Radioimmunoscintigraphy of human colon cancer xenografts in mice with radioiodinated monoclonal antibody B72.3

Monoclonal antibody B72.3 reacts with a tumor-associated antigen that is found on human breast and colon carcinomas in significantly higher concentration than in normal adult tissues. Intact B72.3 IgG was labeled with I-131 or I-125 and injected into athymic mice bearing xenografts of human colon cancer. Whole-body scintiphotos obtained with a pinhole collimator demonstrated tumor localization within 24 hr after intravenous administration, and the tumor-to-background ratio rose continuously for at least 14 days. Progressive antibody accumulation was observed in the tumor during the first 3 days, but no significant normal organ localization was observed at any time. No localization was seen in control tumors, a human melanoma xenograft that lacks the antigen recognized by B72.3. The pharmacokinetics of this antibody in tumor-bearing mice suggest that I-131 B72.3 may be useful for radioimmunotherapy as well as radioimmunoscintigraphy of colon cancer in man.     

Effect of carbohydrate modification on the localization of human polyclonal IgG at focal sites of bacterial infection.

Radiolabeled human polyclonal IgG localizes at focal sites of infection/inflammation. Previous studies have sought to identify the mechanism of localization, but the relative importance of specific antigen recognition by individual antibody molecules, binding to Fc receptors on inflammatory cells and nonspecific processes such as increased tissue permeability remains uncertain. This study was performed to evaluate the specific role of Fc receptor binding as a mechanism of localization. The Fc region of IgG was modified by endoglycosidase-F digestion and periodate oxidation to reduce the binding of IgG to Fc receptors. In-vitro binding was tested in an Fc receptor binding assay using the human monocyte-like cell line, U937. The in-vivo ability of the modified antibodies to localize at focal sites of E. coli infection was tested by biodistribution studied with the 111In-labeled proteins. Modification of the carbohydrate moiety of the Fc region of IgG resulted in a marked decrease in Fc receptor binding in vitro; with antibody concentrations of 1 micrograms/ml (which is presumed to exist at infected sites) showing no binding for endoglycosidase modified IgG and 50% binding for periodate modified IgG. In contrast, in-vivo infection localization as measured by level of accumulation or target-to-background ratio was not significantly effected by carbohydrate modification. These studies suggest that the contribution of Fc receptor binding to IgG localization at sites of infection is minimal.     

124I-labeled engineered anti-CEA minibodies and diabodies allow high-contrast, antigen-specific small-animal PET imaging of xenografts in athymic mice.

Prolonged clearance kinetics have hampered the development of intact antibodies as imaging agents, despite their ability to effectively deliver radionuclides to tumor targets in vivo. Genetically engineered antibody fragments display rapid, high-level tumor uptake coupled with rapid clearance from the circulation in the athymic mouse/LS174T xenograft model. The anticarcinoembryonic antigen (CEA) T84.66 minibody (single-chain Fv fragment [scFv]-C(H)3 dimer, 80 kDa) and T84.66 diabody (noncovalent dimer of scFv, 55 kDa) exhibit pharmacokinetics favorable for radioimmunoimaging. The present work evaluated the minibody or diabody labeled with (124)I, for imaging tumor-bearing mice using a high-resolution small-animal PET system. METHODS: Labeling was conducted with 0.2-0.3 mg of protein and 65-98 MBq (1.7-2.6 mCi) of (124)I using an iodination reagent. Radiolabeling efficiencies ranged from 33% to 88%, and immunoreactivity was 42% (diabody) or >90% (minibody). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA-positive) and C6 rat glioma (CEA-negative) xenografts. Mice were injected via the tail vein with 1.9-3.1 MBq (53-85 microCi) of (124)I-minibody or with 3.1 MBq (85 microCi) of (124)I-diabody and imaged at 4 and 18 h by PET. Some mice were also imaged using (18)F-FDG 2 d before imaging with (124)I-minibody. RESULTS: PET images using (124)I-labeled minibody or diabody showed specific localization to the CEA-positive xenografts and relatively low activity elsewhere in the mice, particularly by 18 h. Target-to-background ratios for the LS174T tumors versus soft tissues using (124)I-minibody were 3.05 at 4 h and 11.03 at 18 h. Similar values were obtained for the (124)I-diabody (3.95 at 4 h and 10.93 at 18 h). These results were confirmed by direct counting of tissues after the final imaging. Marked reduction of normal tissue activity, especially in the abdominal region, resulted in high-contrast images at 18 h for the (124)I-anti-CEA diabody. CEA-positive tumors as small as 11 mg (<3 mm in diameter) could be imaged, and (124)I-anti-CEA minibodies, compared with (18)F-FDG, demonstrated highly specific localization. CONCLUSION: (124)I labeling of engineered antibody fragments provides a promising new class of tumor-specific probes for PET imaging of tumors and metastases.     

