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Gas-filled ultrasound (US) contrast agents easily collapse in the body, and the gas can easily overflow, which limits the effectiveness of US imaging. To address this issue, an injectable gas-generating multi-mode system was developed that carries the MR negative contrast agent Fe3O4, the fluorescent dye Cy5.5, and the CO2 releasing donor (Na2CO3). The nanoparticles can continuously generate carbon dioxide (CO2) gas in acidic tumour tissue in the body, giving the tumour a strong echo signal under ultrasonic imaging. In addition, the nanoparticles confer excellent effects for MR and fluorescence imaging of the tumour tissue. The results indicate that this pH-responsive NP system provides good effects in MR/US/fluorescent imaging. This study provides a useful reference for multi-mode tumour imaging. [ABSTRACT FROM AUTHOR]

Purpose: Conjugation of nanocarriers with antibodies that bind to specific membrane receptors that are overexpressed in cancer cells enables targeted delivery. In the present study, we developed and synthesised two PAMAM dendrimer-trastuzumab conjugates that carried docetaxel or paclitaxel, specifically targeted to cells which overexpressed HER-2. Methods: The 1H NMR, 13C NMR, FTIR and RP-HPLC were used to analyse the characteristics of the products and assess their purity. The toxicity of PAMAM-trastuzumab, PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates was determined using MTT assay and compared with free trastuzumab, docetaxel and paclitaxel toward HER-2-positive (SKBR-3) and negative (MCF-7) human breast cancer cell lines. The cellular uptake and internal localisation were studied using flow cytometry and confocal microscopy, respectively. Results: The PAMAM-drug-trastuzumab conjugates in particular showed extremely high toxicity toward the HER-2-positive SKBR-3 cells and very low toxicity towards to HER-2-negative MCF-7 cells. As expected, the HER-2-positive SKBR-3 cell line accumulated trastuzumab from both conjugates rapidly; but surprisingly, although a large amount of PAMAM-ptx-trastuzumab conjugate was observed in the HER-2-negative MCF-7 cells. Confocal microscopy confirmed the intracellular localisation of analysed compounds. The key result of fluorescent imaging was the identification of strong selective binding of the PAMAM-doc-trastuzumab conjugate with HER-2-positive SKBR-3 cells only. Conclusions: Our results confirm the high selectivity of PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates for HER-2-positive cells, and demonstrate the utility of trastuzumab as a targeting agent. Therefore, the analysed conjugates present an promising approach for the improvement of efficacy of targeted delivery of anticancer drugs such as docetaxel or paclitaxel. [ABSTRACT FROM AUTHOR]

Purpose: Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer I-GLP-1(7-36)amide. Methods: Receptor autoradiography studies with I-GLP-1(7-36)amide agonist or I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. Results: The antagonist I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer I-GLP-1(7-36)amide. For comparison, I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. Conclusion: The GLP-1 receptor antagonist exendin(9-39) labelled with I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients. [ABSTRACT FROM AUTHOR]

Despite their side effect profile, there currently remains a heavy reliance on traditional cytotoxics and particularly tubulin targeting agents in cancer chemotherapy. To address this concern, significant progress has been made in the selective delivery of drugs to the tumour site. This review will examine the published data in support of the hypothesis that forming antibody conjugates of tubulin targeting agents is an effective approach towards their more effective delivery to the tumour site. Particular emphasis will be placed on the diversity of concepts under investigation, the efficacy of resultant conjugates, evidence of decreased resistance and the side effect profiles of the conjugates. [ABSTRACT FROM AUTHOR]

Purpose: A new universal tool for specific, non-covalent and non-destructive attachment of a recombinant antibody fragment to a polymer-modified adenovirus has been utilised to regulate the tropism of adenoviral gene delivery vector. Methods: We have prepared a multivalent reactive N-(2-hydroxypropyl)methacrylamide-based copolymer (PHPMA) bearing an α-bungarotoxin-binding peptide (BTXbp). The copolymer was used for covalent surface modification of adenoviral vectors (Ad). The α-bungarotoxin protein (BTX) has a nanomolar binding affinity for BTXbp, allowing non-covalent linkage of BTX fusion proteins. A single chain variable fragment of anti-PSMA antibody bearing BTX (scFv-BTX) binding to the prostate-specific membrane antigen (PSMA) was conjugated with the copolymer-coated adenovirus to enable specific infection of prostate cancer cells via PSMA receptors. Results: As shown by ELISA, the copolymer-coated virus exhibited much reduced binding to anti-Ad antibodies. Infection of PC-3 and LNCaP prostate cancer cells was ∼100-fold less efficient with copolymer-coated Ad than with un-modified Ad. Conjugation of scFv-BTX with Ad-PHPMA-BTXbp led to 5-10-fold restoration of infection in PSMA-positive LNCaP cells. In PSMA-negative PC-3 cells, the conjugation of scFv-BTX with Ad-PHPMA-BTXbp gave no enhancement of infection. Conclusions: We have shown that the presented Ad-PHPMA-BTXbp/scFv-BTX system can be used as a universal tool for a receptor-specific virotherapy. [ABSTRACT FROM AUTHOR]

