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Competitive binding test for 99m Tc-trastuzumab with BT-474 cells by adding excess nonlabeled trastuzumab (B 0 : radioactivity of 99m Tc on cells without adding nonlabeled trastuzumab; B: radioactivity of 99m Tc on cells with adding nonlabeled trastuzumab).
Source publication
By simply incubating Herceptin (trastuzumab) with [99m Tc(CO)3(OH2)3]+ ion in saline, a significant yield of 99m Tc-labeled trastuzumab was found to be achievable. The effective labeling may be based on that trastuzumab is inherent with endogenous histidine group to which 99m Tc(I) tricarbonyl ion can be strongly bound. For practical 99m Tc labelin...
Context in source publication
Context 1
... cells might be attributed to be nonspecific. The specificity of 99m Tc-trastuzumab to HER-2-overex- pressing cells was clearly confirmed. In a further compe- titive binding test, the binding of 99m Tc-trastuzumab for cultured BT-474 cells would gradually decrease, while nonlabeled trastuzumab for receptor blocking was increasingly added (see Fig. 5). This validated the receptor-mediated binding of 99m Tc(I)-trastuzumab to HER-2-overexpressing cell lines. ...
Citations
... A widely used and versatile method for labeling recombinant proteins with 99m Tc exploits the kinetically inert binding of the [ 99m Tc][Tc(CO) 3 ] + fragment to proteins incorporating a His-tag-a polyhistidine sequence consisting typically of six consecutive His residues often engineered at the N-or C-terminus to facilitate downstream purification by immobilized metal-affinity chromatography. 3 This elegant radiolabeling method, developed by Waibel et al., 4,5 has been used to radiolabel many His-tagged proteins and is also applicable to the βemitting therapeutic congeners, 186 Re and 188 Re. 6,7 Despite its widespread use in molecular imaging research (e.g., labeled Herceptin for breast cancer imaging and Annexin V for apoptosis imaging 8,9 ), use of this method in humans remains rare, and no commercial products are available. This may be in part because its practical utility varies between proteins and is unpredictable, giving quantitative (i.e., >95%) radiochemical yield only rarely. ...
Hexahistidine tags (His-tags), incorporated into recombinant proteins to facilitate purification using metal-affinity chromatography, are useful binding sites for radiolabeling with [99mTc(CO)3]+ and [188Re(CO)3]+ for molecular imaging and radionuclide therapy. Labeling efficiencies vary unpredictably, and the method is therefore not universally useful. To overcome this, we have made quantitative comparisons of radiolabeling of a bespoke Celluspots array library of 382 His-tag-containing peptide sequences with [99mTc(CO)3]+ and [188Re(CO)3]+ to identify key features that enhance labeling. A selected sequence with 10-fold enhanced labeling efficiency compared to the most effective literature-reported sequences was incorporated into an exemplar protein and compared biologically with non-optimized analogues, in vitro and in vivo. Optimal labeling with either [99mTc(CO)3]+ or [188Re(CO)3]+ required six consecutive His residues in the protein sequence, surrounded by several positively charged residues (Arg or Lys), and the presence of phosphate in the buffer. Cys or Met residues in the sequence were beneficial, to a lesser extent. Negatively charged residues were deleterious to labeling. His-tags with adjacent positively charged residues could be labeled as much as 40 times more efficiently than those with adjacent negatively charged residues. 31P NMR of [Re(CO)3(H2O)3]+ and electrophoresis of solutions of [99mTc(CO)3(H2O)3]+ suggest that phosphate bridges form between cationic residues and the cationic metal synthon during labeling. The trial optimized protein, a scFv targeted to the PSMA antigen expressed in prostate cancer, was readily labeled in >95% radiochemical yield, without the need for subsequent purification. Labeling occurred more quickly and to higher specific activity than comparable non-optimized proteins, while retaining specific binding to PSMA and prostate cancer in vivo. Thus, optimized His-tags greatly simplify radiolabeling of recombinant proteins making them potentially more widely and economically available for imaging and treating patients.
