Direct 99mTc labeling of monoclonal antibodies: Radiolabeling and in vitro stability

Direct labeling involves ^99mTc binding to different donor groups on the protein, giving multiple binding sites of various affinities resulting in an in vivo instability. The stability has been considerably improved by activating the antibody using a controlled reduction reaction (using 2-aminoethanethiol). This reaction generates sulfhydryl groups, which are known to strongly bind ^99mTc. The direct ^99mTc antibody labeling method was explored using whole antibodies and fragments. Analytical methods were developed for routine evaluation of radiolabeling yield and in vitro stability.Stable direct antibody labeling with ^99mTc requires the generation of sulfhydryl groups, which show high affinity binding sites for ^99mTc. Such groups are obtained with 2-aminoethanethiol (AET), which induces the reduction of the intrachain or interchain disulfide bond, with no structural deterioration or any loss of immunobiological activity of the antibody. The development of fast, reliable analytical methods has made possible the qualitative and quantitative assessment of technetium species generated by the radiolabeling process. Labeling stability is determined by competition of the ^99mTc-antibody bond with three ligands, Chelex 100 (a metal chelate-type resin), free DTPA solution and 1% HSA solution.Very good ^99mTc-antibody stability is obtained with activated IgG (IgGa) and Fab′ fragment, which makes these substances possible candidates for immunoscintigraphy use.     

Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method

The in vitro labeling and stability of ^99mTc-labeled antibody Fab′ fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed ^99mTc-d-glucarate complex. No loss of radioactivity incorporation was observed for all the ^99mTc-labeled antibody fragments after 24 h incubation at 37 °C. The ^99mTc-labeled antibody fragments (IgG1, N = 2; IgG2a, N = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Furthermore, using the affinity chromatography technique, two of the ^99mTc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 °C. The bonding between ^99mTc and antibody fragments was elucidated by challenging with a diamide ditholate (N₂S₂) compound. The Fab′ with IgG2a isotype displayed tighter binding to ^99mTc in comparison to the Fab′ from IgG1 and IgG3 isotype in N₂S₂ challenge and incubation with human plasma. The in vivo biodistribution of five ^99mTc-labeled fragments were evaluated in normal mice. In conclusion, the direct labeling method allows stable ^99mTc labeling of antibody fragments from three of the major murine isotypes.     

Technetium-99m-labeled platelets: Comparison of labeling with a new lipid-soluble Sn(II)-mercaptopyridine-N-oxide and 99mTc-HMPAO

Platelets pretinned with a neutral Sn(II)-2-mercaptopyridme-N-oxide (SN-MPO) were labeled with ^99mTc and compared to those labeled with ^99mTc-HMPAO. The conditions of labeling platelets, e.g. concentrations of platelets and Sn(II)-MPO, ^99mTc in ACD-saline or ACD-plasma media, pH and incubation time, were optimized using canine platelets. Moderate labeling efficiency was obtained with 20 μg of tin(II) chloride and 30 min incubation with Sn-MPO and pertechnetate. The viability of labeled platelets was determined by platelet recovery and platelet survival times in Beagle dogs. The labeling efficiency with platelets from 43 mL of blood was 62.8 ± 7.6%. The platelet recovery was 35.7 ± 5.0% and exponential survival time was 34.6 ± 3.1 h compared to 43.3 ± 12.0% and 29.5 ± 3.3 h for ^99mTc-HMPAO-labeled platelets. These values were significantly (P < 0.01) lower than ¹¹¹In-labeled platelets. Biodistribution in dogs indicates lower retention in blood, spleen and liver after some initial ^99mTc excretion in urine. The platelet deposition with ^99mTc platelets (Sn-MPO method) on polyurethane angio-catheters was similar to ^99mTc-HMPAO-labeled platelets. This study indicates that the platelets could be successfully labeled with pertechnetate in a cost-effective manner for the evaluation of thromboembolic complications.     