Purpose: The epidermal growth factor receptor (EGFR) is overexpressed in several types of cancer and its inhibition can effectively inhibit tumour progression. The purpose of this study was to design an EGFR-specific imaging probe that combines efficient tumour targeting with rapid systemic clearance to facilitate non-invasive assessment of EGFR expression. Methods: Genetic fusion of a single-chain antibody fragment with the SNAP-tag produced a 48-kDa antibody derivative that can be covalently and site-specifically labelled with substrates containing 0-benzylguanine. The EGFR-specific single-chain variable fragment (scFv) fusion protein 425(scFv)SNAP was labelled with the near infrared (NIR) dye BG-747, and its accumulation, specificity and kinetics were monitored using NIR fluorescence imaging in a subcutaneous pancreatic carcinoma xenograft model. Results: The 425(scFv)SNAP fusion protein accumulates rapidly and specifically at the tumour site. Its small size allows efficient renal clearance and a high tumour to background ratio (TBR) of 33.2 ± 6.3 ( n = 4) 10 h after injection. Binding of the labelled antibody was efficiently competed with a 20-fold excess of unlabelled probe, resulting in an average TBR of 6 ± 1.35 ( n = 4), which is similar to that obtained with a non-tumour-specific probe (5.44 ± 1.92, n = 4). When compared with a full-length antibody against EGFR (cetuximab), 425(scFv)SNAP-747 showed significantly higher TBRs and complete clearance 72 h post-injection. Conclusion: The 425(scFv)SNAP fusion protein combines rapid and specific targeting of EGFR-positive tumours with a versatile and robust labelling technique that facilitates the attachment of fluorophores for use in optical imaging. The same approach could be used to couple a chelating agent for use in nuclear imaging. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinases (MMPs), a group of more than 20 zinc-containing endopeptidases, are upregulated in many diseases, but several attempts to use radiolabelled MMP inhibitors for imaging tumours have proved unsuccessful in mouse models, possibly due to the limited specificity of these agents or their unfavourable pharmacokinetic profiles. In principle, radiolabelled monoclonal antibodies could be considered for the selective targeting and imaging of individual MMPs. We cloned, produced and characterized high-affinity monoclonal antibodies specific to murine MMP-1A, MMP-2 and MMP-3 in SIP (small immunoprotein) miniantibody format using biochemical and immunochemical methods. We also performed comparative biodistribution analysis of their tumour-targeting properties at three time points (3 h, 24 h, 48 h) in mice bearing subcutaneous F9 tumours using radioiodinated protein preparations. The clinical stage L19 antibody, specific to the alternatively spliced EDB domain of fibronectin, was used as reference tumour-targeting agent for in vivo studies. All anti-MMP antibodies and SIP(L19) strongly stained sections of F9 tumours when assessed by immunofluorescence methods. In biodistribution experiments, SIP(SP3), specific to MMP-3, selectively accumulated at the tumour site 24 and 48 h after intravenous injection, but was rapidly cleared from other organs. By contrast, SIP(SP1) and SIP(SP2), specific to MMP-1A and MMP-2, showed no preferential accumulation at the tumour site. Antibodies specific to MMP-3 may serve as vehicles for the efficient and selective delivery of imaging agents or therapeutic molecules to sites of disease. [ABSTRACT FROM AUTHOR]

Overexpression of HER2 receptors is a prognostic and predictive biomarker in breast cancer and a number of other malignancies. Radionuclide molecular imaging of HER2 overexpression may influence patient management making treatment more personalized. Earlier, 111In-DOTA-ZHER2:342-pep2 (ABY-002) Affibody molecule demonstrated excellent imaging of HER2-expressing xenografts in mice shortly after injection. The use of the positron-emitting nuclide 68Ga instead of 111In might increase both the sensitivity of HER2 imaging and accuracy of expression quantification. The goal of this study was to prepare and characterize 68Ga-labelled ABY-002. 68Ga labelling of ABY-002 was optimized. In vitro cell binding and procession of 68Ga-ABY-002 was evaluated. Biodistribution and tumour targeting of 68Ga-ABY-002 and 111In-ABY-002 was compared in vivo by paired-label experiments. ABY-002 was incubated with 68Ga at 90°C for 10 min resulting in a radiochemical labelling yield of over 95%. Capacity for specific binding to HER2-expressing cells was retained. In vivo, both 68Ga-ABY-002 and 111In-ABY-002 demonstrated specific targeting of SKOV-3 xenografts and high-contrast imaging. Background radioactivity in blood, lungs, gastrointestinal tract and muscle fell more rapidly for 68Ga-ABY-002 compared with 111In-ABY-002 favouring imaging shortly after injection. For 68Ga-ABY-002, a tumour uptake of 12.4 ± 3.8%ID/g and a tumour to blood ratio of 31 ± 13 were achieved at 2 h post-injection. 68Ga-ABY-002 is easy to label and provides high-contrast imaging within 2 h after injection. This makes it a promising candidate for clinical molecular imaging of HER2 expression in malignant tumours. [ABSTRACT FROM AUTHOR]