... Waibel et al. [25] directly labeled scFv antibody fragments that contained His-Tag residues resulting in excellent yields using the 99m Tc(CO) 3 þ precursor. Similarly, unmodified trastuzumab was directly radiolabeled with 99m Tc(CO) 3 þ via histidine residues [26]. His-Tag/histidine forms a very stable complex with the 99m Tc(CO) 3 þ core. ...
... Ready to use single vial kits are available commercially for preparing the 99m Tc carbonyl core and the complexes formed are stable over a wide range of pH. In recent years, a number of researchers have successfully demonstrated the feasibility of direct labeling of antibodies using the 99m Tc carbonyl core (Dias et al., 2011;Chen et al., 2008). Maximum success in the arena of radiolabeled antibodies has been achieved in the therapy of Non-Hodgkin 0 s Lymphoma (NHL) for which two radioimmunotherapy agents have been approved by the FDA viz. ...
... The 99m Tc carbonyl ion is a versatile synthon for the 99m Tc labeling of biomolecules. Herein, 99m Tc carbonyl labeling of DTPA conjugated Rituximab was carried out and the percentage radiolabeling yield obtained is comparable to the results obtained by Dias et al. wherein a radiolabeling yield of $98% was obtained by 99m Tc carbonyl labeling of reduced Rituximab (Dias et al., 2011;Chen et al., 2008). Attempts have been made by researchers to radiolabel antibodies directly with 99m Tc carbonyl synthon by coordination with the histidine side chain of the antibody. ...
... Attempts have been made by researchers to radiolabel antibodies directly with 99m Tc carbonyl synthon by coordination with the histidine side chain of the antibody. Direct 99m Tc carbonyl labeling of the inherent histidine groups on the antibody have shown poor radiolabeling yields (Dias et al., 2011;Chen et al., 2008;Misri et al., 2011) probably due to labeling in a non-specific manner. However, purification of the antibody for removal of histidine had resulted in higher yields (Chen et al., 2008) indicating that the labeling yield was dependent on the position/site of the histidine side chains (Waibel et al., 1999). ...
... In this kit (IsoLink 1 , Mallinckrodt), boranocarbonate sodium allows both the production of carbon monoxide and the reduction of technetium. Different antibody radiolabeling strategies using 99m Tc-tricarbonyl have been assessed, with direct antibody labeling [7,10] or using a spacer [11]. Investigations by Egli et al. showed that histidine was the most potent ligand among other amino acids and led to direct radiolabeling of peptides or proteins [12]. ...
... This 99m Tc-tricarbonyl core is an excellent precursor to radiolabel various biomolecules, especially immunoglobulins. Chen et al. [10] radiolabeled native trastuzumab directly with yields of 90%, using commercially available trastuzumab (10 mg). To achieve high synthesis yields with smaller antibody amounts, IgG can be modified before radiolabeling. ...
In recent years, the diagnostic and therapeutic uses of radioisotopes have shown significant progress. Immunoglobulin (Ig) appears to be a promising tracer, particularly due to its ability to target selected antigens. The main objective of this study is to optimize and assess an Ig radiolabeling method with Technetium 99m (99mTc), an attractive radioelement used widely for diagnostic imaging. Monoclonal anti-CD20 IgG was retained to study in vitro and in vivo radiolabeling impact. After IgG derivatization with 2-iminothiolane, IgG-SH was radiolabeled by an indirect method, using a 99mTc-tricarbonyl core. Radiolabeling stability was evaluated over 24h by thin-layer chromatography. IgG integrity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis coupled with Western blot and autoradiography. The radiolabeled Ig's immunoaffinity was assessed in vitro by a radioimmunoassay method and binding experiments with cells (EL4-hCD20 and EL4-WT). Biodistribution studies were performed in normal BALB/c mice. Tumor uptake was assessed in mice bearing EL4-hCD20 and EL4-WT subcutaneous xenografts. With optimized method, high radiolabeling yields were obtained (95.9 ± 3.5%). 99mTc-IgG-SH was stable in phosphate-buffered saline (4°C and 25°C) and in serum (37°C), even if important sensitivity to transchelation was observed. IgG was not degraded by derivatization and radiolabeling, as shown by Western blot and autoradiography results. 99mTc-anti-CD20 IgG-SH immunoaffinity was estimated with Kd = 35 nM by both methods. In vivo biodistribution studies for 48h showed significant accumulation of radioactivity in plasma, liver, spleen, lungs and kidneys. Planar scintigraphy of mice bearing tumors showed a significant uptake of 99mTc-anti-CD20 IgG-SH in CD20+ tumor versus CD20- tumor. Radiolabeling of derivatized IgG with 99mTc-tricarbonyl was effective, stable and required few antibody amounts. This attractive radiolabeling method is "antibody safe" and preserves Ig affinity for antigen, as shown by both in vitro and in vivo experiments. This method could easily be used with noncommercial IgG or other antibody isotypes.
