The effect of circulating antigen and radiolabel stability on the biodistribution of an indium labelled antibody

This study has investigated two of the main problems with radiolabelled antibody imaging, the formation of circulating immune complexes (I.C.) and the non specific binding of radiolabel to the antibody molecule. Patients undergoing immunoscintigraphy with 111In labelled monoclonal antibody ICR2 were divided into three groups who received either the radiolabelled antibody alone (control, n = 12), the radiolabelled antibody which was incubated with the chelating agent diethylene triamine pentacetic acid (DTPA) prior to size exclusion chromatography (n = 6) or whose injectate was treated with DTPA and cold MAb administered intravenously prior to radiolabelled MAb administration (n = 6). Radiolabelled antibody uptake in abdominal organs was measured by region of interest analysis using a gamma camera with online computer and that in tumour and normal tissues by gamma well counting of biopsies. Circulating antigen and immune complex was measured by high pressure liquid chromatography (HPLC). The sensitivity of tumour imaging and the tumour uptake of radiolabelled antibody was not significantly different between the groups. Patients with high circulating antigen levels developed high levels of circulating immune complex but also had high tumour uptakes of radiolabelled antibody. Administration of cold MAb increased the splenic, but did not effect the tumour uptake of radiolabelled antibody and only minimally reduced levels of circulating immune complex. Chelate administration reduced the urinary excretion of radioactivity but increased the liver uptake of radioactivity. These results have shown that successful antibody imaging can be carried out despite high levels of circulating antigen, that large doses of unlabelled antibody are required to prevent immune complex formation and that removal of non specifically bound 111In does not reduce the liver uptake of radioactivity.     

Intraperitoneal radio-localization of tumors pre-targeted by biotinylated monoclonal antibodies

We describe 2-step and 3-step strategies for intraperitoneal tumor radio-localization by means of monoclonal antibodies (MAbs). Nude mice bearing intraperitoneal human colon carcinoma tumors were injected i.p. with biotinylated MAb AUAI, followed 24 hr later by radioiodinated streptavidin (2-step). The uptake of radioactivity in tumor and normal tissues was measured 4 hr after injection of radioactive compound. A 3-step strategy consisted in administering biotinylated antibody, cold avidin after 24 hr and 111In-labelled biotin after a further 4 hr; mice were then killed 2 hr later. Tumor localization of intraperitoneally-administered biotinylated antibody and direct targeting of radioactive streptavidin to biotinylated antibody bound to tumor sites were demonstrated using immunohistochemistry and autoradiography. Our results show that (i) the 2-step approach increased the percentage of radioactivity uptake by tumor with respect to directly labelled antibodies (24% vs. 6%) and improved the tumor/non-tumor ratio; (ii) the 3-step approach allowed faster blood clearance of the radioactive probe (111In-biotin) and yielded high tumor/non-tumor ratios. "Pre-targeting" methods appear to have advantages over the conventional 1-step approach with directly radiolabelled antibody.     

Kinetics, quantitative analysis and radioimmunolocalization using indium-111-HMFG1 monoclonal antibody in patients with breast cancer

HMFG1 tumour associated monoclonal antibody IgG1 and F(ab')2 fragments were radiolabelled with indium-111 and used to study patients with breast cancer. In vitro and in vivo stability of the radiolabelled antibodies was shown to be satisfactory. Thirty patients with primary breast cancer underwent tumour resection and quantitative evaluation of the radioactivity in the tumour and normal tissues following administration of specific and non-specific antibodies. The mean tumour uptake of HMFG1 F(ab')2 fragments at 24 h was significantly higher (P less than 0.05) than the intact IgG but at 48 h there was no difference. The mean tumour uptake with the specific antibody was higher than the non-specific antibody of the same subclass (P less than 0.05). Lymph node metastases showed higher antibody uptake than the corresponding primary tumours (P less than 0.05). Fifteen patients with primary or metastatic breast cancer were investigated by external body scintigraphy using HMFG1 F(ab')2 fragments. Successful localisation was observed in approximately 50% of the primary and metastatic lesions with no false positive results. All the patients had observable concentration of 111In in the liver (20% of the injected dose), the kidneys and the spleen. Following i.v. administration, F(ab')2 fragments cleared from the blood more rapidly than the intact IgG. We conclude that HMFG1 F(ab')2 fragments can localise specifically and faster than intact IgG in breast cancer but the sensitivity of the radioimmunoscintigraphy is relatively low. This method needs further improvement before becoming clinically useful for detecting and staging breast cancer.     