Peptide receptor radionuclide therapy (PRRT) with radiolabelled somatostatin analogues is a novel therapy for patients with somatostatin receptor-positive tumours. We determined the effects of PRRT with [177Lu-DOTA0,Tyr3]octreotate (177Lu-octreotate) on glucose homeostasis and the pituitary-gonadal, pituitary-thyroid and pituitary-adrenal axes. Hormone levels were measured and adrenal function assessed at baseline and up to 24 months of follow-up. In 35 men, mean serum inhibin B levels were decreased at 3 months post-therapy (205 ± 16 to 25 ± 4 ng/l, p < 0.05) and follicle-stimulating hormone (FSH) levels increased (5.9 ± 0.5 to 22.7 ± 1.4 IU/l, p < 0.05). These levels returned to near baseline levels. Total testosterone and sex hormone binding globulin (SHBG) levels decreased (15.0 ± 0.9 to 10.6 ± 1.0 nmol/l, p < 0.05 and 61.8 ± 8.7 to 33.2 ± 3.7 nmol, p < 0.05), respectively, whereas non-SHBG-bound T did not change. An increase (5.2 ± 0.6 to 7.7 ± 0.7 IU/l, p < 0.05) of luteinizing hormone (LH) levels was found at 3 months of follow-up returning to baseline levels thereafter. In 21 postmenopausal women, a decrease in levels of FSH (74.4 ± 5.6 to 62.4 ± 7.7 IU/l, p < 0.05) and LH (26.8 ± 2.1 to 21.1 ± 3.0 IU/l, p < 0.05) was found. Of 66 patients, 2 developed persistent primary hypothyroidism. Free thyroxine (FT4) levels decreased (17.7 ± 0.4 to 15.6 ± 0.6 pmol/l, p < 0.05), whereas thyroid-stimulating hormone (TSH) and triiodothyronine (T3) levels did not change. Reverse triiodothyronine (rT3) levels decreased (0.38 ± 0.03 to 0.30 ± 0.01 nmol/l, p < 0.05). Before and after therapy adrenocorticotropic hormone (ACTH) stimulation tests showed an adequate response of serum cortisol (> 550 nmol/l, n = 18). Five patients developed elevated HbA1c levels (> 6.5%). In men 177Lu-octreotate therapy induced transient inhibitory effects on spermatogenesis, but non-SHBG-bound T levels remained unaffected. In the long term, gonadotropin levels decreased significantly in postmenopausal women. Only a few patients developed hypothyroidism or elevated levels of HbA1c. Therefore, PRRT with 177Lu-octreotate can be regarded as a safe treatment modality with respect to short- and long-term endocrine function. [ABSTRACT FROM AUTHOR]

Background: Hypoxia, which is commonly observed in areas of primary tumours and of metastases, influences response to treatment. However, its characterisation has so far mainly been restricted to the ex vivo analysis of tumour sections using monoclonal antibodies specific to carbonic anhydrase IX (CA IX) or by pimonidazole staining, after the intravenous administration of this 2-nitroimidazole compound in experimental animal models.Methods: In this study, we describe the generation of high-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX, using phage technology.Results: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo. In one of the two animal models of colorectal cancer studied (LS174T), CA IX imaging closely matched pimonidazole staining, with a preferential staining of tumour areas characterised by little vascularity and low perfusion. In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting. We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies.Conclusion: The new human anti-CA IX antibodies are expected to be non-immunogenic in patients with cancer and may serve as broadly applicable reagents for the non-invasive imaging of hypoxia and for pharmacodelivery applications. [ABSTRACT FROM AUTHOR]

Affibody molecules are low molecular weight proteins (7 kDa), which can be selected to bind to tumour-associated target proteins with subnanomolar affinity. Because of rapid tumour localisation and clearance from nonspecific compartments, Affibody molecules are promising tracers for molecular imaging. Earlier, 99mTc-labelled Affibody molecules demonstrated specific targeting of tumour xenografts. However, the biodistribution was suboptimal either because of hepatobiliary excretion or high renal uptake of the radioactivity. The goal of this study was to optimise the biodistribution of Affibody molecules by chelator engineering. Anti-HER2 ZHER2:342 Affibody molecules, carrying the mercaptoacetyl-glutamyl-seryl-glutamyl (maESE), mercaptoacetyl-glutamyl-glutamyl-seryl (maEES) and mercaptoacetyl-seryl-glutamyl-glutamyl (maSEE) chelators, were prepared by peptide synthesis and labelled with 99mTc. The tumour-targeting capacity of these conjugates was compared with each other and with the best previously available conjugate, 99mTc-maEEE-ZHER2:342, in nude mice bearing SKOV-3 xenografts. The tumour-targeting capacity of the most promising conjugate, 99mTc-maESE-ZHER2:342, was compared with radioiodinated ZHER2:342. All novel conjugates demonstrated successful tumour targeting and a low degree of hepatobiliary excretion. The renal uptakes of serine-containing conjugates, 33 ± 5, 68 ± 21 and 71 ± 10%IA/g, for99mTc-maESE-ZHER2:342, 99mTc-maEES-ZHER2:342 and 99mTc-maSEE-ZHER2:342, respectively, were significantly reduced in comparison with 99mTc-maEEE-ZHER2:342 (102 ± 13%IA/g). For 99mTc-maESE-ZHER2:342, a tumour uptake of 9.6 ± 1.8%IA/g and a tumour-to-blood ratio of 58 ± 6 were reached at 4 h p.i. A combination of serine and glutamic acid residues in the chelator sequence confers increased renal excretion and relatively low renal uptake of 99mTc-labelled Affibody molecules. In combination with preserved targeting capacity, this improved imaging of targets in abdominal area. [ABSTRACT FROM AUTHOR]

