Search

Chemical modifications of the biotin carrier in pretargeted avidin–biotin radionuclide therapy may be of paramount importance for tuning the amount of the radioactivity delivered to cancer cells by labelled biotins. We report here the synthesis of a collection of new synthetic DOTA-constructs bearing two (+)-biotin molecules (bis-biotins), designed for the creation of multimeric Av units (tetramers) bonded to the antibody. All the syntheses were carried out following the solid phase strategy and growing the molecules on a Rink Amide resin. The biotin heads are connected through spacers containing PEG or non-PEG residues. Molecular modelling calculations suggested that the Av cross-linking ability of the bis-biotins depends mainly on the spacers length, with the best results being expected for arms affording distances in the range of 10–25 Å between the biotin carboxylate atoms, in the fully extended conformation. SEC-HPLC MALLS analysis of the products of our Av/bis-biotin reaction mixtures have confirmed this hypothesis. The bis-biotin 16, where the non-PEG linker ensured a distance of 26.7 Å between the biotin moieties, gave about 50% of Av oligomers while the shorter analogue 18 (19.5 Å) afforded 100% of an Av polymer containing about 21 protein units. Remarkably, the solubility of both the bis-biotins, i.e.16 and 18, in aqueous solutions was good and they showed excellent stability against the action of peptidases.

Purpose: A novel method for prostate irradiation is investigated. Similarly to (125)I or (103)Pd seed brachytherapy, (90)Y-avidin could be injected via the perineum under ultrasound image guidance. This study inspects the theoretical feasibility with a dosimetric model based on Monte Carlo simulation., Methods: A geometrical model of the prostate, urethra and rectum was designed. The linear-quadratic model was applied to convert (125)I absorbed dose prescription/constraints into (90)Y dose through biological effective dose (BED) calculation. The optimal (90)Y-avidin injection strategy for the present model was obtained. Dose distribution was calculated by Monte Carlo simulation (PENELOPE,GEANT4). Dose volume histograms (DVH) for the prostate, urethra and rectum were compared to typical DVHs of (125)I seed brachytherapy, used routinely in our institute., Results: With (90)Y-avidin, at least 95% of the prostate must receive more than 70 Gy. The absorbed dose to 10% of the urethra (D(10%_urethra)) and the maximum absorbed dose to the rectum (D(max_rectum)) must be lower than 122 Gy. For the present model, the optimum strategy consists in multiple injections of (90)Y-avidin 50 μl drops, for a total volume of 3.1 ml. The minimum activity to deliver the prescribed absorbed dose is 0.7 GBq, which also fully respects urethral and rectal constraints. The resulting dose map has a maximum in the central region with a sharp decrease towards the urethra and the prostate edge. Notably, D(10%_urethra) is 95 Gy and D(max_rectum) is below 2 Gy. Prostate absorbed dose is higher with (90)Y-avidin than (125)I seeds, although the total volume receiving the prescribed absorbed dose is 1-2% lower. Urethral DVH strictly depends on the (90)Y distribution, to be optimized according to prostate shape; in our model, BED(30%_urethra) is 90 Gy with (90)Y-avidin, whereas for patients receiving (125)I seeds it ranges between 150 and 230 Gy. The rectal DVH is always more favourable with (90)Y., Conclusion: The methodology is theoretically feasible and can deliver an effective treatment in T1-T2 prostate cancer. Pharmacokinetic and biodistribution studies in prostate cancer patients are needed for validation.

We recently described an oxidized avidin variant, named AvidinOX(®) , which is a product that chemically links to tissue proteins while maintaining the capacity to uptake intravenously administered biotin. Such product proved to be successful in targeting radionuclide therapy in a mouse model of inoperable breast cancer. Here, we show that the uptake of a single or multiple doses of biotin (up to five times), by the tissue-bound AvidinOX(®) , is stable for 2 weeks. Taking into account that oxidized avidin is the first chemically reactive protein to be proposed for clinical use, we evaluated its tolerability, immunogenicity and mutagenicity. Present in vitro data indicate that AvidinOX(®) (up to 10 μg/5 × 10(5) cells) does not affect cell viability or proliferation of PC3 human prostate cancer or 3T3 mouse fibroblast cell lines as well as primary mouse spleen cells. Safety pharmacology and toxicology studies were conducted using AvidinOX(®) up to the highest concentration compatible with its solubility (about 12 mg/mL), representing four times the product concentration intended for human use, and in the maximum administrable volume compatible with each study system. The intramuscular administration in rat and monkey induced a moderate to strong inflammatory response particularly after a second administration and consistently with the induction of an immune response. Interestingly, the intramuscular administration of AvidinOX(®) to rodents and monkeys exhibiting very high anti-avidin antibody titres was well tolerated with no systemic symptoms of any kind. Intravenous administration of AvidinOX(®) , performed to mimic an accidental injection of the dose intended for a local administration (15 μL of 3.3 mg/mL solution), showed significant localization of the product into the spleen not associated with uptake of the radiolabelled biotin intravenously injected after 24 hr, thus suggesting rapid inactivation. No mutagenic activity was induced by oxidized avidin in prokaryotic and eukaryotic cells. Overall, the present data indicate that AvidinOX(®) is well tolerated in rodents and non-human primates, thus supporting its clinical use within protocols of radionuclide therapy of inoperable tumour lesions., (© 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.)