Monoclonal antibody Po66 uptake by human lung tumours implanted in nude mice: effect of co-administration with doxorubicin.

The efficacy of radioimmunotherapy of tumours with radiolabelled monoclonal antibodies (MAbs) depends on the amount of antibody taken up by the tumour and on its intratumoral distribution. In the case of MAbs directed against intracellular antigens, increasing the permeability of the cytoplasmic membrane may augment the bioavailability of the antigen for the antibody. This raises the question whether the induction of tumour necrosis by chemotherapy can enhance the tumour uptake of radiolabelled monoclonal antibodies. In this work, the effect of doxorubicin on the biodistribution of Po66, an MAb directed against an intracellular antigen, was studied in nude mice grafted with the human non-small-cell lung carcinoma cell line SK-MES-1. After injection on day 0 of 125I-labelled Po66, tumour radioactivity increased up to days 3-5, and then remained unchanged to day 14. The combined administration of 125I-labelled Po66 with 8 mg kg-1 doxorubicin, in two doses separated by 7 days, doubled the radioactivity retained by the tumour. Histological and historadiographic analysis showed, however, that the drug induced cellular damage. In the absence of doxorubicin, the accumulation of Po66 was restricted to some necrotic areas, whereas with doxorubicin the necrosis was more extensive and the antibody more evenly distributed. These results suggest that chemotherapy and immunoradiotherapy combined would enhance tumour uptake of radioisotope and promote more homogenous distribution of the radiolabelled MAb. This would promote eradication of the remaining drug-resistant cells in tumours.     

Immunoscintigraphy of small-cell lung cancer xenografts with anti neural cell adhesion molecule monoclonal antibody, 123C3: improvement of tumour uptake by internalisation.

The efficacy of three murine monoclonal antibodies (MAbs) for immunoscintigraphy of small-cell lung cancer (SCLS) xenografts was studied in a Balb/c nu/nu mouse model. These Mabs, 123C3, 123A8 and MOC191, belong to cluster 1 of anti-SCLC MAbs and bind to the neural cell adhesion molecule (NCAM) with similar affinity. After intraperitoneal injection of these MAbs, labelled with 125I, the highest uptake in tumour tissue was obtained with MAb 123C3. Seven days after the administration of this MAb 13.9% of the injected dose per gram of tumour tissue was retained in the tumour. The corresponding tumour tissue ratios ranged from 3.97 for blood to 31.03 for colon. The imaging results and the tumour uptake were less favourable for the two other MAbs, 123A8 and MOC191 (fractions of injected dose respectively 6.7% and 9.2%), although affinity, biological activity after labelling and uptake in non-tumour tissues were very similar for all three MAbs. These results may be explained by the differences in the interaction between the MAbs and the tumour cells. Mab 123C3 is internalised into tumour cells, whereas both other anti-NCAM Mabs are not. Internalisation into NCI H69 cells was demonstrated in vitro by radioimmunoassay, confocal laser scanning microscopy and electron microscopy. The internalised fraction of MAb 123C3 was 22.3% after 24h, whereas this fraction was only 7.5% for MAb 123A8. Although the internalised radiolabeled Mabs are usually degraded and dehalogenated intracellularly, the retained radioactivity is high. Apparently, intracellular degradation of radiolabelled MAb 123C3 and subsequent secretion of radioactive iodine did not prevent the accumulation of intracellular radioactivity. In conclusion, accumulation and retention of radioactivity in the tumour tissue, due to internalisation of radiolabelled MAbs, may improve the results immunoscintigraphy.     