Virotherapy is currently being developed for many different types of viruses including replication-competent murine leukaemia virus (MLV) as a novel tool in cancer therapy. However, there is the risk of insertional mutagenesis associated with this virus, making careful preclinical studies necessary before its first application in man. We have previously generated conditionally replication-competent MLV variants that require activation by tumour-associated proteases to become infectious. Here we analysed in a comparative study the spreading of non-targeted and of such tumour-targeted MLV variants to tumour and extratumoural organs in immunodeficient mice. Both virus types were able to efficiently infect tumour cells after systemic administration. The non-targeted virus, however, also infected extratumoural organs like bone marrow, spleen and liver efficiently. In contrast, the targeted viruses revealed in a quantitative analysis of virus spreading an up to 500-fold more selective infection of tumour tissue than the non-targeted virus. The data raise serious doubts about a safe clinical use of non-targeted MLV. Engineering the virus to become activatable by tumour-associated proteases can significantly improve the safety of MLV.Gene Therapy (2008) 15, 1500–1510; doi:10.1038/gt.2008.92; published online 29 May 2008 [ABSTRACT FROM AUTHOR]

Respiratory motion of lung lesions is a limiting factor of quantification of positron emission tomography (PET) data. As some important applications of PET such as therapy monitoring and radiation therapy treatment planning require precise quantification, it is necessary to correct PET data for motion artefacts. The method is based on list-mode data. First, the motion of the lesion was detected by a centre of mass approach. In the second step, data were sorted corresponding to the breathing state. A volume of interest (VOI) around the lesion was defined manually, and the motion of the lesion in this VOI was measured with reference to the end-expiration image. Then, all voxels in the VOI were shifted according to the measured lesion motion. After optimisation of parameters and verification of the method using a computer-controlled motion phantom, it was applied to nine patients with solitary lesions of the lung. Fifty percent difference in measured lesion volume and 26% in mean activity concentration were found comparing PET data before and after applying the correction algorithm when simulating a motion amplitude of 28 mm in phantom studies. For patients, maximum changes of 27% in volume and 13% in mean standardised uptake values (SUV) were found. As respiratory motion is affecting quantification of PET images, correction algorithms are essential for applications that require precise quantification. We described a method which improves the quantification of moving lesions by a local motion correction using list-mode data without increasing acquisition time or reduced signal-to-noise ratio of the images. [ABSTRACT FROM AUTHOR]

Monitoring HER2 expression is crucial for selection of breast cancer patients amenable to HER2-targeting therapy. The Affibody molecule ZHER2:342 binds to HER2 with picomolar affinity and enables specific imaging of HER2 expression. Previously, ZHER2:342 with the additional N-terminal mercaptoacetyl-glycyl-glycyl-glycyl (maGGG) sequence was labelled with 99mTc and demonstrated specific targeting of HER2-expressing xenografts. However, hepatobiliary excretion caused high radioactivity accumulation in the abdomen. We investigated whether the biodistribution of ZHER2:342 can be improved by substituting glycyl residues in the chelating sequence with more hydrophilic seryl residues. The Affibody molecule ZHER2:342, carrying the chelators mercaptoacetyl-glycyl-seryl-glycyl (maGSG), mercaptoacetyl-glycyl- D-seryl-glycyl [maG(D-S)G] and mercaptoacetyl-seryl-seryl-seryl (maSSS), were prepared by peptide synthesis and labelled with 99mTc. The differences in the excretion pathways were evaluated in normal mice. The tumour targeting capacity of 99mTc-maSSS-ZHER2:342 was studied in nude mice bearing SKOV-3 xenografts and compared with the capacity of radioiodinated ZHER2:342. A shift towards renal excretion was obtained when glycine was substituted with serine in the chelating sequence. The radioactivity in the gastrointestinal tract was reduced threefold for the maSSS conjugate in comparison with the maGGG conjugate 4 h post injection (p.i.). The tumour uptake of 99mTc-maSSS-ZHER2:342 was 11.5 ± 0.5% IA/g 4 h p.i., and the tumour-to-blood ratio was 76. The pharmacokinetics and uptake characteristics of technetium-labelled ZHER2:342 were better than those of radioiodinated ZHER2:342. The introduction of serine residues in the chelator results in better tumour imaging properties of the Affibody molecule ZHER2:342 compared with glycyl-containing chelators and is favourable for imaging of tumours and metastases in the abdominal area. [ABSTRACT FROM AUTHOR]