Hen egg white avidin is increasingly used in the clinic as part of multifactor treatments such as pretargeted radionuclide therapy of cancer or as an antidote of biotinylated drugs. Taking into account that naturally occurring human antiavidin antibodies (HAVA) are common in humans, the present work investigates avidin immunogenicity as part of risk/benefit evaluations. Sera from 139 oncology patients naive to avidin were confirmed to exhibit HAVA with lognormally distributed titers. HAVA were boosted after avidin treatment, with no correlation with the avidin dose or with the basal titer. No antibody-related clinical symptoms were observed in 21 HAVA-positive patients treated with avidin. In mouse models, high mouse antiavidin antibody titers, induced to simulate the worst human condition, neither reduced the biotin uptake of intratissue-injected avidin nor affected the capacity of intravenously injected avidin to clear a biotinylated drug from circulation. In both models the avidin treatment was well tolerated. Results indicate that avidin immunogenicity does not affect its safety and efficacy, thus encouraging its further use in clinical applications.

Avidin is widely used in vitro for its capacity to bind biotin. However, avidin's in vivo use is limited by its short residence in blood and tissues. An avidin variant, named AvidinOX, has been recently described. This product is obtained by 4-hydroxyazobenzene-2'-carboxylic acid-assisted sodium periodate oxidation of avidin. This method generates aldehyde groups from avidin carbohydrates, sparing biotin-binding sites from inactivation. AvidinOX binds cellular and interstitial protein amino groups through Schiff's bases, resulting in a tissue half-life of 2 weeks, compared with 2 hours of native avidin. Binding of AvidinOX occurs in normal and neoplastic tissues. Data show that AvidinOX, administered intranipple in the breast of transgenic BALB/neuT mice, is highly efficient for capturing (90)Y-biotinDOTA, intravenously injected after 48 hours, leading to eradication of multifocal cancer lesions. Efficacy data, together with good tolerability results, indicate that AvidinOX is a highly innovative reagent for tissue-pretargeted radionuclide therapy.

Purpose: External beam radiotherapy (EBRT) after conservative surgery for early breast cancer requires 5-7 weeks. For elderly patients and those distant from an RT center, attending for EBRT may be difficult or impossible. We investigated local toxicity, cosmetic outcomes, and quality of life in a new breast irradiation technique-intraoperative avidination for radionuclide therapy (IART)-in which avidin is administered to the tumor bed and (90)Y-labelled biotin later administered intravenously to bind the avidin and provide irradiation. Reduced duration EBRT (40 Gy) is given subsequently., Methods: After surgery, 50 (ten patients), 100 (15 patients) or 150 mg (ten patients) of avidin was injected into the tumor bed. After 12-24 h, 3.7 GBq (90)Y-biotin (beta source for therapeutic effect) plus 185 MBq (111)In-biotin (gamma source for imaging and dosimetry) was infused slowly. Whole-body scintigraphy and SPECT/CT images were taken for up to 30 h. Shortened EBRT started 4 weeks later. Local toxicity was assessed by RTOG scale; quality of life was assessed by EORTC QOL-30., Results: Of 35 patients recruited (mean age 63 years; range 42-74) 32 received IART plus EBRT. 100 mg avidin provided 19.5 +/- 4.0 Gy to the tumor bed and was considered the optimum dose. No side-effects of avidin or (90)Y-biotin occurred, with no hematological or local toxicity. Local G3 toxicity occurred in 3/32 patients during EBRT. IART plus EBRT was well accepted, with good cosmetic outcomes and maintained quality of life., Conclusions: IART plus reduced EBRT can accelerate irradiation after conservative breast surgery.

The synthesis of four biotin derivatives carrying two DOTA moieties for each ligand (BisDOTA set) is reported, for increasing radiation/dose ratio and improving efficiency in the pretargeted avidin-biotin radioimmunotherapy. The biotin-containing scaffold of two BisDOTA was similar to the mono-DOTA derivative previously described. Then the scaffold was elongated by trifunctionalized spacers of different length and conjugated with one of the COOH groups of two DOTA. Two others were prepared starting from a on-resin lysine residue. The lysine alpha-NH2 was bonded to biotin, and then spacers were appended to the epsilon-NH2 and conjugated with two DOTA molecules. One compound contained a p-aminobenzoic acid spacer, which ensured higher head-to-tail distance and increased rigidity of the chain. These last two compounds had a very high ability to bond avidin and were labeled with 90Y at high specific activity. All the compounds were resistant to the action of serum biotinidases.