Biodistribution of lymphokine-activated killer (LAK) cells in Wag rats after hepatic-artery or jugular-vein infusion.

This study deals with the biodistribution of syngeneic radiolabeled lymphokine-activated killer (LAK) cells in Wag rats after infusion via the hepatic artery or the jugular vein. The biodistribution of 111Indium-labeled LAK cells was evaluated using serial whole-body gamma camera imaging. Furthermore, we investigated 2 factors that might influence the biodistribution of these effector cells: purity of LAK cells and administration of interleukin-2 (IL-2). After injection of 111Indium-labeled LAK cells via the hepatic artery or via the jugular vein we could detect important differences in the biodistribution pattern up to 5 hr after injection. LAK cells administered via the jugular vein were all found in the lungs up to 2 hr after injection and then redistributed to the liver and to the spleen. LAK cells administered via the hepatic artery were all found in the liver after injection and redistributed after 2 hr mainly to the spleen. About 8 hr after injection we could no longer detect any differences in the biodistribution pattern according to the route of administration. Biodistribution was followed for up to 72 hr after injection but the pattern showed no change after 8 hr, whichever the route of administration. A purified adherent-LAK population, a large granular lymphocyte culture with only 6% T cells, showed the same distribution pattern as standard LAK cells (40% T cells). Infusions of 40,000 units of IL-2 per day, starting 3 days before and continuing after administration of radio-labeled LAK cells, accelerated the redistribution of these cells by both routes of administration. We conclude that up to 2 hr after local infusion, a high concentration of LAK cells in the first capillary bed can be obtained. Therefore, local administration of LAK cells may be more effective against tumors.     

In vivo formation of aflatoxin B1-DNA adducts in parenchymal and non-parenchymal cells of rat liver.

The induction of hepatocellular carcinoma from liver parenchymal cells in laboratory animals by aflatoxin B1 (AFB1) is well documented. In contrast no tumours arising from the sinusoidal cell population have been reported after exposure to AFB1. The apparent resistance of the latter cell type was investigated at the level of DNA adduct formation in vivo in male Sprague-Dawley rats. Liver parenchymal and non-parenchymal cell populations were isolated from rats at 20 min and 1, 24 and 72 h after administration of 240 microCi (0.6 mg) [G-3H]AFB1/kg. AFB1-DNA binding was observed in both liver cell subpopulations and was 3- to 5-fold higher in parenchymal cells than in non-parenchymal cells. The major DNA adduct found in parenchymal cells at 1 h after AFB1 administration was 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-gua), whereas at later time points the persistent secondary adduct, AFB1-formamidopyrimidine, predominated. In contrast, AFB1-gua was not observed at any time in DNA from non-parenchymal cells and the secondary adducts predominated throughout. These observations are discussed with reference to the susceptibility of different liver cell types to AFB1-carcinogenesis and the possible roles of the major AFB1-DNA adduct species.     

The low uptake of an abrin A-chain immunotoxin by rat hepatic cells in vivo and in vitro

The therapeutic value of antibody-ricin A-chain conjugates (immunotoxins) as antineoplastic agents is limited by their rapid removal from the circulation, primarily by cells of the liver which take up the immunotoxin through receptor mediated recognition of mannose-containing oligosaccharides in the toxin A-chain. We have therefore examined the uptake by rat hepatic cells of a monoclonal antibody (LICR-LOND Fib 75) conjugate assembled with the ricin related, but carbohydrate free, A-chain of the plant toxin abrin. The abrin A-chain immunotoxin was very poorly taken up in vivo and in vitro by both hepatic parenchymal and non parenchymal cells whereas a comparable conjugate assembled with ricin A-chain was actively accumulated by liver cells particularly the hepatic non-parenchymal cells. Furthermore, the abrin A-chain immunotoxin uptake by non-parenchymal cells in vitro was unaffected by mannose and the immunotoxin bound less readily to liver cells than did the ricin A-chain conjugate, consistent with a proposal that its accumulation by hepatic cells is brought about by endocytosis following non-specific binding or by fluid phase pinocytosis. These results suggest abrin A-chain immunotoxins might be further explored as anti-cancer agents since in some cases they could have an improved therapeutic efficacy over immunotoxins constructed with ricin A-chains.     