To investigate the in vitro binding properties of a novel radiolabelled bombesin analogue, 177Lu-AMBA, in human neoplastic and non-neoplastic tissues selected for their expression of the bombesin receptor subtypes GRP-R, NMB-R and BRS-3. In vitro receptor autoradiography was performed in cancers expressing the various bombesin receptor subtypes. The novel radioligand 177Lu-AMBA was used and compared with established bombesin radioligands such as 125I-Tyr4-bombesin and 125I-[ D-Tyr6,β-Ala11,Phe13,Nle14]-bombesin(6–14). In vitro incidence of detection of each of the three bombesin receptor subtypes was evaluated in each tumour. 177Lu-AMBA identified all GRP-R-expressing tumours, such as prostatic, mammary and renal cell carcinomas as well as gastrointestinal stromal tumours. 177Lu-AMBA also identified all NMB-expressing tumours, but did not detect BRS-3-expressing tumours or BRS-3-expressing pancreatic islets. GRP-R-expressing peritumoural vessels were heavily labelled with 177Lu-AMBA. In contrast to the strongly GRP-R-positive mouse pancreas, the human pancreas was not labelled with 177Lu-AMBA unless chronic pancreatitis was diagnosed. In general, the sensitivity was slightly better with 177Lu-AMBA than with the conventional bombesin radioligands. The present in vitro study suggests that 177Lu-AMBA may be a very useful in vivo targeting agent for GRP-R-expressing tumours, NMB-R-expressing tumours and GRP-R-expressing neoangiogenic vessels. [ABSTRACT FROM AUTHOR]

Agents able to bind tightly and selectively to disease markers can greatly benefit disease diagnosis and therapy. Aptamers are functional molecules, usually DNA or RNA oligonucleotides, with the appropriate sequence and structure to form a complex with a target molecule. MUC1 is a well-known tumour marker present in a variety of malignant tumours and it has been a target of interest for many years. In this work we report the selection of DNA aptamers that bind with high affinity and selectivity to the MUC1 peptides. Combinatorial chemistry techniques based on the SELEX methodology were used for the identification of the specific aptamers. These were selected from an initial library containing a 25-base-long variable region, resulting in 425 random sequences of single-stranded DNA molecules, for their ability to bind to synthetic forms of MUC1. Ten rounds of in vitro selection were performed enriching for MUC1 binding. By round ten more than 90% of the pool of sequences consisted of MUC1-binding molecules. Selected aptamer families were cloned, sequenced and found to be unique, sharing no sequence consensus. The binding properties of these aptamers were quantitated by enzyme-linked immunosorbent assay and surface plasmon resonance, whereas their specificity for MUC1-expressing cancer cells has been validated using fluorescent microscopy. Aptamers offer significant advantages over existing antibody-based recognition procedures in that they offer higher binding affinity (higher retention/reduced dissociation) and specificity to the target (ability to determine variations on the protein target down to single amino acid changes), higher selectivity against mutated protein epitopes and potentially reduced immunogenicity and increased tumour penetration associated with their size. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Purpose: For the assessment of folate-based radiopharmaceuticals, human nasopharyngeal KB carcinoma cells are traditionally used although nasopharyngeal cancer is rare. On the other hand, the folate receptor (FR) is frequently overexpressed on diverse cancer types, the highest frequency (>90%) being on ovarian carcinomas. The goal of our study was the in vitro and in vivo assessment of different FR-positive human carcinoma cells. In addition, a murine sarcoma cell line was assessed as a pre-clinical alternative to human xenograft models. Methods: FR-positive human nasopharyngeal, cervical, ovarian and colorectal cancer cell lines and the transgenic mouse sarcoma (24JK-FBP) cell line were targeted with a novel 99mTc-tricarbonyl folate derivative 2. Comparative in vitro cell binding studies were carried out under standardised folate-deficient conditions. In vivo studies were performed in nude mice and C6 black mice. Results: The in vitro cell experiments revealed only FR-specific binding (unspecific <0.02%), ranging from 3.5% to 52% of complex 2 owing to variable levels of FR expression of the cell lines. In vivo tumour uptake of radiotracer 2 varied less than in vitro. It ranged from 0.66± 0.17% ID/g (LoVo) through 1.16±0.64% ID/g (IGROV-1) and 1.55±0.43% ID/g (24JK-FBP) to 2.33±0.36% ID/g (KB) 4 h p.i. Conclusion: These pre-clinical studies indicate that in vitro data obtained in FR-positive cancer cells do not necessarily correspond with or predict in vivo radiofolate uptake in corresponding (xeno)grafts. In addition, the murine 24JK-FBP cell line proved to be a valuable preclinical alternative to human tumour models. [ABSTRACT FROM AUTHOR]