Avidin is a glycoprotein from hen egg white that binds biotin with very high affinity. Here we describe OXavidin, a product containing aldehyde groups, obtained by ligand-assisted sugar oxidation of avidin by sodium periodate. OXavidin chemically reacts with cellular and tissue proteins through Schiff's base formation thus residing in tissues for weeks while preserving the biotin binding capacity. The long tissue residence of OXavidin as well as that of OXavidin/biotinylated agent complex occurs in normal and neoplastic tissues and immunohistochemistry shows a strong and homogenous stromal localization. Once localized in tissue/tumor, OXavidin becomes an "artificial receptor" for intravenous injected biotin allowing tumor targeting with biotinylated therapeutics like radioisotopes or toxins. Moreover, present data also suggest that OXavidin might be useful for the homing of biotinylated cells. Overall, OXavidin exhibits a remarkable potential for many different therapeutic applications.

Purpose: In a continuous effort to seek for anticancer treatments with minimal side effects, we aim at proving the feasibility of the Intraoperative Avidination for Radionuclide Therapy, a new procedure for partial breast irradiation., Experimental Design: To assess doses of 90Y-DOTA-biotin to target (i.e., breast tumor bed) and nontarget organs, we did simulation studies with 111In-DOTA-biotin in 10 candidates for conservative breast surgery. Immediately after quadrantectomy, patients were injected with 100-mg avidin in the tumor bed. On the following day, patients were given 111In-DOTA-biotin (approximately 111 MBq) i.v. after appropriate chase of biotinylated albumin (20 mg) to remove circulating avidin. Biokinetic studies were done by measuring radioactivity in scheduled blood samples, 48-h urine collection, and through scintigraphic images. The medical internal radiation dose formalism (OLINDA code) enabled dosimetry assessment in target and nontarget organs., Results: Images showed early and long-lasting radioactive biotin uptake in the operated breast. Rapid blood clearance (<1% at 12 h) and urine excretion (>75% at 24 h) were observed. Absorbed doses, expressed as mean+/-SD in Gy/GBq, were as low as 0.15+/-0.05 in lungs, 0.10+/-0.02 in heart, 0.06+/-0.02 in red marrow, 1.30+/-0.50 in kidneys, 1.50+/-0.30 in urinary bladder, and 0.06+/-0.02 in total body, whereas in the targeted area, they increased to 5.5+/-1.1 Gy/GBq (50% ISOROI) and 4.8+/-1.0 Gy/GBq (30% ISOROI)., Conclusion: Our preliminary results suggest that Intraoperative Avidination for Radionuclide Therapy is a simple and feasible procedure that may improve breast cancer patients' postsurgical management by shortening radiotherapy duration.

A new procedure, known as Intraoperative Avidination for Radionuclide Therapy (IART), is described in breast cancer patients. In this paper, we provide proof of the principle that intraoperative injection of avidin in the tumour bed after quadrantectomy allows homing in of intravenously (IV) administered radioactive biotin to the target site. This approach of targeted therapy consists of two steps: (i) "avidination" of the anatomical area of the tumour with avidin injected by the surgeon, into and around the tumour bed; (ii) targeting the anatomical area of the tumour by IV injection of radiolabelled biotin. The scintigraphic images demonstrated fast and stable uptake of labelled biotin at the site of operated breast. The radiation dose released to the index quadrant was more than 5 Gy/GBq, consistent with a boost of 20 Gy for an activity of 3.7 GBq 90Y-biotin (100mCi). A further large clinical trial facing IART in combination with reduced external-beam radiotherapy is, in our opinion, fully justified.

Avidin was modified with poly(ethylene glycol) in the presence of a biotin binding site protective agent synthesised by imminobiotin conjugation to branched 20 kDa PEG. Avidin was incubated with imminobiotin-PEG and reacted with high amounts of 5, 10 or 20 kDa PEG to modify the protein amino groups. Circular dichroism demonstrated that the extensive PEGylation does not alter the protein conformational structure. The affinity of avidin-PEG conjugates for biotin and biotinylated antibodies depended on the PEG size or the use of a protective agent. Avidin-PEG 10 and 20 kDa prepared in the presence of imminobiotin-PEG maintained 100% of the native affinity for biotin. The 5 kDa PEG derivative and the ones obtained without biotin site protection maintained 79-85% of the native affinity. The affinity for biotinylated antibodies decreased to 35% when the conjugation was performed without imminobiotin-PEG, while the conjugates obtained with high-molecular-weight PEGs in the presence of protective agent displayed high residual affinity. All conjugates possessed negligible antigenicity and immunogenicity. PEGylation greatly prolonged the avidin permanence in the circulation, reduced its disposition in the liver and kidneys and promoted accumulation into solid tumors. PEGylation was found to prevent the protein cell uptake, either by phagocytosis or pinocytosis.