High-dose therapy with 90Yttrium-labeled monoclonal antibody CC49: a phase I trial.

A Phase I trial of increasing administered activities of 90yttrium (90Y)-labeled monoclonal antibody (MAb) CC49 was conducted to determine whether extrahematopoietic toxicity occurred with this radioimmunoconjugate. Twelve patients with various gastrointestinal tract cancers were administered a tracer dose of 111In-labeled MAb CC49 for biodistribution and pharmacokinetic studies. Patients then underwent a single treatment with increasing administered activities of 90Y-labeled MAb CC49 (0.3, 0.4, and 0.5 mCi/kg). Biodistribution studies, using 111In-labeled MAb CC49 as a surrogate, were determined using planar and single photon emission computed tomography imaging. Pharmacokinetic studies were performed by measuring radioactivity in blood samples taken at intervals after radioimmunoconjugate infusions. Tissue biopsies of tumor metastases and related normal tissues (liver and bone marrow) were obtained for radioactivity measurements. Radiation dosimetry estimates were calculated using these data. Toxicity was evaluated using the National Cancer Institute Common Toxicity Criteria. No dose limiting extrahematopoietic toxicity was identified in the range of administered activities used in this study. Radioimmunolocalization based on planar and single photon emission computed tomography images 111In-labeled MAb CC49 showed heterogeneous (nonspecific) liver and splenic uptake. Liver metastases were usually photopenic, and extrahepatic metastases showed faint to moderate uptake. The alpha and beta half-lives of 111In-labeled MAb CC49 and 90Y-labeled MAb CC49 in the blood were similar. Absorbed radiation dose estimates in metastatic tumor sites ranged from 180 to 3000 cGy. The percentage of injected dose/kg of tumor ranged from 1.12 to 18.14; however, tumor:normal liver ratios were consistently <1. No objective responses were observed. Doses of up to 0.5 mCi/kg could be administered with reversible grade IV myelotoxicity. Absorbed radiation dose in tumor was suboptimal, even at the highest administered activity level. Deposition of 90Y in liver was high, and estimates of absorbed dose in liver equaled or exceeded that which could be achieved in metastatic tumor sites. Strategies to enhance access of radioimmunoconjugates in tumor and diminish deposition in the liver need to be developed for effective treatment using MAb CC49 with chelated radiometals.     

Neovascular targeting with cyclic RGD peptide (cRGDf-ACHA) to enhance delivery of radioimmunotherapy

Radioimmunotherapy (RIT) has been hampered by delivery of only a small fraction of the administered dose of radiolabeled MAb to tumor. A strategy for creating and controlling tumor vascular permeability would enable more effective RIT. The alpha v beta 3 integrin receptor is an appealing target for strategies designed to enhance permeability of tumor vessels because it is highly and preferentially expressed in most tumors. In human tumor mouse models, apoptosis of neovascular endothelial cells has been demonstrated after treatment with alpha v beta 3 antagonists. Since this apoptotic effect could transiently increase permeability of tumor blood vessels, radiolabeled antibodies (MAb) circulating during this period would have increased access to extravascular tumor. To determine if this hypothesis was correct, a pharmacokinetic study of an immunospecific MAb given after an alpha v beta 3 antagonist was performed in nude mice bearing human breast cancer xenografts. The alpha v beta 3 antagonist, cyclic RGD pentapeptide (c-RGDf-ACHA; cyclo arginine glycine aspartic acid D-phenylalanine -1 amino cyclohexane carboxylic acid), inhibits alpha v beta 3 binding to its vitronectin ligand at nanomolar levels. Cyclic RGD peptide (250 micrograms i.p.) given 1 hour before 111In-ChL6 MAb resulted in a 40-50% increase in tumor uptake (concentration), when compared to the control tumor uptake, of MAb 24 hours after administration. When cyclic RGD peptide was given as a continuous infusion (17.5 micrograms/hr) for 1 or 24 hours before 111In-ChL6, tumor uptake of 111In-ChL6 was increased less, and, these data were not statistically different from the control data. There were no differences for any of the groups in the groups in the concentrations of 111In-ChL6 in normal organs or blood when compared to the control group. The results suggest that cyclic RGD peptide provided a temporary, selective increase in tumor vascular permeability, that allowed a larger fraction of the 111In-ChL6 to accumulate in the tumor.     