... and/or 99m TcO 4 suspended in the supernatant. The radiochemical yield of the 99m Tc(I)-HSA-NPs was calculated from the obtained radioactivities, as below Eq. 2 [20,21]: Radio-labeled yield ð%Þ ¼ radioactivity in precipitate total radioactivity ...
The purpose of this study was to develop a convenient and robust method for preparation of 99mTc(I)-labeled human serum albumin nanoparticles (HSA-NPs) as a SPECT agent for tumor imaging. Efficient radiolabeling 99mTc to HSA-NPs was achieved by simply reacting [99mTc(OH2)3(CO)3]+ with the reconstituted HSA-NPs solution. The size and morphology of 99mTc(I)-HSA-NPs were analyzed and found not to significantly change after the preformed HSA-NPs were lyophilized or labeled with 99mTc(I). By incubating 99mTc(I)-HSA-NPs in rat plasma at 37 °C for 30 h, 75 % of the radioactivity were found to be remained in the particles, suggesting good in vitro stability.
... Targeting NBs through surface modifications is considered a promising theranostic approach. In preclinical medical imaging studies, Herceptin and its analogs have been shown to be effective targeting tools [15][16][17][18][19]. Thus, breast cancers that overexpress Her-2 are an attractive target for continued development of specific theranostic approaches. ...
There is an unmet need for specific and sensitive imaging techniques to assess the efficacy of breast cancer therapy, particularly Her-2-expressing cancers. Ultrasonic microbubbles are being developed for use as diagnostic and therapeutic tools. However, nanobubbles circulate longer, are smaller, and diffuse into extravascular tissue to specifically bind target molecules. Here, we characterize a novel Herceptin-conjugated nanobubble for use against Her-2-expressing tumors.
Phospholipid-shelled nanobubbles conjugated with Herceptin (NBs-Her) were fabricated using a thin-film hydration method and characterized in vitro in breast cancer cell lines and in vivo in a mouse model.
The average size of the unconjugated nanobubbles (NBs-Blank) and NBs-Her was 447.1 ± 18.4 and 613.0 ± 25.4 nm, respectively. In cell culture, the NBs-Her adhered to Her-2-positive cells significantly better than to Her-2-negative cells (p < 0.05). In vivo, the peak intensity and the half-time to washout of the NBs-Her were significantly greater than those of the NBs-Blank (p < 0.05). In addition, contrast-enhanced ultrasound imaging quality was improved through the use of the NBs-Her. The nanobubbles were able to penetrate into tumor tissue to allow extravascular imaging, but did not penetrate normal skeletal muscle.
The Herceptin-conjugated nanobubble had many properties that made it useful for in vivo imaging, including longer circulation time and better tumor selectivity. This platform may be able to provide targeted delivery of therapeutic drugs or genes.
... The low spin d 6 configuration of Tc(I) in the 99m Tc carbonyl synthon imparts high stability to it over a wide pH range. Reports indicate that a number of antibodies have been directly radiolabeled with the 99m Tc carbonyl synthon [23,24]. ...