Objective: To verify whether native avidin, made radioactive through the binding with technetium-99m labeled biotin (99mTc-biotin), selectively accumulated in superficial tumor tissues following intravesical administration., Methodology: A total of fifteen patients with transitional cell bladder cancer were instilled intravesically with radiolabeled avidin. Cold biopsies were obtained from macroscopically normal and tumor tissues before transurethral resection (TUR) and the radioactivity in the samples was measured., Results: Increased accumulation of radiolabeled avidin was observed in tumor tissue compared to normal bladder tissue and in some cases, remarkably high quotients of uptake (q) in tumor versus normal tissues were determined (86 and 44). The three patients instilled with a deglycosylated avidin at neutral pI, who served as a control, showed no significant uptake in either tumor or normal urothelium and no difference in relative uptake (q=1.0)., Conclusion: This pilot study indicated that intravesical administration of radiolabeled avidin resulted in a preferential accumulation in tumor tissue compared to normal urothelium. The instillation of radiolabeled avidin warrant further investigations in order to explore the possibility to treat superficial bladder neoplasms locally by replacing 99mTc with high energy beta emitting radionuclides associated with biotin.

Chicken avidin and bacterial streptavidin are proteins used in a wide variety of applications in the life sciences due to their strong affinity for biotin. A new and promising use for them is in medical pretargeting cancer treatments. However, their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in these applications. To search for potentially beneficial new candidates, we screened egg white from four different poultry species for avidin. Avidin proteins, isolated from the duck, goose, ostrich and turkey, showed a similar tetrameric structure, similar glycosylation and stability against both temperature and proteolytic activity of proteinase K as chicken avidin. Biotin-binding properties of these avidins, measured using IAsys optical biosensor, were similar to those found in avidin from the chicken. Three of these novel avidins, however, showed different immunological cross-reactivities when compared with chicken avidin. The patient sera responses to duck, goose and ostrich avidins were also lower than those observed for chicken and turkey avidins. Our findings suggest that the use of these proteins offers advantages over chicken avidin and bacterial streptavidin in pretargeting applications.

Avidin-poly(ethylene glycol) (PEG) conjugates were obtained by derivatization of about 10% of the protein amino groups (four amino groups per protein molecule) with linear 5 kDa PEG or branched 10 or 20 kDa PEGs. Circular dichroism analysis showed that the polymer conjugation neither altered the protein structure nor the environment of the aromatic amino acids which are present at the level of the biotin binding site. Spectroscopic studies were carried out to evaluate the biotin recognition activity of the conjugates either in terms of number of biotin binding sites or avidin/biotin affinity. Avidin-PEG 5 kDa and avidin-PEG 10 kDa displayed over 90% of the native protein biological activity while a reduction in the recognition of biotinylated antibodies of about 25% was found with PEG 20 kDa. In vivo studies demonstrated that the protein immunogenicity was in the order: wild type avidin>avidin-PEG 5 kDa>avidin-PEG 10 kDa>avidin-PEG 20 kDa. By intravenous injection into mice bearing a solid tumor, all conjugates displayed prolonged permanence in the circulation with respect to the native protein. The area under the curve values of avidin-PEG 5 kDa, avidin-PEG 10 kDa and avidin-PEG 20 kDa were about 3-, 7- and 30-times higher than the wild type avidin with reduced accumulation in kidneys and liver. Interestingly, all conjugates accumulated significantly in the tumor mass. In particular, in the case of avidin-PEG 20 kDa, 8% of the injected dose (ID)/g of tissue accumulated in the tumor after 5 h from the administration and over 6% of the ID/g was maintained throughout 72 h., (Copyright 2002 Elsevier Science B.V.)

In a previous study we applied a three-step avidin-biotin pretargeting approach to target 90Y-biotin to the tumour in patients with recurrent high grade glioma. The encouraging results obtained in this phase I-II study prompted us to apply the same approach in an adjuvant setting, to evaluate (i) time to relapse and (ii) overall survival. We enrolled 37 high grade glioma patients, 17 with grade III glioma and 20 with glioblastoma, in a controlled open non-randomized study. All patients received surgery and radiotherapy and were disease-free by neuroradiological examinations. Nineteen patients (treated) received adjuvant treatment with radioimmunotherapy. In the treated glioblastoma patients, median disease-free interval was 28 months (range=9-59); median survival was 33.5 months and one patient is still without evidence of disease. All 12 control glioblastoma patients died after a median survival from diagnosis of 8 months. In the treated grade III glioma patients median disease-free interval was 56 months (range=15-60) and survival cannot be calculated as only two, within this group, died. Three-step radioimmunotherapy promises to have an important role as adjuvant treatment in high grade gliomas, particularly in glioblastoma where it impedes progression, prolonging time to relapse and overall survival. A further randomized trial is justified., (Copyright 2002 The Cancer Research Campaign)

We have shown previously that T cells, tagged with biotinylated anti-CD3 antibody fragments, can exert avidin-dependent cytolytic activity on suitably biotinylated tumor cells in vitro. In this study, we demonstrate that avidin-driven CTL-tumor bridging in vivo leads to growth inhibition of murine tumors WEHI-164 fibrosarcoma and RMA lymphoma. The biodistribution of biotin-tagged 111In-labeled T cells demonstrated a selective avidin-dependent and time-dependent accumulation of radioactivity at tumor sites. The specificity of lymphocyte tumor localization was demonstrated by the concurrent time-dependent decrease of radioactivity in the blood and in all other organs. Furthermore, we documented a therapeutic effect of the adoptively transferred T cells, i.e., a significant delay of tumor growth at early stages. All of the experiments included a control group of mice, which received all of the reagents, except avidin. These avidin-minus mice showed no specific localization and no delay in tumor growth, indicating that avidin bridging was essential for T-cell activity at tumor sites.