Localization and imaging of radiolabelled monoclonal antibody against squamous-cell carcinoma of the head and neck in tumor-bearing nude mice

Nude mice carrying human squamous-cell carcinoma xenografts were given i.v. injections of radiolabelled monoclonal antibodies (MAbs). MAb E 48, which reacts with squamous-cell carcinomas, was labelled with 131I, while a second control MAb of similar immunoglobulin subclass was labelled with 125I. Both antibodies were injected simultaneously, then the mice were scanned with a gamma camera or their tissues were removed and antibody uptake was calculated as a percentage of the injected dose. Uptake of E 48 reached a peak value of 16%/g on day 3, while uptake of the control antibody was less than 1.8%/g. By 24 hr after injection tumor could be visualized without subtraction techniques. At days 3 and 7, only xenografts were visible on imaging. These findings suggest that E 48 is capable of high specificity in targeting isotopes to squamous-cell carcinomas in an experimental setting.     

Radioimmunoimaging in malignant melanoma patients with the use of indium-111-labeled antimelanoma monoclonal antibody (ZME-018) to high-molecular-weight antigen

Radioimmunolocalization of an 111In-labeled, mouse antimelanoma monoclonal antibody (MAb), ZME-018, was examined in 30 patients with metastatic malignant melanoma. Each patient received a single iv infusion of MAb at concentrations ranging from 0.6 to 40 mg, coupled to 5 mCi 111In by the chelating agent pentetic acid. No toxicity was observed in any patient. Total-body and region of interest scans performed at 4, 24, and 72 hours following MAb administration revealed uptake in 110 of 171 previously diagnosed metastases for a sensitivity of 64%. Nonspecific uptake of radioactivity was consistently observed in the liver and spleen, and less frequently in the bowel, testes, axillae, and bone. Sensitivity of detection increased significantly at doses of MAb above 2.5 mg, with 74% of the lesions imaging at 20 mg/5 mCi compared with 29% at 2.5 mg/5 mCi (P less than .005). Sensitivity actually decreased slightly at the 40-mg dose. There was a significant correlation between tumor uptake of MAb-111In-conjugate and increasing tumor size. Soft tissue lesions, such as skin and lymph node metastases, were imaged to a greater extent (77%) than were visceral metastases (40%). Mean plasma clearance of ZME-018 was prolonged with a half-life of 33.6 hours in patients receiving 40 mg, compared with 17.8 hours in patients given 2.5 mg (P less than .01). Urinary excretion of the isotope averaged 11.4% of the injected dose over 48 hours. Hence radioimmunolocalization of melanoma with 111In-ZME-018 appeared feasible. The sensitivity of the technique varies with MAb dose, specific activity of 111In-MAb conjugate, tumor size, and disease site.     

Localization of radiolabelled F(ab')2 fragments of monoclonal antibodies in nude mice bearing intraperitoneally growing human ovarian cancer xenografts