Enhancement of clearance of plant lectins as radiopharmaceuticals by chemically glycosylated antilectin antibody

The influence of chemical glycosylation on clearance of plant lectins, employed as radiopharmaceuticals, was assessed. Controlled chemical modification of the antibody to concanavalia ensiformis agglutinin did not affect its immunologic activity, but led to rapid clearance from circulation. In tumor-bearing mice, notable increases of selected tumor/non tumor ratios even after only 1 h of presence of the galactosylated antibody to the lectin in circulation were observed. Similarly, galactosylated antibody to pisum sativum agglutinin caused significant increases of tumor/non tumor ratios.     

Enhancement of clearance of plant lectins as radiopharmaceuticals by chemically glycosylated antilectin antibody

The influence of chemical glycosylation on clearance of plant lectins, employed as radiopharmaceuticals, was assessed. Controlled chemical modification of the antibody to concanavalia ensiformis agglutinin did not affect its immunologic activity, but led to rapid clearance from circulation. In tumor-bearing mice, notable increases of selected tumor/non tumor ratios even after only 1 h of presence of the galactosylated antibody to the lectin in circulation were observed. Similarly, galactosylated antibody to pisum sativum agglutinin caused significant increases of tumor/non tumor ratios.     

1-11C-acetate as a PET radiopharmaceutical for imaging fatty acid synthase expression in prostate cancer.

Although it is accepted that the metabolic fate of 1-(11)C-acetate is different in tumors than in myocardial tissue because of different clearance patterns, the exact pathway has not been fully elucidated. For decades, fatty acid synthesis has been quantified in vitro by the incubation of cells with (14)C-acetate. Fatty acid synthase (FAS) has been found to be overexpressed in prostate carcinomas, as well as other cancers, and it is possible that imaging with 1-(11)C-acetate could be a marker for its expression. METHODS: In vitro and in vivo uptake experiments in prostate tumor models with 1-(11)C-acetate were performed both with and without blocking of fatty acid synthesis with either C75, an inhibitor of FAS, or 5-(tetradecyloxy)-2-furoic acid (TOFA), an inhibitor of acetyl-CoA carboxylase (ACC). FAS levels were measured by Western blot and immunohistochemical techniques for comparison. RESULTS: In vitro studies in 3 different prostate tumor models (PC-3, LNCaP, and 22Rv1) demonstrated blocking of 1-(11)C-acetate accumulation after treatment with both C75 and TOFA. This was further shown in vivo in PC-3 and LNCaP tumor-bearing mice after a single treatment with C75. A positive correlation between 1-(11)C-acetate uptake into the solid tumors and FAS expression levels was found. CONCLUSION: Extensive involvement of the fatty acid synthesis pathway in 1-(11)C-acetate uptake in prostate tumors was confirmed, leading to a possible marker for FAS expression in vivo by noninvasive PET.     

Preclinical radioimmunotargeting of folate receptor alpha using the monoclonal antibody conjugate DOTA-MORAb-003