Purpose: To further improve tumour targeting and delivery of imaging agents by long-circulating liposomes via the coupling of the anti-cancer monoclonal antibody 2C5 with nucleosome-restricted activity, which can recognize the surface of various tumours but not normal cells and can specifically target pharmaceutical carriers to tumour cells in vitro and in vivo. Methods: The 2C5 antibody was attached to the surface of long-circulating PEG-liposomes (LCL) by the post-insertion technique after antibody modification with a single-terminus activated PEG-lipid derivative to yield nucleosome-specific tumour-targeted liposomes. Tumour cell binding of the targeted liposomes was verified both by fluorescence microscopy and by flow cytometry in several cell lines using fluorescently labelled liposomes. 111In-radiolabelled liposomal formulations (prepared using membrane-anchored chelating groups) were used to examine in vivo biodistribution and tumour accumulation of liposomes by direct gamma scintigraphy. Results: The 2C5 antibody-modified LCL demonstrated a three- to eightfold increase in in vitro specific cell binding to various cancer cell lines of diverse origin. 111In-labelled tumour-targeted liposomes demonstrated prolonged circulation and doubled tumour accumulation compared with that of control formulations. Whole-body gamma scintigraphic imaging of mice implanted with different tumours revealed markedly faster (6 h post injection for 2C5-LCL vs 24 h for non-specific analogues) and superior in vivo tumour visualization with 111In-2C5-LCL than with the 2C5-free formulations in tested tumour models. Conclusion: The 2C5 antibody-modified LCL effectively and specifically accumulate in various tumours and can serve as delivery vehicles for imaging agents, allowing for fast and efficient tumour visualization. [ABSTRACT FROM AUTHOR]

Purpose: The folate receptor (FR) is a valuable tumour marker, since it is frequently overexpressed on various cancer types. The purpose of the present study was to pre-clinically evaluate novel site-specifically modified 99mTc(CO)3 folate (γ-derivative 4, α-derivative 5) and pteroate (6) conjugates for FR targeting. Methods: The 99mTc(CO)3 radiotracers 4-6 were prepared by a kit-like procedure. In vitro characterisation (KD and Bmax) of the radiotracers was performed with FR-positive KB cells. Tissue distribution was studied in tumour-bearing mice. SPECT/CT experiments were performed with a dedicated small animal SPECT/CT scanner. Results: The complexes 4-6 were formed in high yields (>92%). Binding constants of the radiotracers (KD in nM: 4: 2.09; 5: 2.51; 6: 14.52) were similar to those of 3H-folic acid (KD in nM: 7.22). In vivo the folate derivatives showed significantly better tumour uptake (4: 2.3±0.4% ID/g and 5: 1.2±0.2% ID/g, 4 h p.i.) than the pteroate derivative (6: 0.4±0.2% ID/g, 4 h p.i.). Clearance of all radiotracers from the blood pool and from non-targeted tissues was efficient (tumour to blood ratio approx. 200-350, 24 h p.i.). FR-positive tissue and organs were successfully visualised via small animal SPECT/CT. Conclusion: Radiotracers 4-6 are the first 99mTc(CO)3 tracers prepared via a kit formulation which exhibit full biological activity in vitro and in vivo. Folate derivatives 4 and 5 revealed significantly better pharmacokinetic properties than the pteroate derivative 6. Promising preclinical SPECT results warrant further assessment of 99mTc (CO)3 radiofolates for detection of FR-positive tumours. [ABSTRACT FROM AUTHOR]

This study assessed the clinical usefulness of a new technetium-99m labelled somatostatin analogue from the standpoint of oncological diagnostics. The study group comprised 40 patients in whom malignant neoplasms (32 primary and 8 metastatic) had been diagnosed. Among the primary tumours there were 21 cases of lung cancer (2 small cell and 19 non-small cell), seven pituitary adenomas (five hormonally active and two inactive), one liposarcoma, two carcinoids and one breast carcinoma. The metastatic tumours consisted of three malignant melanomas, one phaeochromocytoma, one prostatic cancer, one leiomyosarcoma, one pancreatic carcinoma ectopically secreting ACTH and one carcinoid of the thymus. The radiopharmaceutical [sup99m]Tc-EDDA/HYNIC-Tyr³-octreotide was administered i.v. at an activity of 740­925 MBq. The imaging comprised a whole-body scan and a single-photon emission tomography acquisition. Positive scintigrams were obtained in all cases of small cell and non-small cell lung cancer, four out of five hormonally active pituitary adenomas, one out of two cases of carcinoid, the liposarcoma and the breast cancer. Neoplastic metastases were visualised in two out of three patients with melanoma and in patients with phaeochromocytoma, ACTH-secreting pancreatic carcinoma and thymic carcinoid. Scintigrams were negative in both hormonally inactive pituitary adenomas, in one case of metastatic malignant melanoma, in the leiomyosarcoma and in the case of metastasis from prostatic carcinoma. The results of this pilot study indicate that [sup99m]Tc-EDDA/HYNIC-TOC is a potentially useful radio-pharmaceutical for imaging of a wide range of primary and metastatic tumours. Special attention should be paid to the successful imaging of all cases of non-small cell lung cancer. [ABSTRACT FROM AUTHOR]