Evaluation of the phytic acid effect on the labeling of blood elements with technetium-99m and on the survival of a strain of Escherichia coli treated with stannous fluoride

The labeling of red blood cells with technetium-99m(^99mTc) depends on a reducing agent and stannous ions, as chloride or fluoride, are widely utilized. This labeling may also be altered by drugs. Moreover, some authors have reported that the survival of Escherichia coli (E. coli) cultures decreases in presence of stannous ions. Phytic acid is present in the daily diet and we evaluated its influence on: (i) the labeling of blood elements with ^99mTc and (ii) on the survival of an E. coli strain treated with stannous fluoride. Heparinized whole blood was withdrawn from Wistar rats and it was incubated with stannous chloride and with ^99mTc, as sodium pertechnetate, centrifuged and plasma (P) and blood cells (BC) were isolated. Samples of P and BC were also precipitaded with trichloroacetic acid, centrifuged and soluble (SF) and insoluble fractions (IF) isolated. E. coli culture was treated with stannous fluoride in presence of phytic acid. As phytic acid altered the fixation of ^99mTc on BC, on IF-P and on IF-BC and, moreover, it abolished the lethal effect of stannous fluoride on the E. coli culture, we can suggest that, probably, phytic acid would have chelating properties to the stannous ions.     

Technetium-99m labeled monoclonal antibodies: Evaluation of reducing agents

We have evaluated five compounds, stannous chloride (SnCl₂), 2-mercaptoethanol (2-ME), dithiothreitol (DTT), dithioerythritol (DTE), and ascorbic acid (AA) to reduce monoclonal antibody MoAb (disulfide groups and compared their efficacy for labeling MoAbs with ^99mTc. The reduction of ^99mTc with dithionite at pH 11 was nearly quantitative. The use of AA, at a molar ratio of 3500:1, for three IgG and three IgM antibodies examined, gave a labeling efficiency greater than 95%. Hence no purification was needed. The immunospecificity of AA preparations determined by specific antigen assay was 84 ± 1% for an IgM and 82.6 ± 1.1% for an IgG, highest among all agents tested. The stability of the tracer was evaluated by challenging the product with such ^99mTc avid agents as cysteine, DTPA, and human serum albumin. By HPLC analysis, no ^99mTc was transchelated using chelating agent to protein molar ratios as high as 500:1. In two separate groups of five mice each, the liver uptake at 4 h post injection averaged 6.8 ± 2.9% per gram for ¹²⁵I-TNT-1 (IgG) and 6 ± 5.1% per gram for the same MoAb labeled with ^99mTc using AA. The AA technique promises to label antibodies with ^99mTc and perhaps with ¹⁸⁶Re, by a simple “kit” procedure.     

Recent developments in 99mTc and 123I-radiopharmaceuticals for SPECT imaging

Availability of ¹²³I of high radionuclidic purity has encouraged the development of ¹²³I-based radiopharmaceuticals for the assessment of myocardial fatty acid metabolism, myocardial neuronal activity, and for receptor and antibody imaging. Advances in the chemistry of technetium have resulted in the development of novel agents for myocardial and cerebral perfusion and renal function studies. Monoclonal antibodies labeled with ^99mTc show promise for imaging neoplastic lesions, myocardial infarcts, and thrombus localization. Recent developments in ¹²³I and ^99mTc agents for myocardial and brain imaging studies are discussed.     

Technetium-99m-labeled antibodies

It is essential in any method for radiolabeling antibody with^99mTc that the labeling procedure is rapid and reliable, producing a highly stable^99mTc-antibody complex with minimal effect on the immunoreactivity of the antibody. In the present study, analysis of the stability and homogeneity of radiolabeled (^99mTc and¹²⁵I) antibodies (HMFG1 and PR1A3) was carried out by fast protein liquid chromatography (FPLC) using superose-6 and S-200 columns, and by polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. Superose 6 and S-200 gel filtration analysis showed the radiolabel (^99mTc or¹²⁵I) eluting with a retention time identical to that of native antibody. No peaks of relative molecular size (Mr) corresponding to possible antibody fragments were seen in either the UV or the radioactive FPLC elution profiles. PAGE analysis of^99mTc labeled antibody, however, revealed the presence of a number of radiolabeled antibody fragments (Mr<IgG) that were not detected by the same analysis of¹²⁵I labeled antibody. The stability of the radiolabeled antibodies in serum in vitro was also studied. FPLC (superose-6) analysis after 45 h incubation in normal serum in vitro revealed 3.3% (HMFG1), and 20% (PR1A3) of the^99mTc on a molecule or aggregate with a Mr greater than that of IgG. There is also the appearance of small amounts of^99mTc-labeled material with a Mr<IgG in the later fractions (2.2% for HMFG1 and 4.9% for PR1A3). Similar results were obtained using radioiodinated antibody, although the small amount of low molecular size material detected as a single peak with a longer retention time than the^99mTc equivalent corresponds to free iodide.     