The biodistribution and pharmacokinetics of 2 monoclonal antibodies (MAbs) specific for ovarian carcinoma, OC125 and OV-TL3, were studied in nude mice bearing intraperitoneally (i.p.) growing human ovarian carcinoma xenografts of NIH:OVCAR-3. The ovarian carcinoma xenografts grew as non-adherent cells in ascites and as solid implants in the peritoneal cavity of injected mice. The biodistribution and pharmacokinetics were determined by measurement of radioactivity in tumor masses, ascites, blood and other tissues after intravenous (i.v.) and i.p. injection of radioiodinated F(ab')2 fragments of MAbs. The specificity of the observed tumor localization was then evaluated by comparing the uptake of the anti-ovarian carcinoma antibodies OC125 and OV-TL3 with the uptake of a radioiodinated non-ovarian carcinoma-specific MAb A2C6. The results of the study indicate that uptake of the anti-ovarian carcinoma antibodies was highest in the non-adherent tumor cells in the ascites after i.p. injection. The observed uptake was 85% injected dose/g for OV-TL3 and 22% injected dose/g for OC125. This compares to the observed antibody uptake of 9% injected dose/g for OV-TL3 and less than 1% injected dose/g for OC125 in solid tumor masses after i.p. injection. After i.v. injection, uptake of OC125 and OV-TL3 was less than 3% injected dose/g, both for nonadherent tumor cells and for solid tumor masses. The data support the conclusion that OV-TL3 is superior to OC125 and that i.p. administration of radiolabelled MAb F(ab')2 fragments is superior to their i.v. administration for immunotherapy of ovarian carcinoma.     

Intravesical administration of indium-111-labelled hmfg2 monoclonal antibody in superficial bladder carcinomas

Tumour-associated HMFG2 monoclonal antibody (MAb) was labelled with indium-111 and administered intravesically to 20 patients with known or suspected superficial bladder carcinoma. The antibody solution was kept in the bladder for 1 hr and was then washed out. Cystoscopy was performed at 2 and 24 hr after instillation. Radioactivity of tumour and normal tissue obtained from the bladder during cystoscopy and cells recovered from urine after the instillation were counted in a gamma-counter. Conventional histology, immunocytochemistry and autoradiography were also performed. Mean uptake at 2 and at 24 hr was higher in tumours than in normal samples. Autoradiography showed selective accumulation of radioactivity in cells which expressed the antigen detected by the HMFG2 MAb. There was no correlation of tumour uptake with the grade of tumour. No radioactivity was found in the blood of patients after the instillation. Based on dosimetric calculations, however, the radiation dose that can be delivered to the tumours is not sufficient to be cytotoxic, possibly due to inadequate penetration and retention by tumour tissue. Nevertheless, the significant difference between antibody uptake by the tumours and that by normal urothelium, observed in our study, allow for the possibility of using this approach therapeutically.     

Genotoxic effects of intragastrically administered benzo[a] pyrene in rat liver and intestinal cells

The genotoxic effects of intragastrically administered benzo[a]pyrene (BaP) were studied in isolated rat liver and intestinalcells. We found that BaP was unable to induce unscheduled DNAsynthesis in rat parenchymal liver cells in vivo. In contrast,alkaline elution showed that at 5 h after administration ofBaP a considerable number of alkali-labile sites was presentin the DNA of both intestinal cells and parenchymal liver cells,but not in that of non-parenchymal liver cells. The possibilityis discussed that these sites were induced by radical intermediates,generated during the metabolization of BaP.     

Three-step monoclonal antibody tumor targeting in carcinoembryonic antigen-positive patients

We describe a method to postlabel, in vivo, biotinylated monoclonal antibodies pretargeted onto tumor deposits when most of the non-tumor-bound antibodies have already been cleared as avidin-bound complexes. The application of this principle to tumor detection by immunoscintigraphy was tested in 20 patients with histologically documented cancer and increased circulating carcinoembryonic antigen levels. One mg of biotinylated anti-carcinoembryonic antigen monoclonal antibody (FO23C5) was administered i.v. (first step). After 3 days, 4-6 mg of cold avidin were injected i.v. (second step), followed 48 h later by 0.2-0.3 mg of a biotin derivative labeled with 111In (2-3 mCi) (third step). No evidence of toxicity was observed. Whole body radioactivity distribution was measured in five patients at various intervals postinjection by the conjugate counting technique. Tumors and metastases were detected in 18 of 19 patients (the remaining patient was a true negative) within 3 h after administration of 111In-biotin by planar or single photon emission tomography imaging. At the time of imaging, tumor/blood pool ratio was 5.5 +/- 3.2, and tumor/liver ratio was 6.7 +/- 3.9. Blood clearance of 111In-biotin was multiexponential, with the fast component having a t1/2 of 5 +/- 3 min. Urinary excretion of radioactivity over 3 h was 63.5 +/- 4.9% of the injected dose. Radioactivity at 3 h was 6.5 +/- 1.8% in blood, 1.6 +/- 0.3% in the kidney, and 2.4 +/- 0.6% in the liver. This approach represents an improvement in immunoscintigraphic techniques for tumor localization. The potential use for radioimmunotherapy is discussed.     