Earlier studies have shown that modification of the octapeptide octreotide in positions 3 and 8 may result in compounds with increased somatostatin receptor affinity that, if radiolabelled, display improved uptake in somatostatin receptor-positive tumours. The aim of a recent research study in our laboratory was to employ the parallel peptide synthesis approach by further exchanging the amino acid in position 3 of octreotide and coupling the macrocyclic chelator DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to these peptides for labelling with radiometals like gallium-67 or -68, indium-111, yttrium-90 and lutetium-177. The purpose was to find radiopeptides with an improved somatostatin receptor binding profile in order to extend the spectrum of targeted tumours. A first peptide, [[sup111]In,[sup90]Y-DOTA]-1-Nal3-octreo- tide (111In,90Y-DOTA-NOC), was isolated which showed an improved profile. In[supIII]-DOTA-NOC exhibited the following IC[sup50] values (nM) when studied in competition with [sup[125I]][Leu[sup8], D-Trp[sup22], Tyr[sup25]]somatostatin-28 (values for Y[supIII]-DOTA-NOC are shown in parentheses): sstr2, 2.9±0.1 (3.3±0.2); sstr3, 8±2 (26±1.9); sstr5, 11.2±3.5 (10.47plusmn;1.6). Affinity towards sstr1 and 4 was very low or absent. In[supIII]-DOTA-NOC is superior to all somatostatin-based radiopeptides having this particular type of binding profile, including DOTA-lanreotide, and has three to four times higher binding affinity to sstr2 than In[supIII],Y[supIII]-DOTA-Tyr3-octreotide (InIII,YIII-DOTA-TOC). In addition, [[sup111]In]DOTA-NOC showed a specific and high rate of internalization into AR4–2J rat pancreatic tumour cells which, after 4 h, was about two times higher than that of [[sup111]In]DOTA-TOC and three times higher than that of [[sup111]In]DOTA-octreotide ([[sup111]In]DOTA-OC). The internalized radiopeptides were externalized intact upon 2 h of internalization followed by an acid wash. After 2–3 h of externalization a plateau is reached, indicating a steady-state situation explained by reactivation of the receptors followed by re-endocytosis. Biodistribution studies in CA 20948 tumour-bearing rats showed rapid clearance from all sstr-negative tissues except the kidneys. At 4 h the up-take of [[sup111]In]DOTA-NOC in the tumour and sstr-positive tissues, such as adrenals, stomach and pancreas, was three to four times higher than that of [[sup111]In]DOTA-TOC. Differential blocking studies indicate that this is at least partially due to the uptake mediated by sstr3 and sstr5. These very promising preclinical data justify the use of this new radiopeptide for imaging and potentially internal radiotherapy studies in patients. [ABSTRACT FROM AUTHOR]

The purpose of this investigation was to characterise the in vivo chemistry and binding mechanisms of technetium-99m dimercaptosuccinic acid [Tc(V)DMSA]. Biodistribution was studied in mice by frozen section whole-body autoradiography and microautoradiography in selected tissues. Binding to bone mineral analogues was studied in vitro using various forms of calcium phosphate and hydroxyapatite under varied conditions. Similar studies withTc-hydroxymethylene diphosphonate (HDP) were also carried out for comparison. The in vivo stability ofTc(V)DMSA was monitored by high-performance liquid chromatographic analysis of blood and urine samples taken over 24 h from patients injected with the tracer. Whole-body autoradiography shows thatTc(V)DMSA has highest affinity for bone (cortical rather than medullary) in mice. Substantial uptake of the tracer was also observed in the kidney (cytoplasm of cortical renal tubular cells). No specific localisation was observed in the liver at either the microscopic or the macroscopic level. WhileTc-HDP bound strongly to calcium phosphates under all conditions,Tc(V)DMSA binding was inhibited in the presence of phosphate and was stronger at pH 6.0 than at pH 7.4. In non-phosphate buffers, however, the binding ofTc(V)DMSA remained high across the pH range 4-7.4.Tc(V)DTVISA binds to calcium phosphates chemically unaltered, and no radioactive species other than the three isomers ofTc(V)DMSA were detected in blood or urine samples taken from patients up to 24 h after injection.Tc(V)DMSA is stable in vivo, and no conversion of the complex to other chemical species needs to be invoked to explain its uptake in bone metastases or soft tissue tumour. Bone affinity may be due to reversible binding of the unaltered complex to the mineral phase of bone. [ABSTRACT FROM AUTHOR]

Various tumours, classically specified as either neuroendocrine or non-neuroendocrine, contain high numbers of somatostatin receptors, which enable in vivo localization of the primary tumour and its metastases by scintigraphy with the radiolabelled somatostatin analogue octreotide. In addition granulomas and autoimmune processes can be visualized because of local accumulation of somatostatin receptor-positive activated mononuclear leucocytes. In many instances a positive scintigram predicts a favourable response to treatment with octreotide. It is tempting to speculate that octreotide labelled with an appropriate radionuclide might be used in cancer therapy. The successful application of radiolabelled octreotide in scintigraphy indicates the possible usefulness of other radiolabelled peptides, either native peptides or derivatives of these, in, for example, nuclear oncology. The small size of these peptides, e.g. bombesin and substance P, is of the utmost importance for a relatively fast blood clearance, thus leading to low background radioactivity. In this way peptides are powerful alternatives to (fragments of) monoclonal antibodies, the application of which to scintigraphic localization of specific cell surface antigen-bearing tumours is plagued by slow blood clearance and, hence, high background levels. [ABSTRACT FROM AUTHOR]