INTRODUCTION: The in vitro and in vivo behavior of the radiolabeled monoclonal antibody MORAb-003 was investigated as a prelude to a clinical trial. METHODS: The cellular retention of 111In- and 131I-labeled MORAb-003 was investigated using IGROV1 and SW620 cells. Biodistribution studies in tumor-bearing mice were performed with the more favorable agent. RESULTS: Five 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA) molecules were conjugated to MORAb-003 with no apparent loss of immunoreactivity. Radiolabeled MORAb-003 had a high affinity for the folate receptor alpha (FRA) expressed by both IGROV1 and SW620 cells and was found to bind to around 8 x 10(5) and 7 x 10(5) sites/cell, respectively. Both cancer cell lines were found to internalize both 131I- and 111In-labeled MORAb-003, but 111In was retained and 131I was released as iodide. In athymic mice, 111In-DOTA-MORAb-003 was cleared from the blood with a single exponential biological clearance rate of 110 h. The uptake in SW620 tumors was 32+/-5%ID/g after 4 days. The clearance rate of activity from normal organs such as liver, kidney and spleen was similar to the blood clearance and was 5.36%ID/g, 4.03%ID/g and 4.36%ID/g at 1 day postinjection and 2.14%ID/g, 1.65%ID/g and 3.74%ID/g after 8 days, respectively. In a pilot clinical study, the biodistribution and tumor targeting of 111In-MORAb-003 was assessed in three patients undergoing treatment with cold MORAb-003. CONCLUSION: MORAb-003 is an attractive antibody for radioimmunoscintigraphy and possibly radioimmunotherapy of FRA-expressing cancers in addition to its potential direct therapeutic effects.     

Tumor targeting and SPECT imaging properties of an 111In-labeled galectin-3 binding peptide in prostate carcinoma

IntroductionGalectin-3 (gal-3) is a carbohydrate binding protein that has been implicated in cell adhesion, tumor invasion and metastasis. The objective of this study was to evaluate the tumor targeting and imaging properties of a gal-3 binding peptide selected by phage display in a mouse model of metastatic human prostate carcinoma expressing gal-3.MethodsA gal-3 binding peptide, ANTPCGPYTHDCPVKR, was synthesized with a Gly–Ser–Gly (GSG) spacer and 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) and then radiolabeled with ¹¹¹In. The in vitro cell binding properties of ¹¹¹In-DOTA-(GSG)-ANTPCGPYTHDCPVKR were determined in metastatic human PC3-M prostate carcinoma cells. The pharmacokinetics and single-photon emission computed tomographic (SPECT/CT) imaging with the radiolabeled peptide were evaluated in SCID mice bearing human PC3-M prostate carcinoma tumor xenografts.ResultsThe radiolabeled peptide bound with a 50% inhibitory concentration of 191±10.2 nM to cultured PC3-M prostate carcinoma cells. In vivo tumor uptake and retention coupled with fast whole-body clearance of the peptide were demonstrated in PC3-M tumor-bearing SCID mice. The tumor uptake rates of the radiolabeled peptide were 1.27±0.10%ID/g at 30 min, 0.82±0.15%ID/g at 1 h and 0.57±0.09%ID/g at 2 h. MicroSPECT/CT studies revealed good tumor uptake of ¹¹¹In-DOTA-(GSG)-ANTPCGPYTHDCPVKR 2 h postinjection, while uptake in normal organs was low, with the exception of the kidneys.ConclusionsIn vitro cell binding along with tumor uptake of ¹¹¹In-DOTA-(GSG)-ANTPCGPYTHDCPVKR in PC3-M human prostate carcinoma tumor-bearing SCID mice suggests the potential of this peptide as a radiopharmaceutical for imaging of gal-3-expressing prostate tumors.     

Rapid background reduction of circulating sodium iodide I 125-labelled Pisum sativum agglutinin used as a tumor-imaging radiopharmaceutical by the chemically galactosylated antibody

Influence on the clearance of background radioactivity from the blood was studied by using a chemically galactosylated antibody to the radiopharmaceutical. Sodium iodide I 125-labelled Pisum sativum agglutinin (125I-PSA) was used as the model tumor-imaging radiopharmaceutical in this series of experiments. Rabbit (anti-PSA) immunoglobulin G (IgG) was chemically galactosylated with varying amounts of cyanomethylgalactose. Galactose concentration ranged from 11 to 17 mol/mol protein. Antibody activity was not affected by chemical galactosylation under the experimental conditions used. Blood clearance of the galactosylated anti-PSA (GAP) in normal mice was enhanced to varying degrees, depending on the degree of galactosylation; similarly, liver uptake was increased with the degree of galactosylation. Following injection of 125I-PSA in normal mice, the lectin was rapidly removed from the blood by subsequent injection of GAP. Increased hepatic uptake of the complex (lectin-galactosylated antibody) via protein-carbohydrate recognition caused the pronounced decrease in the 125I-PSA blood level. The effective time for 125I-PSA removal was as short as 15 min. The potency was dependent on the degree of galactosylation of the antibody. In sarcoma 180 (S-180) tumor-bearing mice, the capacity for blood clearance of 125I-PSA was also positively correlated to the degree of galactosylation. Moreover, the variation in the delivered dose ratio of antibody to lectin proved to lead to a further increase in background clearance. As a result, especially the tumor:blood ratio was significantly improved by a single administration of chemically galactosylated antibody, as compared with the value measured in the presence of unmodified antibody. These initial studies suggest that administration of GAP may improve nuclear imaging with radiopharmaceuticals.     