We report here the results of a validation study of the avidin/indium-111 biotin approach in patients with skeletal lesions. This study involved 54 patients with orthopaedic conditions: 20 patients with intermediate suspected osteomyelitis of the trunk, 19 patients with infection/inflammation of prosthetic joint replacements, and 15 patients with suspected osteomyelitis of appendicular bones. Avidin (3 mg) was injected as an i.v. bolus, followed 4 h later by 111In-biotin; imaging was acquired 30 min and 16-18 h after administration of 111In-biotin. Technetium-99m hexamethylpropylene amine oxime (99mTc-HMPAO)-labelled leucocyte scintigraphy was performed in 39/54 patients. The overall sensitivity of the avidin/111In-biotin scan was 97.7% (versus 88.9% for 99mTc-HMPAO leucocyte scintigraphy). While the diagnostic performance of avidin/111In-biotin scintigraphy was similar to that of 99mTc-HMPAO leucocyte scintigraphy in patients with prosthetic joint replacements or osteomyelitis of appendicular bones, the avidin/111In-biotin approach clearly performed better than 99mTc-HMPAO leucocyte scintigraphy in patients with suspected osteomyelitis of the trunk (100% sensitivity, specificity and accuracy versus 50% sensitivity, 100% specificity and 66.7% accuracy for 99mTc-HMPAO-leucocyte scintigraphy). These results demonstrate the feasibility of the avidin/111In-biotin approach for imaging sites of infection/inflammation in the clinical setting. Although no systematic advantages of avidin/111In-biotin scintigraphy were found versus 99mTc-HMPAO leucocyte scintigraphy, the newer scintigraphic method is more practicable and involves lower biological risk for the operators.

The mature hen avidin encoded by a synthetic cDNA was expressed in Escherichia coli in an insoluble form. After resolubilization, renaturation and purification, a recovery of about 20 mg/l cell culture was obtained. ELISA assays indicated no apparent differences in biotin binding between the natural and recombinant avidins. In addition, an acidic avidin mutant, bearing the substitutions Lys3-->Glu, Lys9--> Glu, Arg26-->Asp and Arg124-->Leu of four exposed basic residues, was produced. The protein, expressed and renatured as wild-type avidin, showed unaltered biotin-binding activity. The acidic pI (approximately 5.5) and lack of aggregation of the mutant allowed easy electrophoretic analysis under non-denaturing conditions of the protein alone and of its complexes with biotin, biotinylated transferrin or peroxidase. Analysis of the sera from sensitized subjects revealed that the avidin mutant has altered antigenicity. Both recombinant avidins were crystallized and the three-dimensional structures solved by molecular replacement and refined to 0.22 nm resolution. The three-dimensional structures of the two recombinant molecules, in the absence of biotin and of glycosylation, are fully comparable with those of the natural hen avidin previously reported.

Pretargeting techniques using the avidin-biotin system have shown encouraging results in both diagnostic and therapeutic clinical trials. It has been shown that in cancer therapy the ideal agent to be used for pretargeting should have a plasma half-life longer than avidin and lower immunogenicity than streptavidin in order for these procedures to be applied safely and repeatedly in patients. We prepared a recombinant form of avidin with no carbohydrates and avidins, biochemically modified either by decreasing the positive charges with succinic anhydride or by linking polyethylene glycol (PEG) at three different molar ratios and evaluated their in vivo behaviour after i.p. administration in mice. The succinylation and PEGylation of avidin increased the plasma half-life proportionally to the degree of protein modification. The procedures, however, affected the biotin binding to some extent. The biodistribution studies showed that, for all six time points (ranging from 20 min to 18 h post-injection), the liver and kidney to blood ratios were lower for PEGylated avidins than native, recombinant and succinyl avidin. Recombinant and low PEGylated avidin evoked an immune response in all mice after at least three injections. Native, recombinant and succinyl avidins showed higher serum titres than PEGylated avidins. In conclusion, the conjugation of avidin to PEG chains (n = 7) originates a compound with a suitable blood clearance, low immunogenicity and concurrent low cross-reactivity with avidin.