Objective:
The objective of this study was to explore the potential of (99m)Tc carbonyl labeled DTPA-bevacizumab as a tumor imaging agent. Bevacizumab (Avastin) is a humanized monoclonal antibody (MoAb) that inhibits the vascular endothelial growth factor (VEGF).
Methods:
Bevacizumab was conjugated with paraisothiocyanatobenzyl diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA) and subsequently radiolabeled with (99m)Tc via the (99m)Tc carbonyl synthon. The radioconjugate after purification was characterized by SE-HPLC and its in vitro stability was determined by histidine challenge experiments. Biodistribution studies to determine the uptake by tumors were carried out in melanoma model.
Results:
The radiochemical purity of (99m)Tc carbonyl labeled antibody was >98 %. The radiolabeled antibody exhibited good stability in the histidine challenge experiments up to 24 h when stored at 37 °C. Biodistribution studies in mice bearing melanoma showed significant tumor uptake (6.9 ± 2.2 % ID/g at 24 h p.i.) which was reduced to 1.6 ± 0.4 % ID/g on co-injection with cold Bevacizumab.
Conclusions:
The (99m)Tc carbonyl-DTPA-bevacizumab conjugate with good radiochemical purity, excellent stability and good specificity for VEGF indicates its potential as a radioimmunoscintigraphy agent for various cancers.
... Histidine has been demonstrated to be the favoured [ 99m Tc(CO) 3 ] + -binding ligand among amino acids and labelling efficiency and stability increase with increased number of engineered histidines [2]. Direct labelling of non-His-tag proteins with [ 99m Tc(CO) 3 ] + has previously resulted in poor stability and low labelling efficiency and specific activity, indicating that other potential amino acid side chains donor groups such as thiol, thioether, carboxylate and amine do not make a significant contribution in the absence of histidines [3][4][5][6][7][8][9]. Modification of the His-tag sequence from HHHHHH to HEHEHE in order to improve tracer biodistribution also resulted in reduced labelling efficiency [10,11]. ...
... Our group has recently shown that the complex specifically binds to the His-tag, demonstrated by the fact that rhenium tricarbonyl labelled His-tag protein post-tryptic digest is only bound to peptide fragments containing a His-tag [12]. Previous reports have indicated that non-His-tag proteins can be radiolabelled with [ 99m Tc(CO) 3 ] + [4] albeit with relatively low efficiency, but little or no challenge or stability data was reported. By using a competitive binding assay, here we demonstrated that the technetium tricarbonyl indeed binds selectively to a His-tagged protein in the presence of a non-His-tagged protein and little or no (<2% ± 0.9%) non-specific labelling was observed. ...
To date, the majority of protein-based radiopharmaceuticals have been radiolabelled using non-site-specific conjugation methods, with little or no control to ensure retained protein function post-labelling. The incorporation of a hexahistidine sequence (His-tag) in a recombinant protein can be used to site-specifically radiolabel with 99mTc-tricarbonyl ([99mTc(CO)3]+). This chemistry has been made accessible via a technetium tricarbonyl kit; however, reports of radiolabelling efficiencies and specific activities have varied greatly from one protein to another. Here, we aim to optimise the technetium tricarbonyl radiolabelling method to produce consistently >95% radiolabelling efficiencies with high specific activities suitable for in vivo imaging.
Four different recombinant His-tagged proteins (recombinant complement receptor 2 (rCR2) and three single chain antibodies, alpha-CD33 scFv, alpha-VCAM-1 scFv and alpha-PSMA scFv), were used to study the effect of kit volume, ionic strength, pH and temperature on radiolabelling of four proteins.