Targeting of ganglioside GD2 monoclonal antibody to neuroblastoma

The biodistribution of 3F8, and IgG3 murine monoclonal antibody (MoAb) specific for the disialoganglioside GD2, was studied in nude mice xenografted with human neuroblastoma (NB). 3F8 conjugated to radioactive iodine and injected intravenously localized selectively to seven human NB when compared with Ewing's sarcoma and Hela cell xenograft controls. Uptake in NB was shown to be specific for MoAb 3F8 when contrasted with pooled mouse IgG or irrelevant IgG3 MoAb controls. Both small (50 mg) and large tumors greater than 2 g) showed radiolocalization. The percent injected dose uptake per gram tumor ranged from 8 to 50% and was inversely correlated with tumor size. Optimal tumor to normal tissue ratios were reached by 24-48 hr. There was no abnormal uptake in the reticuloendothelial system and the MoAb did not cross the blood-brain barrier. Based on the kinetics of the amount of radioactivity deposited in tissues, the relative radiation dose to normal organs was estimated to be 1% to 20% of the tumor dose. The MoAb 3F8 is useful for targeting radioactivity to human NB in vivo and the nude mice xenograft model may allow optimization of parameters that influence such biodistribution.     

氯氧喹对Lewis肺癌细胞及荷瘤小鼠的辐射增敏作用

目的 研究氯氧喹联合X射线对Lewis肺癌细胞及荷瘤小鼠的辐射增敏作用.方法 细胞及动物实验均设置为对照组、给药组、照射组及给药+照射组(联合组)4个组别.采用体外细胞培养MTT检测及荷瘤动物模型观察方法,分别予以不同处理因素后,细胞实验用MTT法检测各组别细胞吸光度(A)值;小鼠皮下接种Lewis肺癌细胞,予以相关处理因素后,观察记录肿瘤生长情况并测量其长、宽径从而计算出肿瘤抑制率及肿瘤生长延迟.照射结束后眼静脉采血,检测各组小鼠血液标本中细胞成分数量的变化情况.结果 MTT实验结果显示,细胞照射24 h后,给药+照射组SF值较单纯照射组有一定的降低,但差异无统计学意义(P＞0.05),而照射48 h后,在10、20、40和80 mg/L浓度下,两组间差异有统计学意义(t=3.22、4.23、4.46和4.58,P＜0.05).动物实验结果显示,给药+照射组的肿瘤生长曲线明显较单纯照射组低平,但差异无统计学意义;给药+照射组的肿瘤控制率及肿瘤生长延迟远高于单独给药组及照射组(t=3.15～3.59,P＜0.05).另外,氯氧喹并未增加辐射对小鼠血液细胞成分的不良反应.结论 氯氧喹对Lewis肺癌细胞及荷瘤小鼠均具有明显的辐射增敏作用,对小鼠血液系统未见明显不良反应,值得进一步深入研究.

