 and 9.1 X 10(8) M-1, respectively. Serial blood sampling indicated that blood clearance of 125I activity decreased with increasing protein size. A comparison of tumor-to-tissue ratios at two doses of 125I-B6.2 IgG and F(ab')2 suggests that the optimal dose of antibody may depend on the imaging time. A comparison of the blood clearance of 125I activity following injection of 125I-B6.2 and nonspecific 125I-MOPC 21 demonstrated that the specific antibody cleared much more rapidly. When 125I-B6.2 was injected into mice bearing either breast or melanoma tumors, the thyroid uptake of 125I was significantly greater in mice with breast tumors. These results suggest that the catabolism of radiolabel from the specific antibody in the presence of tumor is greater and that this may be an important factor in determining the optimal radiolabel for immunoscintigraphy.     

An in vitro model for the scintigraphic detection of thrombi using a 99Tcm-labelled antifibrin monoclonal antibody

Because of their specific targeting properties, monoclonal antibodies have found widespread use in nuclear medicine. In this paper, a method is described for the evaluation of immunoscintigraphic parameters for the detection of thrombi, using a 99Tcm-labelled antifibrin monoclonal antibody (designated as Y22). An in vitro model was developed to evaluate the effects of various environmental conditions on uptake by plasma clots of 99Tcm-Tc-Y22 in circulating plasma on a gamma camera. The clots became visible as hotspots after approximately 1 h of circulation of 99Tcm-Y22 containing citrated plasma at 37 degrees C. Circulation of 99Tcm-fibrinogen, 99Tcm-HSA or 99Tcm-control MoAb did not show visible uptake by the clots under the same conditions. At 37 degrees C, 99Tcm-Y22 accumulated approximately four times faster than at 20 degrees C. Heparin did not affect binding of the antibody to clots. To assess the feasibility of thrombus detection in vivo, an extracorporeal rat thrombus model was used. A thrombus in a shunt between a carotid artery and a jugular vein became visible 1 h after injection of the labelled Y22 and, more clearly, after 3 h.     

A prospective evaluation of OC125 and magnetic resonance imaging in patients with ovarian carcinoma

Eighteen patients with suspected primary or recurrent ovarian carcinoma have been investigated in each case by the assay of serum levels of the antigen CA125, immunoscintigraphy using¹³¹I-OC125 antibody and magnetic resonance imaging using a 0.15 Tesla-system. The final diagnosis was confirmed by laparotomy or laparoscopy. Serum levels of CA125 ranged between 5 and 780 units/ml (normal range < 35). Antibody images and MRI were truly positive in 11 patients, 2 of whom were subsequently found to have bowel tumours. MRI showed greater detail of smaller lesions whilst immunoscintigraphy was more suited to the detection of distant metastases. In 7 patients the antibody images were positive whilst the serum marker levels were normal. This pilot study provides a preliminary comparison of the more recent techniques currently being evaluated for the detection of ovarian carcinoma.     

A prospective evaluation of OC125 and magnetic resonance imaging in patients with ovarian carcinoma

Eighteen patients with suspected primary or recurrent ovarian carcinoma have been investigated in each case by the assay of serum levels of the antigen CA125, immunoscintigraphy using 131I-OC125 antibody and magnetic resonance imaging using a 0.15 Tesla system. The final diagnosis was confirmed by laparotomy or laparoscopy. Serum levels of CA125 ranged between 5 and 780 units/ml (normal range less than 35). Antibody images and MRI were truly positive in 11 patients, 2 of whom were subsequently found to have bowel tumours. MRI showed greater detail of smaller lesions whilst immunoscintigraphy was more suited to the detection of distant metastases. In 7 patients the antibody images were positive whilst the serum marker levels were normal. This pilot study provides a preliminary comparison of the more recent techniques currently being evaluated for the detection of ovarian carcinoma.     

Use of PET in neuroendocrine tumors. In vivo applications and in vitro studies.