Apart from its use in endocrinology and rheumatology, therapeutic nuclear medicine is developing rapidly as an additional treatment modality in oncology. Many different specific tumour-seeking radiopharmaceuticals are being applied both for diagnostic scintigraphy and treatment, using multiple routes and mechanisms to target radionuclides at tumours. After a brief introduction of some basic principles of radionuclide targeting, the therapeutic radiopharmaceuticals available are reviewed according to the accumulation site in relation to the cell nucleus; the results of their current clinical use for therapy are also reviewed. The response observed to a number of these applications, the non-invasiveness of the procedure and the relative lack of toxicity and late effects in comparison with chemotherapy and external beam radiotherapy make radionuclide therapy an attractive and realistic alternative in the management of malignant disease, as well as in the treatment of a few benign disorders. [ABSTRACT FROM AUTHOR]

At our laboratory we are currently evaluating the suitability of mouse/human chimeric monoclonal antibodies (cmAb) for use in radioimmunotherapy of patients with head and neck squamous cell carcinoma (HNSCC). We have developed cmAb containing the human constant IgG1 domain and the variable domains of murine mAb (mmAb) E48 and U36 respectively. We considered the tumour-bearing nude mouse to be a well-validated model for a first testing of the targeting capabilities of these cmAb in comparison with the mmAb. Therefore, 3 μg cmAb E48 (labelled with 125I) and 3 μg mmAb E48 (labelled with 131I) were simultaneously injected into HNSCC-bearing nude mice and, at various assay times, mAb uptake in blood and other tissues was assessed. Remarkably, while in roughly 50% of the animals the biodistribution of the conjugates was similar, in the other animals cmAb E48 showed a much higher blood clearance than mmAb E48. This resulted in a lower tumour uptake of cmAb E48 in comparison with mmAb E48. To determine whether this phenomenon was related to mAb E48 or to the animal model, other cmAb-mmAb combinations were evaluated in the same way: cmAbs SF-25, 17-1A and U36 (all IgG1) were tested and all showed a rapid elimination in about 50% of the animals. Besides a decrease in blood concentration, an increase of cmAb levels in liver and spleen was observed within 24 h after injection. Isotype-specific enzyme-linked immunosorbent assays showed that mice that demonstrated a rapid elimination of cmAb from the blood had much lower endogenous IgG1, IgG2b and IgG3 titres than mice showing normal clearance. IgG2a levels were low in all mice. Biodistribution experiments with 3 μg chimeric 17-1A isoforms showed high blood clearance in a proportion of the mice for IgG1, IgG3 and IgG4, but not for IgG2. Increase of the cmAb dose to 100 μg resulted in a similar cmAb and mmAb biodistribution in all mice. Moreover, the biodistribution of the F(ab′)2 fragment of an IgG1 cmAb was similar for all mice in contrast to that of coinjected whole IgG. On the basis of these results it can be hypothesized that, in mice with low endogenous IgG titres, cmAb with specific isotypes are rapidly removed from the blood (and ultimately from the body) by mediation of Fc-binding receptors. Apparently, in mice with high endogenous IgG titres or in mice receiving a high cmAb dose, these receptors are saturated. Furthermore, the rapid elimination of cmAb from nude mice, which may occur after injection at a low dose, is a phenomenon related to the nude mouse model. [ABSTRACT FROM AUTHOR]

In previous reports we have described the development of mAb K984, reactive with an epitope expressed on the outer cell surface of undifferentiated, proliferating cells in normal stratified squamous epithelia and their neoplastic counterparts [28, 30]. The K984 antigen was also found to be homogeneously expressed by in vitro cultured squamous cell carcinoma (SCC) cell lines. In the present study we demonstrate that mAb K984 induces a significant, dose-dependent growth inhibition when SCC cells are grown in vitro as monolayer cultures in the presence of mAb K984. These data seem to indicate that mAb K984 has potential for tumour targeting, especially in a therapeutic setting. As a first approach to evaluate the suitability of mAb K984 for tumour targeting in vivo, radiolabelled mAb K984 was administered to SCC-xenografted nude mice. Selective tumour accumulation of mAb K984 was observed. Tumour to blood ratios and tumour to non-tumour ratios, as based on the biodistribution data, were at least ten times higher in case of the specific mAb K984 when compared to another non-specific, isotypematched control antibody. mAb K984 was also capable of visualizing tumour deposits in xenografted nude mice. The corollary of these findings is that the mAb K984-defined antigen probably is involved in the regulation of proliferation of stratified squamous epithelium and squamous cell carcinoma and that mAb K984 has potential for specific tumour targeting. [ABSTRACT FROM AUTHOR]