Abstract：

Objective To study whether Chloroxoquinoline could enhance the radiation sensitivity of Lewis lung cancer cells and xenograft tumors in tumor-bearing mice.Methods Both cell and animal experiments were divided into control group,drug group,irradiation group and combination group.MTT assay was used to detect the optical density of Lewis lung cancer cells cultured in vitro.The model of tumor-bearing mice was established first and then the tumor diameters of different groups were measured to figure out the tumor control rates and tumor growth delay.After irradiation,the blood samples in each group were collected to detect the amount of blood cells.Results MTT showed 24 hours after irradiation,the cell survival fraction of combination group was lower than that of irradiation group,but no statistical differences (P ＞ 0.05).However,48 hours later,the cell survival fraction of combination group was obviously lower than irradiation group with statistically significant differences( t = 3.22,4.23,4.46,4.58,P ＜ 0.05).Animal experiments showed the tumor growth curve of combination group was significantly lower than irradiation group.The tumor control rate and tumor growth delay of combination group was more above the other groups ( t = 3.15-3.59,P ＜ 0.05 ).Additionally,according to the analysis of blood samples,the hematological toxicity of chloroxoquinoline on mice was not found.Conclusion Chloroxoquinoline shows its radiation sensitizing effect on Lewis lung cancer cells and xenograft tumors in mice without hematological toxicity.     

Synthesis and evaluation of a macrocyclic bifunctional chelating agent for use with bismuth radionuclides

The detailed synthesis of the bifunctional chelating agent 2-(p-isothiocyanatobenzyl)-1,4,7,10,13-pentaazacyclopentadecane-N,N',N",N"',N"-pentaacetic acid (BF_PEPA) is reported. This ligand was conjugated to monoclonal antibody B3 and the resultant immunoconjugate radiolabeled with (205,206)Bi. The in vivo stability of the radiolabeled immunoconjugate, and targeting characteristics were determined by biodistribution studies in A431 xenograft tumor-bearing mice sacrificed at 0.5, 1, 2, 4, and 24 hr. Results indicate that BF_PEPA appears to not be a suitable bifunctional chelating agent for sequestering isotopes of Bi(III) for radioimmunotherapy applications.     

In vitro and in vivo evaluation of 64Cu-TETA-Tyr3-octreotate. A new somatostatin analog with improved target tissue uptake.

Radiolabeled somatostatin analogs have demonstrated potential as cancer therapeutic agents. Many of these agents are based on the analog octreotide (OC). Recently it has been shown that substitution of a tyrosine for phenylalanine in the 3-position and changing the C-terminus from an alcohol to an acid improves the targeting of somatostatin-rich tissues. The compound, 1,4,8,11-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid-Tyr3-octreotate (TETA-Y3-TATE), was synthesized and radiolabeled with 64Cu. The receptor binding properties of 64Cu-TETA-Y3-TATE showed an estimated Kd value of 549 pM in somatostatin receptor-positive CA20948 tissue membrane. High tumor uptake was observed in two animal tumor models. Tumor uptakes of 2.37 %ID/g in CA20948 tumor-bearing rats and 21.60 %ID/g in AR42J tumor-bearing SCID mice were observed at 1 h, compared with 1.09 %ID/g and 11.24 %ID/g for 64Cu-TETA-OC. Higher uptake in other somatostatin-receptor rich tissues was also observed, compared with 64Cu-TETA-OC. Positron emission tomography (PET) imaging with 64Cu-TETA-Y3-TATE in a baboon showed significant uptake in the pituitary and adrenals, and clearance through the kidneys. 64Cu-TETA-Y3-TATE, a new OC analog for binding somatostatin receptors, demonstrated significantly greater uptake in somatostatin-rich tissues in two tumor-bearing animal models, and demonstrated great potential as a radiopharmaceutical for imaging and therapy of somatostatin receptor-positive tissues.     

Use of specific antibody for rapid clearance of circulating blood background from radiolabeled tumor imaging proteins

A major problem that arises when radiolabeled serum proteins are used for tumor imaging is the presence of a large amount of circulating background activity that persists for several days. This delays imaging for at least 2 days following injection and necessitates computer subtraction of simulated background (second radiopharmaceutical injection) which introduces artifacts that are difficult to control. We propose here the injection of specific antibody immediately before imaging as an alternate way of reducing blood background through clearance of the immune complex by the liver. 111In-alkyl human transferrin and IgG were injected IV in BALB/c tumor mice, and followed in 18 h by anti-human transferrin and anti-human IgG antibody IV. Two hours later, the tumor and organ distribution of activity was compared with control mice not receiving antibody. 111In-transferrin blood activity was reduced to 1/48 of control with no decrease in tumor concentration: as a result, the tumor to blood ratio increased from 1.4:1 to 78:1. 111In-IgG blood activity was reduced to 1/17 of control, again with no decrease in tumor. The tumor to blood ratios increased from 0.7:1 to 17:1. The liver picked up most of the blood activity with none of the complex going to spleen, bone marrow, or kidney. Dog experiments showed clearance of blood was 90% complete in less than 15 min following antibody injection. Simultaneous scintillation images showed complete clearance of activity from the heart and great vessels in the chest and neck, and over the abdomen, with a concomitant increase in liver activity but no increase in spleen, kidney, or bone marrow activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved radioimmunolocalization of human tumor xenografts following subcutaneous delivery of monoclonal antibodies