Unlabelled: Radioimmunodetection of tumor can be improved by introducing a two-step system in which radiolabeled streptavidin is administrated after the injection of a biotinylated monoclonal antibody (MAb) (two-step) or radiolabeled biotin is injected after biotinylated MAb and avidin (three-step). The anti-carcinoembryonic antigen (CEA) MAb FO23C5 has been recently exploited in a three-step protocol based on the avidin-biotin system. The anti-folate receptor (FR) MAb MOv18 has proven suitable for radioimmunodetection of ovarian cancer using directly radiolabeled MAb or in a two-step method. In this study, we analyzed the suitability of MOv18 in a three-step protocol in ovarian carcinoma patients and the internalization events after formation of the MOv18-avidin complex., Methods: Selected patients with documented metastatic lesions were enrolled in a three-step radioimaging analysis with biotinylated MOv18 and FO23C5, avidin and (111)In-labeled biotin. Two-step internalization experiments were conducted in vitro with MOv18 and MOv19 MAbs on the FR-overexpressing IGROV1 cell line and with the anti-CEA MAb FO23C5 on the LS174T cell line. Cells were incubated sequentially with biotinylated MAb and 125I-labeled streptavidin or with 125I-biotinylated MAb and cold streptavidin., Results: In the in vivo study, SPECT revealed the majority of metastatic lesions in patients injected with biotinylated MOv18; however, the tumor-to-background ratio was relatively low. In the in vitro study, a consistent internalization was induced by antigen-biotinylated MAb-streptavidin complex formation at the cell surface in both antigenic systems analyzed. However, the extent of internalization was lower in the CEA model., Conclusion: The internalization ability of avidin suggests its potential clinical application for delivering toxic agents in a two-step approach (biotinylated MAb + avidin conjugate). The suitability of a given MAb for three-step clinical applications (biotinylated MAb + avidin + biotin) should be previously investigated by using appropriate in vitro experiments.

Tumor pretargeting with biotinylated antibodies and avidin, followed by a delayed delivery of radioactive-labeled biotin, is currently used for in vivo diagnosis and therapy in cancer patients. Herein, we describe the use of a three-step antibody/avidin targeting approach to increase the local concentration and the persistence of biotinylated human tumor necrosis factor alpha (bio-TNF) on a mouse tumor. Mouse RMA lymphoma cells were transfected with the Thy 1.1 allele (RMA-Thy 1.1) to generate a unique tumor-associated antigen. In vitro pretargeting of RMA-Thy 1.1 cells with the biotinylated anti-Thy 1.1 monoclonal antibody 19E12 (bio-19E12) and NeutrAvidin increased the amount of bio-TNF that bound to the cell (10-20 times in comparison with non-pretargeted cells), as well as its half-life on the surface (>30 times). Furthermore, cell pretargeting reduced by more than 2 orders of magnitude the LD50 of bio-TNF in a cytolytic assay with actinomycin D. Finally, RMA-Thy 1.1 cells, pretreated in vitro with bio-TNF according to the three-step procedure and injected into syngeneic C57/BL6 mice, were less tumorigenic than controls. These results indicate that the three-step targeting approach markedly increases the amount and the persistence of bio-TNF on the cell surface and that cell-bound bio-TNF can trigger cytolytic effects in vitro and antitumor effects in vivo. Tumor pretargeting with biotinylated antibodies and avidin could be a novel strategy for increasing the therapeutic index of TNF.

Unlabelled: Prosthetic vascular graft infection, though rare, carries high morbidity and mortality rates; therefore, timely diagnosis is important. Patients, however, often present with vague symptoms, and radiological investigations are frequently inconclusive. These factors may lead to prolonged periods of observation and hospitalization, with the resultant increase in costs and complication rates, before reaching a final diagnosis. This prospective study evaluates the use of nonspecific avidin/111In-biotin imaging in diagnosing prosthetic vascular graft infection., Methods: Twenty-five patients with a total of 29 grafts were investigated. Eighteen patients (19 grafts) had low probability of disease, whereas the remaining 7 patients (10 grafts) warranted surgical exploration based on clinical, laboratory or radiological evidence. Avidin was first injected intravenously and then followed 24 hr later by administration of 111In-biotin. Whole-body images were obtained 10 min and 2 hr postinjection of 111In-labeled biotin. SPECT imaging was performed 1 hr postinjection. Increased uptake along part or the whole length of the graft was considered evidence of graft infection., Results: Avidin/111In-biotin scintigraphy correctly identified all infected grafts, as confirmed by culturing surgical specimens. In contrast, infection was correctly excluded in all but one of the grafts, and long-term follow-up was used to assess the presence of infection in patients who did not undergo surgical intervention., Conclusion: Avidin/111In-biotin scintigraphy is a simple and accurate imaging method for the routine diagnosis of vascular graft infection, and it may have a role in identifying the disease process in its initial stages, thus improving prognosis.