Technetium-99m labeled 50H.19 antibody fragments: Interaction of the antibody with platelets

The monoclonal antibody 50H.19 recognized three antigens (M_r = 31-, 40-, 45-K) on normal and thromboasthenic platelets, but only one (M_r = 31-K) on Bernard-Soulier platelets. The intact antibody and its F(ab′)₂ fragments, had direct platelet-aggregating activity, and induced the platelet release reaction. The intact antibody potentiated platelet aggregation induced by platelet-activating factor or thrombin. Additions of indomethacin did not inhibit aggregation: addition of PGI₂, or a calcium channel blocker completely inhibited aggregation. A reduced amount of platelet-aggregating activity was observed with antibody fragments prepared for labeling with ^99mTc by pre-exposure to stannous ions, and herein used in biodistribution studies and elsewhere in thrombus imagining studies (J. Nucl. Med. 27: 1315; 1986). Antibody fragments radiolabeled with ^99mTc bound to isolated platelets and to clots containing platelets.     

Preparation,radiolabeling and biodistribution of a new class of bisaminothiol (BAT) ligands as possible imaging agents

In developing new ligands as potential brain and heart perfusion imaging agents two ligands based upon N₂S₂ donor atoms with the biphenyl backbone were synthesized. Biphenyl-2,2′-bis(N-1-amino-2-methyl-propane-2-thiol) (BP-BAT-TM) and biphenyl-2,2′-bis(N-1-amino-2-ethyl-butane-2-thiol) (BP-BAT-TE) form stable, neutral and lipid soluble complexes with [^99mTc]pertechnetate in the presence of tin(II) tartarate as a reducing agent. The [^99mTc]BP-BAT-TM complex penetrates the blood-brain barrier following i.v. injection into rats. Washout from the brain is fast, indicating no retention. The biodistribution of [^99mTc]BP-BAT-TE in rats showed an intitial heart uptake (0.8% /organ, at 2 min) and a slow washout (0.74% at 15 min). No brain uptake was found (0.05%). Significant uptake and retention in liver was observed. An imaging study of [^99mTc]BP-BAT-TE in a monkey showed no brain uptake and a clear indication of liver uptake and gall bladder clearance. These results indicate that this ligand system may be suitable as the basic core structure for the development of new imaging agents. Further studies with structural variations in the biphenyl backbone are warranted to develop new ^99mTc imaging agents for clinical applications.     

Use of Technetium (99mTc) as a Bacterial Label in Lung Clearance Studies

The suitability of technetium (^99mTc), a gamma emitter, for labeling of Diplococcus pneumoniae in studies of lung bacterial clearance was examined. A killed bacterial slurry with high specific activity was obtained with a ferric ascorbate reducing system. Approximately 5.5% of radioactive counts dissociated from labeled bacteria in 6 h. Rats were exposed to a uniformly mixed aerosol of untagged, viable pneumococci and killed, ^99mTc-tagged pneumococci. The aerodynamic behavior of labeled and unlabeled pneumococci was similar. Viable bacterial counts and radioactive counts were determined in lung homogenates at intervals following exposure, and rates of bacterial killing and disappearance of radioactive counts were plotted. Radioactive counts did not increase in the liver during the period of observation, suggesting that the decrease in lung radioactivity represents mucociliary clearance and not release of isotope to the systemic circulation. The use of ^99mTc for bacterial labeling provides advantages of technical simplicity and personnel safety compared to the use of beta-emitting isotopes.     