Positron emission tomography (PET) performed with various radiolabelled compounds facilitates the study of tumor biochemistry. If the tumor uptake of an administered tracer is greater than that of surrounding normal tissue, it is also possible to localize the tumor. In initial studies, 18F-labeled deoxyglucose (FDG) was attempted to visualize the tumors, since this tracer had been successfully used in oncology, reflecting increased glucose metabolism in cancerous tissue. However, this tracer was not to any significant degree taken up by the neuroendocrine tumors. Instead, the serotonin precursor 5-hydroxytryptophan (5-HTP) labeled with 11C was used and showed an increased uptake and irreversible trapping of this tracer in carcinoid tumors. The uptake was selective and the resolution so high that we could detect more liver and lymph node metastases with PET than with CT or octreotide scintigraphy. One problem was, however, the high renal excretion of the tracer producing streaky artifacts in the area of interest. Using the decarboxylase inhibitor carbidopa, given as peroral premedication, the renal excretion decreased 6-fold and at the same time the tumor uptake increased 3-fold, hence improving the visualization of the tumors. When patients were followed during treatment with PET using 5-HTP as a tracer, a > 95% correlation between changes in urinary 5-hydroxyindoleacetic acid (U-5-HIAA) and changes in the transport rate constant for 5-HTP was observed. Thus, PET can be used to monitor treatment effects. Elevation of U-5-HIAA is considered to be uncommon in endocrine pancreatic tumors (EPTs). Initially, 11C-labeled L-DOPA was attempted as another amine important in the APUD system. With L-DOPA about half of the EPTs, mainly functioning tumors, could be detected. Recently, 5-HTP was explored as a universal tracer also for EPT and foregut carcinoids, extending the PET-examination to both thorax and abdomen (whole-body PET-examination). With this method we were able to visualize small lesions in the pancreas and thorax (e.g. ACTH-producing bronchial carcinoids) not detectable by any other method including octreotide scintigraphy, MRI and CT. Several other tracers have been investigated, e.g. the monoamineoxidase (MAO-A) inhibitor harmine with promising results in non-functioning EPTs. We are currently exploring a wide range of biochemical systems, including enzymes and receptors, both for neurotransmitters and for peptides and proteins in in vitro assays with the potential to use some of the developed tracers for in vivo visualization and tumor biological studies. In conclusion, PET is a valuable tool in the diagnosis of neuroendocrine tumors. It can detect small lesions in the thorax and abdomen not detected by other methods, which has been of great value preoperatively in several cases. It detects more lesions in the liver and lymph nodes than other methods and furthermore, it can be used to monitor treatment effects.     

In vivo and in vitro characterizations of three 99mTc-labeled monoclonal antibody G250 preparations.

In previous clinical studies, excellent visualization of tumor lesions has been observed with 131I-labeled monoclonal antibody (mAb) G250 in patients with renal cell carcinoma (RCC). In several cases, 131I-cG250 immunoscintigraphy disclosed tumor lesions that were not visualized by radiography or CT. To improve image quality, we aimed to develop a 99mTc-labeled mAb G250 preparation for radioimmunodetection of RCC. We studied in vitro stability, biodistribution and imaging potential of three 99mTc-labeled G250 preparations in nude mice with subcutaneous RCC xenografts.125I-G250 and the nonspecific mAb 131I-MN14 were used as control antibodies. METHODS: The mAb G250 was labeled with 99mTc according to three methods using: (a) S-hydrazinonicotinamide (HYNIC), (b) S-benzoylmercaptoacetyltriglycine (MAG3) and (c) a direct labeling method (Schwarz method). The stability of all preparations was tested in serum at 37 degrees C during 48 h. In addition, diethylenetriamine pentaacetic acid, cysteine and glutathione challenge assays were performed. RESULTS: All preparations showed good stability in serum during the 48-h incubation period. 99mTc-G250 (Schwarz) showed release of the radiolabel at a 100-fold or higher molar excess of cysteine and at a 10,000-fold or higher molar excess of glutathione. 99mTc-MAG3-G250 showed release of the radiolabel at a 10,000-fold molar excess of cysteine. 99mTc-HYNIC-G250 was stable under all conditions. Tumors were clearly visualized with all preparations. 99mTc-G250 (Schwarz) showed significantly lower blood levels (3.8 %ID/g) compared with all other preparations (11.2, 13.4 and 13.4 %ID/g for 99mTc-HYNIC-G250, 99mTc-MAG3-G250 and 125I-G250, respectively, 48 h postinjection). At 48-h postinjection, mean tumor uptake was very high with all mAb G250 preparations: 92.4 (99mTc-HYNIC-G250), 125.9 (99mTc-MAG3-G250), 29.4 (99mTc-G250 Schwarz) and 75.4 (125I-G250) %ID/g. Mean tumor uptake of the nonspecific 131I-MN14 mAb was 6.6 %ID/g. CONCLUSION: In this study, 99mTc-HYNIC-G250 showed excellent in vitro stability and tumor targeting. Moreover, this preparation could be labeled with high efficiency (>95%) at room temperature within 15 min. Therefore, 99mTc-HYNIC-G250 seems to be an ideal candidate for radioimmunodetection of RCC.     