The localization of a radiolabeled murine monoclonal antibody reactive with choriocarcinomas to human choriocarcinoma xenografts following intravenous and subcutaneous injection was evaluated by gamma scanning and tissue sampling. Tumor xenografts were established in the popliteal node region of athymic nude mice after repeated innoculations of the hind foot pads with BEWO choriocarcinoma cells. In dual label specific antibody studies, tumor/non tumor uptake ratios following subcutaneous (resulting in considerable intralymphatic uptake) injection of 131I-5F9.3 were significantly higher than those achieved post simultaneous intravenous injection of 125I-5F9.3. Double label experiments with 131I-5F9.3 and a nonspecific antibody, 125I-UPC-10, following subcutaneous injection, demonstrated that the high localization to popliteal region tumors was largely due to antibody specificity. Gamma scans following subcutaneous antibody administration of specific antibody to tumor bearing animals showed tumors soon after subcutaneous injection, at times earlier than those typically seen following intravenous delivery. Similar subcutaneous injections showed little normal nodal uptake in BALB/c control animals on gamma scans. No correlation was seen between tumor localization by specific antibody between the intravenous and intralymphatic routes, implying a difference in the mechanisms of tumor uptake of antibody by these two routes. The subcutaneous approach to antibody delivery offers advantages over intravenous delivery in tumors of human origin, including higher tumor/non tumor ratios and earlier imaging times. This was true even though these tumors were many times larger than normal lymph nodes. This subcutaneous delivery advantage should be exploitable in imaging nodal metastases of human tumors.     

Conjugation with (111)In-DTPA-poly(ethylene glycol) improves imaging of anti-EGF receptor antibody C225.

Significant liver uptake often limits the clinical application of radiolabeled antibodies in radioimmunodetection. The purpose of this study was to evaluate the gamma-imaging properties of an antiepidermal growth factor receptor (EGFR) antibody, C225, conjugated with heterofunctional poly(ethylene glycol) (PEG) with 1 terminus of the polymer attached to a radiometal chelator, diethylenetriaminepentaacetic acid (DTPA). METHODS: Two preparations of PEG-modified C225, one with 20% and the other with 60% amine substitution, were labeled with (111)In. The conjugates, (111)In-DTPA-PEG-C225, were injected intravenously into nude mice with EGFR-positive A431 tumors. For comparison, C225 directly labeled with (111)In was also injected. In a competitive study, mice with A431 tumors were pretreated intravenously with 100-fold excess of native C225, followed by an injection of (111)In-DTPA-PEG-C225 30 min or 20 h later. In addition, (111)In-DTPA-PEG-C225 was injected into mice with EGFR-positive MDA-MB-468 tumors and EGFR-negative MDA-MB-435 tumors. Images were acquired at 5 min and at 2, 6, 24, and 48 h after injection of the radiotracers. Regions of interest (ROIs) were drawn on the computer images around the whole body, liver, muscle, and tumor. The counts per pixel in the tumor and normal tissues were calculated. At 48 h, the mice were killed and dissected. Blood, liver, muscle, and tumor samples were removed and the radioactivity of each sample was measured. RESULTS: In A431 tumor xenografts, the tumor uptake of C225 modified with PEG was not significantly different than the uptake of unmodified (111)In-DTPA-C225. Uptake in the liver, however, was reduced by 38%-45%, and the reduction increased with increasing degree of PEG substitution. Tumors of A431 and MDA-MB-468 xenografts were clearly visualized with (111)In-DTPA-PEG-C225, whereas tumors of the MDA-MB-435 xenograft, which expresses low levels of EGFR, were not as readily visible. The tumor-to-blood ratios of (111)In-DTPA-PEG-C225 in A431 and MDA-MB-468 xenografts were about 3 fold higher than in MDA-MB-435 xenografts. Blocking EGFR by pretreatment with native C225 significantly reduced the uptake of (111)In-DTPA-PEG-C225 in the liver. The tumor-to-blood ratios in mice with A431 tumors were decreased 2.5-2.7 fold after pretreatment with a large excess of C225. Similar results were obtained with MDA-MB-468 tumor xenografts. In contrast, the tumor-to-blood ratios in mice with MDA-MB-435 tumor xenografts were not significantly different in C225-pretreated mice than in nonpretreated mice. CONCLUSION: These findings indicate that (111)In-DTPA-PEG-C225 selectively localized to the tumors expressing high levels of EGFR. PEG-modification of C225 significantly reduced its liver uptake, resulting in improved visualization of EGFR-positive tumors. Using PEG as a linker between the monoclonal antibody and metal chelator is a useful strategy to optimize the imaging characteristics of antibody-based scintigraphic agents.     