Objectives: To evaluate a new imaging technique, for diagnosis of prosthetic vascular graft infection. Avidin is a protein which accumulates nonspecifically at sites of inflammation or infection. Due to its extremely low dissociation constant with biotin sites of infection can be imaged, using avidin as a pre-target, followed by injection of 111In-labelled biotin. This technique is much simpler than the common scintigraphic methods which employ labelling of blood components and its target-to-background ratio is greater than the methods employing radiolabelled proteins., Design: Prospective clinical study., Setting: A single department of vascular surgery and one of nuclear medicine of a Northern Italian hospital., Materials: Between May 1993 and May 1994, 31 grafts in 26 patients were studied; the series included 23 men and three women with a mean age of 65.5 years (range 54-76 years). The prosthetic graft (Dacron -16, ePTFE -15) were: aortoaortic 5, aortobifemoral 15, aortoiliac 1, and femoropopliteal 10. Sixteen patients were suspected of having a vascular graft infection (Group A), the other 10 patients served as controls (Group B). 20 mg of Avidin were injected iv, followed 24 h later by i.v. injection of 500 micrograms of Biotin labelled with 74 MBq of 111In., Chief Outcome Measures: Whole-body imaging was performed at 10 min and 2 h post-injection, along with SPECT imaging when indicated. Scan results were correlated with the traditional imaging modalities and the clinical outcome of the patients., Main Results: In Group A: two patients (three grafts) were excluded from the study, there were six true-positives, one false-positive and 11 true-negatives. Results in Group B: 10 true-negatives. The overall sensitivity was 100%, the specificity 95%, the accuracy 96%, the positive predictive value 86% and the negative predictive value 100%., Conclusions: These data suggest that Avidin/111In-labelled Biotin scintigraphy is a useful non invasive diagnostic method for early diagnosis of suspected prosthetic vascular graft infection.

Purpose: Radiolabeled monoclonal antibodies (MAbs) have been reported to allow tumor intraoperative detection by means of a gamma-detecting probe. The technology is called the Radioimmunoguided Surgery (RIGS) system. The main inconveniences of the method are 1) the long interval needed for clearance of unattached MAbs from the patient's body, between the injection of the MAb and surgery, and 2) the low sensitivity of current MAbs used in detecting small tumors. We describe a new method to overcome these inconveniences using biotinylated MAbs and avidin in order to obtain a rapid blood clearance of the radiolabeled MAbs both anticarcinoembryonic antigen and antitumor-associated glycoprotein-72 MAbs., Methods: Twenty patients with primary and recurrent colorectal cancer have been enrolled in the study; 125I-biotinylated MAbs FO23C5 (anticarcinoembryonic antigen) and B72.3 (antitumor-associated glycoprotein-72) followed by cold avidin were injected in 13 patients and 7 patients, respectively., Results: A decrease of 94 +/- 3 percent of circulating radioactivity was achieved in 3 to 5 days. Patients underwent surgery approximately seven days after MAb injections rather than after four weeks. Tumors were localized in 14/20 (70 percent) patients (true positive), 2 (10 percent) were false negative, and 4 (20 percent) were true negative. The overall sensitivity level in early-stage primary cancers was 37 percent when related to the presence of disease and 75 percent when related to antigenic expression. The sensitivity for more advanced cancer and for recurrences was 100 percent. Moreover, the in vivo tumor targeting of biotinylated MAb was demonstrated in frozen tumor section by direct streptoavidin-peroxidase staining., Conclusions: The avidin-biotin system may enhance applicability and effectiveness of radioimmunoguided surgery (RIGS).

Radiolabelled MoAbs have been used in both the diagnosis and the treatment of a variety of tumors. Recently, a three-step tumor pre-targeting strategy has been proposed to overcome one of the major limiting factors in radioimmunodetection: the low tumor-to-background ratio. We evaluated this pre-targeting protocol in 10 patients diagnosed as having pulmonary carcinoma. One milligram of biotinylated anti-CEA MoAb (FO23C5) was administered i.v. (1st step); 24 hours later 5 mg of avidin was injected i.v. (2nd step) followed by 100-500 ng of 111In-biotin (5 mCi) the day after (3rd step). Imaging was performed using single photon emission tomography (SPET). No toxicity and no adverse reactions were observed. Tumor was detected in 8 out of 10 patients. Mediastinal metastases were also localised in 2 out of 3 patients, and adrenal gland recurrency in 1 out of 2 patients. The tumor/background (normal lung), heart, liver and spine ratios were respectively 2.0, 1.0, 1.3 and 4.1 at 90 minutes post-injection. These preliminary data show that the three-step pretargeting method is safe and allows SPECT tumor localization soon after the injection of the radiolabel. In the future, the use of MoAbs with higher specificity could result in improved tumor-targeting, and in the possibility of lung cancer radioimmunotherapy.