In vitro and in vivo comparison of DTPA- and DOTA-conjugated antiferritin monoclonal antibody for imaging and therapy of pancreatic cancer

Pancreatic cancer has a very poor prognosis with a less than 5% survival rate at 5 years. Neither external beam radiation nor chemotherapy, alone or in combination, have given encouraging results so far. A possible solution might come from the use of targeted therapy such as radioimmunotherapy. We present here the results obtained from the preclinical development of a new monoclonal antiferritin antibody (Ab), AMB8LK. Ferritin is overexpressed in pancreatic cancer and could thus be used as a target for the delivery of radioactivity at the tumour sites. The AMB8LK Ab was conjugated to three chelating agents: the 2-(4-isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid (PSCN-Bz-DTPA), the (R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid (p5CN-Bz-CHX-A"-DTPA) and the 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (pSCN-Bz-DOTA). Radiolabelling of the three immunoconjugates with indium 111 and yttrium 90 as well as in vitro stability and immunoreactivity against pure ferritin and cells expressing ferritin were analysed. In vivo biodistribution studies were conducted on normal and on human pancreatic adenocarcinoma CAPAN-1 tumour bearing mice. These experiments demonstrated good radiolabelling (>95%), stability and immunoreactivity of the three compounds. In the biodistribution studies, differences between the three immunoconjugates were apparent in the rate of blood clearance and in tumour, liver and bone uptake. A very good pancreatic adenocarcinoma tumour targeting was observed especially with the Bz-DTPA-AMB8LK: 20% of the injected dose of the indium-labelled compound 3 days after injection; 15% of the injected dose 5 days after that of the yttrium-labelled Ab. Altogether, these results in animal models suggest that (90)Y-Bz-DTPA-AMB8LK is a good candidate for further therapeutic efficacy studies.     

Immunoscintigraphy of human ovarian cancer xenografts using a radiolabelled monoclonal antibody to human pregnancy serum-derived immune complexes

The in vivo imaging of xenografted human ovarian cancer in nude mice with a specific and control radiolabelled monoclonal antibody (MoAb) is described. The specific MoAb was previously raised by immunizing mice with immune complexes derived from late human pregnancy serum. In the first group of mice the specific MoAb 131I-5E3 F(ab')2 was injected, while a second group received equivalent amounts of a control MoAb 131I-UJ13A F(ab')2. The mice were imaged at various times up to a maximum of 2 weeks using a gamma camera, and the tumour to non-tumour (T/NT) ratio was recorded for each group. The T/NT ratio rose to 2.02 in the specific group, while the corresponding ratio in the control group was 0.70. In addition, the count rate in the tumour and non-tumour regions was determined on each imaging occasion. Biological half-lives of the divalent fragments of 5E3 and UJ13A in the tumour were 7.53 days and 0.62 days, respectively. Following sacrifice, the tumours were excised and counted relative to the rest of the animal, and the T/NT ratio was calculated. In vitro results were in direct agreement with those recorded in vivo using the gamma camera. From the results it would appear that the divalent fragment of 5E3, which has been raised to immune complexes derived from late human pregnancy serum, is specific for human ovarian tumour xenografts in the nude mouse model.     

In vitro and in vivo properties of human/mouse chimeric monoclonal antibody specific for common acute lymphocytic leukemia antigen

A human/mouse chimeric monoclonal antibody specific for a common acute lymphocytic leukemia antigen was efficiently obtained by ligating human heavy-chain enhancer element to the chimeric heavy- and light-chain genes. Cell binding and competitive inhibition assays of both radioiodine and indium-111- (111In) labeled chimeric antibodies demonstrated in vitro immunoreactivity identical with that of the parental murine monoclonal antibodies. The biodistribution of the radiolabeled chimeric antibody in tumor-bearing nude mice was similar to that of the parental murine antibody. Tumor accumulation of radioiodinated parental and chimeric antibodies was lower than that of 111In-labeled antibodies, probably because of dehalogenation of the radioiodinated antibodies. Indium-111-labeled chimeric antibody clearly visualized xenografted tumor. These results suggest that a human/mouse chimeric antibody can be labeled with 111In and radioiodine without the loss of its immunoreactivity, and that chimeric antibody localizes in vivo in the same way as the parental murine antibody.     

Evaluation of therapeutic response using iodine-131-B72.3 monoclonal antibody in patients with ovarian carcinoma

We used radiolabelled monoclonal antibodies (MoAbs) to prove disease persistence after treatment for ovarian carcinoma. Twelve patients with histologically confirmed ovarian carcinoma were studied. They received 5 mCi (1 mg) of iodine-131-B72.3 by intravenous injection before and after a complete course of chemotherapy. Images were obtained with a LFOV gamma camera 2 h after MoAb administration and daily up to 6 days. Before treatment 8 patients had a true positive scan. Questionable antibody uptake was observed in 2 patients while 1 had a true negative scan and 1 a false-negative examination. After treatment the therapeutic response was evaluated. Five patients had partial remission and antibody scan showed persistence of disease in all patients except 1. Four patients showed progression of the disease and 1 no change. The antibody scan was positive in 4 and questionable in 1. Two patients had complete remission and negative antibody scans. Computed tomography (CT) could not always discriminate postoperative fibrosis from tumour lesions, especially when the peritoneum was involved in the disease. High serum levels of tumour markers were constantly associated with the presence of tumour lesions, but normal values did not guarantee absence of disease. We conclude than the antibody scan is complementary to CT and serum tumor markers in the definition of therapeutic response.     