111In-labeled galectin-3-targeting peptide as a SPECT agent for imaging breast tumors.

Galectin-3 is a member of the galectin family of beta-galactoside-binding animal lectins. Galectin-3 is overexpressed in a wide range of neoplasms and is associated with tumor growth and metastases. Given this fact, radiolabeled galectin-3-targeting molecules may be useful for the noninvasive imaging of tumors expressing galectin-3, as well as for targeted radionuclide therapy. In this study, the tumor cell-targeting and SPECT properties of a galectin-3-avid peptide identified from bacteriophage display were evaluated in human breast carcinoma cells and in human breast tumor-bearing mice. METHODS: The galectin-3-avid peptide G3-C12 (ANTPCGPYTHDCPVKR) was synthesized with a Gly-Ser-Gly (GSG) linker at the amino terminus. After conjugation with 1,4,7,10-tetra-azacyclododecane-N,N',N'N'-tetraacetic acid (DOTA), the peptide was labeled with (111)In. The radiochemical purity and stability of the compound was assessed by high-performance liquid chromatography. MDA-MB-435 human breast carcinoma cells expressing galectin-3 were used to characterize the in vitro binding properties of the radiolabeled compound. SCID mice bearing MDA-MB-435 xenografts were used as an in vivo model for biodistribution and imaging studies with the (111)In-labeled peptide. RESULTS: (111)In-DOTA(GSG)-G3-C12 bound specifically to galectin-3-expressing MDA-MB-435 cells. The radiolabeled peptide was stable in serum and was found intact in excreted urine for at least 1 h. Competitive binding experiments indicated that the radiolabeled peptide exhibited an inhibitory concentration of 50% of 200.00+/-6.70 nM for cultured breast carcinoma cells. In vivo biodistribution studies revealed that tumor uptake was 1.2+/-0.24, 0.75+/-0.05, and 0.6+/-0.04 (mean +/- SD) percentage injected dose per gram at 30 min, 1.0 h, and 2.0 h after injection of the radiotracer, respectively. SPECT/CT studies with (111)In-DOTA(GSG)-G3-C12 showed excellent tumor uptake and contrast in the tumor-bearing mice. Specificity of peptide binding was demonstrated by successful blocking (52%) of in vivo tumor uptake of (111)In-DOTA(GSG)-G3-C12 in the presence of its nonradiolabeled counterpart at 2 h after injection. CONCLUSION: This study demonstrated the successful use of a new radiolabeled peptide for the noninvasive imaging of galectin-3-positive breast tumors. This peptide may be a promising candidate for future clinical applications.     