Tumor-necrosis factor can enhance radio-antibody uptake in human colon carcinoma xenografts by increasing vascular permeability

Marked differences in the tumor uptake of a ¹²⁵I-labeled monoclonal antibody (MAb) directed against carcinoembryonic antigen (CEA) were observed in 4 serially transplanted human colorectal carcinomas in nude mice. A comparative study showed that elevated values of measurable tumor vascular parameters, such as permeability, blood flow and blood volume, correlated better with high MAb tumor uptake than the concentration of target antigen in the tumor. In an attempt to modify the vascular parameters and to determine if this could increase antibody uptake by the tumor, rhTNFα (TNF) was injected i.t. or i.v. and antibody localization experiments were performed immediately thereafter. Results showed that the permeability of the tumor vessels increased 8 to 10 fold 1 hr after i.t. injection of TNF as compared to control tumors injected with saline. Tumor uptake of ¹²⁵I-labeled anti-CEA MAb, was 3 times higher 2 hr after i.v. injection and still 27% higher 22 hr later, as compared to results from controls. Intravenous injection of TNF simultaneously with the ^l25I- labeled anti-CEA MAb also resulted in a 2-fold increase in tumor uptake 4 hr after injection, but the increase was no longer significant 24 hr after injection. Interestingly after i.v. injection of TNF, the MAb concentration in the blood and other normal tissues, such as liver, kidneys, lungs and heart was decreased, resulting in significantly higher ratios of tumor to normal tissue. Taken together the results demonstrate that injection of TNF can increase tumor vascular permeability and improve radioantibody uptake. This raises the possibility of increasing the radiation dose delivered by antibody to the tumor in the course of radioimmunotherapy.     

Concentrations of vinblastine in human intracerebral tumor and other tissues

Uptake of vinblastine into human cerebrospinal fluid, intracerebral tumor and autopsy tissues was quantitated radiochemically after separating vinblastine from its metabolites by high pressure liquid chromatography. Only low concentrations of vinblastine were found in cerebrospinal fluid from a single patient. A second patient who received a tracer dose of radiolabelled vinblastine prior to surgical resection of an intracerebral tumor had slightly less radioactivity in tumor than in temporalis muscle, but more in tumor than in edematous brain surrounding the tumor. The radioactivity in tumor increased gradually and exceeded concurrent plasma radioactivity by 2 hr after drug administration. A third patient died 4 hr into a planned 24-hr infusion of radiolabeled vinblastine. Highest vinblastine concentrations were found in organs with high blood flow such as kidney and heart. Intermediate concentrations were found in liver and lung, and low concentrations were found in prostate, gastrointestinal tract, spleen, muscle, bladder, and hepatic and lymph node metastases. A fourth patient died one month after receiving radiolabeled vinblastine. Highest concentrations were in liver and next highest concentrations were in intracerebral tumor. Moderately high concentrations were found in pancreas, thyroid, lung, spleen, ovary, kidney, and kidney metastases. Lowest concentrations were found in omental metastases, heart, breast, and brain. Vinblastine concentration decreased with increasing distance into brain from the brain metastases. Thus, vinblastine was not selectively localized in tumors. The concentrations in tumor did not reflect the concentration in the organ in which the tumor was located. There was no indication that uptake into intracerebral tumor was impaired. Cerebrospinal fluid and brain concentrations of vinblastine did not give any indication of the concentration attainable in intracerebral tumor.     