Diagnosis and experimental therapy of cancer have been performed with encouraging results using radiolabelled monoclonal antibodies. However, the high background due to non-specific uptake by normal tissue and blood is a major drawback in antibody-guided tumor detection. Various strategies have been proposed to overcome this problem, such as computed background subtraction, use of a second antibody, and local delivery. An antibody is a slow "bullet" for tumor targeting, since in many lesions it requires two or three days to accumulate. The use of fragments such as F(ab')2 or Fab, which display a faster blood clearance than whole antibody, improves tumor localization to a sufficient extent (hours) to allow the use of the most suitable radionuclides, e.g. 99m-Tc. In therapeutic applications we are still far away from the optimal condition in terms of the absolute amount of radioactivity delivered to the tumor. The high specificity of antibodies could be exploited at its best by delaying the delivery of the label to a time when the ratio tumor-bound to non-tumor-bound antibody has reached its maximum value. To obtain this goal, the label should display a fast clearance and should be captured by the antibody already targeted onto tumor cells. These considerations have led to strategies of tumor pretargeting where antibody and label are administered separately. One of these strategies, based on the avidin-biotin system, has already been used extensively for several years in immunohistochemistry and in ELISA. Due to the flexibility of this system, several alternative protocols are possible.(ABSTRACT TRUNCATED AT 250 WORDS)

Several monoclonal antibodies (MAbs), reactive with tumor-associated antigens, selectively persist on tumor sites in vivo for many days. If biotinylated, such highly specific tags on tumor cells could become targets for radioactive avidin, administered after suitable intervals. The proposed strategy is based on a number of assumptions concerning the ability of avidin to preserve its biological properties in heterologous in vivo environments, on its lack of toxicity and on its biodistribution. A preliminary study has been carried out in rabbits, using biotinylated nitrocellulose and polystyrene targets. The results of this study indicate that in rabbits 1) avidin can be administered i.v. and i.p. without adverse reactions, 2) it does not show any preferential localization, 3) it is eliminated with a biological half-life of 24 hr, 4) its biological properties are not impaired by in vivo conditions, since it accumulates at biotinylated targets only, 5) CEA-bearing targets can be biotinylated in vivo by biotin-labelled anti-CEA MAbs and 6) the biotin-avidin chain can be further extended in vivo since bound avidin is still able to bind biotinylated radioactive proteins.

Background: Breast conserving surgery (BCS) plus external beam radiotherapy (EBRT) is considered the standard treatment for early breast cancer. We have investigated the possibility of irradiating the residual gland, using an innovative nuclear medicine approach named IART® (Intra-operative Avidination for Radionuclide Therapy). Aim: The objective of this study was to determine the optimal dose of avidin with a fixed activity (3.7 GBq) of 90Y-biotin, in order to provide a boost of 20 Gy, followed by EBRT to the whole breast (WB) at the reduced dose of 40 Gy. Local and systemic toxicity, patient's quality of life, including the cosmetic results after the combined treatment with IART® and EBRT, were assessed. Methods: After tumour excision, the surgeon injected native avidin diluted in 30 ml of saline solution into and around the tumour bed (see video). Patients received one of three avidin dose levels: 50 mg (10 pts), 100 mg (15 pts) and 150 mg (10 pts). Between 12 to 24 h after surgery, 3.7 GBq 90Y-biotin spiked with 185 MBq 111In-biotin was administered intravenously (i.v.). Whole body scans and SPECT images were performed up to 30 h post-injection for dosimetric purposes. WB-EBRT was administered four weeks after the IART® boost. Local toxicity and quality of life were evaluated. Results: Thirty-five patients were evaluated. No side effects were observed after avidin administration and 90Y-biotin infusion. An avidin dose level of 100 mg resulted the most appropriate in order to deliver the required radiation dose (19.5 ± 4.0 Gy) to the surgical bed. At the end of IART®, no local toxicity occurred and the overall cosmetic result was good. The tolerance to the reduced EBRT was also good. The highest grade of transient local toxicity was G3, which occurred in 3/32 pts following the completion of WB-EBRT. The combination of IART®+EBRT was well accepted by the patients, without any changes to their quality of life. Conclusions: These preliminary results support the hypothesis that IART® may represent a valid approach to accelerated WB irradiation after BCS. We hope that this nuclear medicine technique will contribute to a better management of breast cancer patients. [ABSTRACT FROM AUTHOR]

The diagnosis of recurrent ovarian carcinoma is usually determined at surgical re-exploration since the main non-invasive diagnostic tests have low accuracy. It would be desirable to have a high accuracy non-invasive diagnostic procedure. With this aim, we have assessed the utility of three-step immunoscintigraphy. Thirty patients were intravenously injected with biotinylated monoclonal antibodies MOv18 and B72.3, followed by avidin-streptavidin injection and finally by 111In-biotin. Tumour recurrences were imaged 2 h post radioactivity injection. All patients underwent surgical re-exploration 3-4 days after immunoscintigraphy; the presence of tumour in the area of immunoscintigraphic uptake was evaluated in the biopsied material. Twenty-one patients studied were true-positive, five were true-negative, four were false-positive and none was false-negative. The diagnostic accuracy, positive predictive value and negative predictive value were 87%, 84% and 100% respectively. If these findings are confirmed in a larger number of patients, we expect immunoscintigraphy to be introduced as a cost-effective procedure in the follow-up of patients who have received surgery for ovarian carcinoma, since it promises to reliably identify patients who do not require surgical re-exploration, and guide biopsies when they are indicated. [ABSTRACT FROM AUTHOR]