Improved radioimmunolocalization of human tumor xenografts following subcutaneous delivery of monoclonal antibodies

The localization of a radiolabeled murine monoclonal antibody reactive with choriocarcinomas to human choriocarcinoma xenografts following intravenous and subcutaneous injection was evaluated by gamma scanning and tissue sampling. Tumor xenografts were established in the popliteal node region of athymic nude mice after repeated innoculations of the hind foot pads with BEWO choriocarcinoma cells. In dual label specific antibody studies, tumor/non tumor uptake ratios following subcutaneous (resulting in considerable intralymphatic uptake) injection of ¹³¹I-5F9.3 were significantly higher than those achieved post simultaneous intravenous injection of ¹²⁵I-5F9.3. Double label experiments with ¹³¹I-5F9.3 and a nonspecific antibody, ¹²⁵I-UPC-10, following subcutaneous injection, demonstrated that the high localization to popliteal region tumors was largely due to antibody specificity. Gamma scans following subcutaneous antibody administration of specific antibody to tumor bearing animals showed tumors soon after subcutaneous injection, at times earlier than those typically seen following intravenous delivery. Similar subcutaneous injections showed little normal nodal uptake in BALB/c control animals on gamma scans. No correlation was seen between tumor localization by specific antibody between the intravenous and intralymphatic routes, implying a difference in the mechanisms of tumor uptake of antibody by these two routes. The subcutaneous approach to antibody delivery offers advantages over intravenous delivery in tumors of human origin, including higher tumor/non tumor ratios and earlier imaging times. This was true even though these tumors were many times larger than normal lymph nodes. This subcutaneous delivery advantage should be exploitable in imaging nodal metastases of human tumors.     

Small-animal PET of tumor angiogenesis using a (76)Br-labeled human recombinant antibody fragment to the ED-B domain of fibronectin.

The aim of this study was to image the extra domain B (ED-B) of fibronectin, an angiogenesis-related target, in solid tumors using small-animal PET. Toward this aim, an ED-B fibronectin-binding human antibody derivative (L19-SIP) was labeled with (76)Br via an enzymatic approach. Biodistribution and imaging studies were performed in human teratoma-bearing mice for up to 48 h after injection. METHODS: L19-SIP was labeled with (76)Br using bromoperoxidase/H(2)O(2). The stability of the labeled antibody was tested both in vitro and in vivo. Biodistribution and small-animal imaging studies (PET and CT) were performed in F9-bearing 129/sv mice (n = 3 or 4). RESULTS: The enzymatic radiobromination approach afforded the labeled antibody in high yield (>55%) under mild reaction conditions. (76)Br-L19-SIP stability in mouse serum proved to be similar to that of the (125)I-labeled analog (>80% of intact material at 48 h after injection). Fast and specific in vivo targeting was obtained in tumors and other organs expressing ED-B fibronectin (i.e., ovaries and uterus). However, slow renal clearance and persistent activity predominately in blood and stomach suggests partial (76)Br-L19-SIP debromination in vivo. This debromination was confirmed in a metabolism study in normal mice. The F9 tumors were clearly imaged by small-animal PET at each considered time point, starting at 5 h up to 48 h after injection. CONCLUSION: (76)Br-L19-SIP specifically accumulated at the target site, enabling detailed small-animal PET of tumor neovasculature. Therefore, targeting the angiogenesis-associated expression of ED-B fibronectin can be a valuable tool for tumor detection using molecular imaging with PET.     

Sequential imaging of indium-111-labeled monoclonal antibody in human mammary tumors hosted in nude mice

Using a bifunctional chelating agent, indium-111 was attached to a monoclonal antibody 10- 3D2 , specific for a 126-kilodalton phosphoglycoprotein antigen associated with human mammary carcinoma, and was then used to localize and visualize human mammary tumors hosted in nude mice. Simultaneous tumor concentration of In-111-10- 3D2 was eight times greater than that of control I-125-MOPC-21. Uptake of F(ab')2 and Fab of 10- 3D2 was also compared. the scintigrams demonstrated that intact antibody provided the best images. Control In-111-labeled MOPC-21 and plasma did not show specific localization in the tumor. Uptake of In-111-labeled 10- 3D2 was also compared in two lines of human mammary tumors, BT-20 and HS- 578T . Imaging with 10- 3D2 was better for BT-20 than for HS- 578T . These studies demonstrated that (a) In-111-10- 3D2 can be utilized to image human mammary tumors hosted in nude mice; (b) intact antibody provided the best tumor images, although F(ab')2 had optimal target-to-background ratios for earlier imaging; and (c) different mammary tumor lines with possibly different concentrations of tumor-associated antigen showed different rates of uptake and apparent saturation with 10- 3D2 .