In vitro evaluation of the astatinated chimeric monoclonal antibody U36, a potential candidate for treatment of head and neck squamous cell carcinoma

Purpose The purpose of this study was to analyse the properties of the astatinated chimeric MAb (cMAb) U36 as a conjugate to selectively target and eradicate head and neck squamous cell carcinoma (HNSCC).Methods cMAb U36 was labelled with ²¹¹At via the linker N-succinimidyl 4-(trimethylstannyl)benzoate (SPMB). The quality of the conjugate was extensively evaluated for binding and internalisation capacity, and compared with ¹²⁵I-SPMB-cMAb U36. The cellular toxicity of the astatinated conjugate was assessed in two types of in vitro growth assay and compared with ¹³¹I-labelled cMAb U36 (directly labelled).Results Comparisons between ²¹¹At-cMAb U36 and ¹²⁵I-cMAb U36 demonstrated an optimal functional capacity of the labelled products. Immunoreactivity and affinity assays showed high immunoreactive fractions (>93%), and an affinity in good agreement between the astatinated and iodinated antibodies. For both conjugates, specific binding to HNSCC cells could be demonstrated, as well as some internalisation. Retention of the astatinated conjugate was just slightly lower than for the iodinated conjugate and still reasonable for therapeutic use (31±2% vs 42.6±1.0% at 22 h), demonstrating no adverse effects from astatination of the antibody. Studies on cellular toxicity demonstrated a dose-dependent and antigen-specific cellular toxicity for ²¹¹At-cMAb U36, with about 10% cell survival at 50 decays per cell. The ¹³¹I-labelled conjugate was not as efficient, with a surviving cell fraction of about 50% at 55 decays per cell.Conclusion These results indicate that ²¹¹At-cMAb U36 might be a promising future candidate for eradicating HNSCC micrometastases in vivo.     

Evaluation of therapeutic response using iodine-131-B72.3 monoclonal antibody in patients with ovarian carcinoma

We used radiolabelled monoclonal antibodies (MoAbs) to prove disease persistence after treatment for ovarian carcinoma. Twelve patients with histologically confirmed ovarian carcinoma were studied. They received 5 mCi (1 mg) of iodine-131-B72.3 by intravenous injection before and after a complete course of chemotherapy. Images were obtained with a LFOV gamma camera 2 h after MoAb administration and daily up to 6 days. Before treatment 8 patients had a true positive scan. Questionable antibody uptake was observed in 2 patients while 1 had a true negative scan and 1 a false-negative examination. After treatment the therapeutic response was evaluated. Five patients had partial remission and antibody scan showed persistence of disease in all patients except 1. Four patients showed progression of the disease and 1 no change. The antibody scan was positive in 4 and questionable in 1. Two patients had complete remission and negative antibody scans. Computed tomography (CT) could not always discriminate postoperative fibrosis from tumour lesions, especially when the peritoneum was involved in the disease. High serum levels of tumour markers were constantly associated with the presence of tumour lesions, but normal values did not guarantee absence of disease. We conclude than the antibody scan is complementary to CT and serum tumor markers in the definition of therapeutic response.Preliminary results were presented at the 2nd International Conference on Diagnosis and Therapy with Monoclonal Antibodies, Naples, Italy, 26–28 April 1990     

In vitro and in vivo effects of diethylene triamine penta-acetic acid on the distribution of indium-111 monoclonal antibody metabolism

The effects of diethylene triamine penta-acetic acid (DTPA) on indium-111 monoclonal antibody (MoAb) metabolism were examined. Sequential analysis of ¹¹¹In-MoAb incubated in serum at 37°C by high performance liquid chromatography (HPLC) and electrophoresis revealed that the radioactivity gradually moved from the MoAb to a 70–90 kDa molecular weight fraction. DTPA inhibited the transchelation of ¹¹¹In to this fraction. It also decreased ¹¹¹In uptake by isolated rat heptocytes but did not remove ¹¹¹In incorporated in hepatocytes. The daily in vivo administration of DTPA (0.5–2.0 mg/mouse daily) to athymic mice after ¹¹¹In-MoAb injection significantly reduced the ¹¹¹In uptake in the liver and kidney. The tumour uptake was decreased somewhat but not significantly. The serum radioactivity in the 70–90 kDa fraction was also decreased. Scintigraphic examination demonstrated a decreased liver uptake in the DTPA-treated group of mice. Our results show that ¹¹¹In released from the DTPA-MoAb conjugate in serum binds to molecules of 70–90 kDa and that DTPA decreases the ¹¹¹In uptake in this fraction, which induces a decrease of ¹¹¹In accmulation in normal tissues. Offprint requests to: Y Kimura