Immunospecific saturable clearance mechanisms for indium-111-labeled anti-melanoma monoclonal antibody 96.5 in humans

Liver uptake of 111In-labeled monoclonal antibodies (MoAb) remains a significant problem in radioimaging studies to date. To determine if the observed liver uptake of an 111In-labeled anti-melanoma antibody 96.5 (111In-96.5) was dependent on the presence of hepatic antigen or on recognition of circulating murine antibody, escalating doses of an unlabeled nonimmunoreactive MoAb (NIR-MoAb) were administered to 18 patients with metastatic malignant melanoma either 1 or 24 h prior to an infusion of 1 mg of 111In-96.5. The number of metastases imaged, pharmacokinetics, and the ratio of radioactivity (expressed as average counts/pixel) in liver (L), spleen (S), bone (B), and kidney (K) compared to blood pool (heart = H) were examined. Results were prospectively compared with data from six patients who received immunoreactive unlabeled 96.5 prior to 111In-96.5. Increasing dose or changes in the preinfusion time of NIR-MoAb had no significant effect on the biodistribution of 111In-96.5. In contrast, patients who received unlabeled, immunoreactive 96.5 prior to 111In-96.5 infusion demonstrated a significant drop [P less than 0.001] in the liver/heart ratio of radioactivity [2.81 +/- 0.35 (SEM)] compared to patients receiving the identical dose of NIR-MoAb [10.35 +/- 1.33]. Significant decreases in spleen/heart and bone/heart ratios were also observed. Pharmacokinetic studies showed that the volume of distribution (Vd) and the plasma t1/2 both decreased when 96.5 was administered compared to NIR-MoAb. In addition, a 4-fold increase in concentration X time was obtained after 96.5 antibody was administered compared to NIR-MoAb. More metastases were imaged in patients receiving preinfusions of 96.5 (23 of 28) than in patients receiving NIR-MoAb (10 of 18; P less than 0.05). Although tissue distribution of 111In-labeled antibody can be ascribed to nonspecific organ clearance of murine antibodies, a substantial component of tissue disposition of antibody 96.5 was shown to be a consequence of specific clearance of immunoreactive antibody which may cross-react with tissue antigens.     

Evaluation of a cathepsin-cleavable peptide linked radioimmunoconjugate of a panadenocarcinoma MAb, m170, in mice and patients

PURPOSE: Radioimmunotherapy (RIT) delivered by radiometal immunoconjugates (RICs) is dose limited by deposition and retention of radioactivity in normal tissues. In order to increase elimination of radioactivity from the liver and body, a peptide having a specific cathepsin B cleavage site was placed between the radiometal chelate, 111In-DOTA, and the panadenocarcinoma monoclonal antibody (MAb), m170. Experimental design: Indium-111 (111In)-1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-2-iminothiolane (2IT)-m170 and 111In-DOTA-peptide-m170, representing the same MAb and chelate without and with a cleavable linkage, were studied in athymic mice and patients with breast or prostate cancer. Pharmacokinetics, cumulated activities and therapeutic indices (TI), were evaluated. Cumulated activities in the liver and tumors were calculated and used as a surrogate for radiation dose. RESULTS: Except for liver, the pharmacokinetics of 111In-DOTA-peptide-m170 were similar to those of the 111In-2IT-2-[p(bromoacetamido)benzyl]-1,4,7,10-tetraazocyclododecane-N,N',N",N"'-tetraacetic acid-m170 (111In-2IT-BAD-m170) in mice and patients. Liver cumulated activities for 111In-DOTA-peptide-m170 were consistently decreased when compared to those for 111In-2IT-BAD-m170, reductions varying between 22-30%. Cumulated activities for 111In-DOTA-peptide-m170 in the malignant tumors of the patients were as great as those for 111In-2IT-BAD-m170, so that the tumor-to-liver cumulated activity ratios (therapeutic indices) were better for 111In-DOTA-peptide-m170. CONCLUSIONS: A cathepsin-B-cleavable peptide used to link chelated 111In to MAb, m170, reduced liver cumulated activity (radiation dose) and improved the TI. This novel linker illustrates the importance of linker technology in the development of safer RICs for cancer therapy.