We used 260 and 350 muL [99mTc(CO)3]+ kits enabling us to radiolabel at higher [99mTc(CO)3]+ and protein concentrations in a smaller volume and thus increase the rate at which maximum labelling efficiency and specific activity were reached. We also demonstrated that increasing the ionic strength of the reaction medium by increasing [Na+] from 0.25 to 0.63 M significantly increases the rate at which all four proteins reach a >95% labelling efficiency by at least fourfold, as compared to the conventional IsoLink(R) kit (Covidien, Petten, The Netherlands) and 0.25 M [Na+].
We have found optimised kit and protein radiolabelling conditions suitable for the reproducible, fast, efficient radiolabelling of proteins without the need for post-labelling purification.
... Ready to use single vial kits are available commercially for preparing the 99m Tc carbonyl core and the complexes formed are stable over a wide range of pH. In recent years, a number of researchers have successfully demonstrated the feasibility of direct labeling of antibodies using the 99m Tc carbonyl core (Dias et al., 2011;Chen et al., 2008). Maximum success in the arena of radiolabeled antibodies has been achieved in the therapy of Non-Hodgkin 0 s Lymphoma (NHL) for which two radioimmunotherapy agents have been approved by the FDA viz. ...
... The 99m Tc carbonyl ion is a versatile synthon for the 99m Tc labeling of biomolecules. Herein, 99m Tc carbonyl labeling of DTPA conjugated Rituximab was carried out and the percentage radiolabeling yield obtained is comparable to the results obtained by Dias et al. wherein a radiolabeling yield of $98% was obtained by 99m Tc carbonyl labeling of reduced Rituximab (Dias et al., 2011;Chen et al., 2008). Attempts have been made by researchers to radiolabel antibodies directly with 99m Tc carbonyl synthon by coordination with the histidine side chain of the antibody. ...
... Attempts have been made by researchers to radiolabel antibodies directly with 99m Tc carbonyl synthon by coordination with the histidine side chain of the antibody. Direct 99m Tc carbonyl labeling of the inherent histidine groups on the antibody have shown poor radiolabeling yields (Dias et al., 2011;Chen et al., 2008;Misri et al., 2011) probably due to labeling in a non-specific manner. However, purification of the antibody for removal of histidine had resulted in higher yields (Chen et al., 2008) indicating that the labeling yield was dependent on the position/site of the histidine side chains (Waibel et al., 1999). ...
... Technetium-99m is an ideal radionuclide for diagnostic organ imaging due to its optimum γ-energy (140 keV), short half-life (6 hr), low cost, and wide availability. After the introduction of a 99m Tc-tricarbonyl precursor with a low oxidation state (I) , many approaches have been attempted to label a 99m Tc-tricarbonyl precursor to a biomolecule, from glucose to an antibody (Schibli et al., 2000;Alberto et al., 1996;Chen et al., 2008;Taylor et al., 2010). ...
99mTc tricarbonyl glycine monomers, trimers, and pentamers were synthesized and evaluated for their radiolabeling and in vivo distribution characteristics. We synthesized a 99mTc-tricarbonyl precursor with a low oxidation state (I). 99mTc(CO)3(H2O)3
+ was then made to react with monomeric and oligomeric glycine for the development of bifunctional chelating sequences for biomolecules. Labeling yields of 99mTc-tricarbonyl glycine monomers and oligomers were checked by high-performance liquid chromatography. The labeling yields of 99mTc-tricarbonyl glycine and glycine oligomers were more than 95%. We evaluated the characteristics of 99mTc-tricarbonyl glycine oligomers by carrying out a lipophilicity test and an imaging study. The octanol-water partition coefficient of 99mTc tricarbonyl glycine oligomers indicated hydrophilic properties. Single-photon emission computed tomography imaging of 99mTc-tricarbonyl glycine oligomers showed rapid renal excretion through the kidneys with a low uptake in the liver, especially of 99mTc tricarbonyl triglycine. Furthermore, we verified that the addition of triglycine to prototype biomolecules (AGRGDS and RRPYIL) results in the improvement of radiolabeling yield. From these results, we conclude that triglycine has good characteristics for use as a bifunctional chelating sequence for a 99mTc-tricarbonyl- based biomolecular imaging probe.