A study of the concept of non-radioactive unit-dosed reagent kits [cold unit doses (CUDs)] as an efficient and cost-saving method for 99mTc radiopharmaceutical preparation

Traditionally, when preparing ^99mTc-labeled radiopharmaceuticals, [^99mTc]pertechnetate is added to the entire contents of a vial of reagent kit, and patient doses are subsequently withdrawn from the vial. This technique of compounding can be potentially wasteful for two reasons: (1) once reconstituted with ^99mTc, most reagent kits have a relatively short shelf-life, and thus the entire contents may not be used before expiration and (2) due to a need to conserve radioactivity in many hospitals, enough [^99mTc]pertechnetate is added to the reagent kit in order to retrieve only 1–2 patient doses, even though adequate chemicals (ligand, reducing agent, etc.) are present in the reagent kit to supply as many as 5–10 doses. Hence, a method for optimizing the efficient use of reagent kits would be desirable. The purpose of this study was to determine the feasibility of unit-dosing non-radioactive reagent kits and storing these cold unit doses (CUDs) for eventual labeling with ^99mTc. To evaluate this concept, unit doses were prepared from reagent kits of medronate (MDP) and pentetate (DTPA). The specific variables studied in this research were the effects of storage time, storage temperature and reconstitution volume (dilution) on the unit doses. These effects were monitored by measuring the radiochemical and biodistribution properties of the unit doses following their final reconstitution with [^99mTc]pertechnetate. The labeling efficiency was determined using instant thin layer chromatography (ITLC), and the biodistribution patterns of these radiolabeled CUDs were studied in mice. The results showed that MDP- and DTPA-CUDs stored at −18 °C retained the properties which resulted in acceptable radiochemical purity and biodistribution in mice for as long as 30 days. On the other hand, the radiochemical purity of MDP and DTPA unit doses stored at 25 °C deteriorated rapidly. Mean radiochemical purities as low as 0.58–19.4% were observed on day 30. Altered biodistributions were observed in a manner consistent with the decreased labeling efficiencies. The CUDs of lower dilution (3 mL) appeared to be more stable than the CUDs of higher dilution (10 mL). However, the effect of reconstitution volume was much less significant than the temperature effect on the CUDs. In conclusion, the concept of unit-dosing non-radioactive reagent kits appears to provide an efficient and cost-saving method for preparing infrequent and emergency radiopharmaceutical doses. The study also showed that the storage temperature of these unit doses is critical to the success of the procedure. The volume of reconstitution has a minimal impact on the stability of CUDs if stored at the appropriate temperature.     

Development,optimization, and biovalidation of <Superscript>99m</Superscript>Tc–insulin complex

Human biosynthetic insulin is a polypeptide hormone that plays an important and essential role in control of the level of carbohydrate, protein, and fat metabolism in the blood. Human pancreatic insulin was labeled with ^99mTc to form a new radiopharmaceutical with a labeling yield of 99 ± 1% under optimum conditions: 0.1 mL insulin, pH 7, 25 μg stannous chloride, 1 mL (19 mCi) of pertechnetate, room temperature, and 10 min reaction time. The ^99mTc–insulin complex was examined using paper chromatography, ITLC, electrophoresis, and HPLC. In addition, in vitro and in vivo study of ^99mTc–insulin complex was performed at different time intervals.     

Similarity of copper and technetium binding sites in human IgG

Kits for direct labeling of IgG with ^99mTc were used without modification for the preparation of [⁶⁷Cu]IgG. The IgG was pre-treated to generate thiolate groups which would bind ⁶⁷Cu. The direct labeling of reduced IgG with ⁶⁷Cu was highly efficient, resulting in approx. 95% ⁶⁷Cu binding. Non-reduced IgG (negative control) had labeling efficiencies of less than 10%. IgG pre-exposed to Cu(II) had reduced amounts of ^99mTc bound to it. The results demonstrate a direct relationship between copper- and ^99mTc-binding sites in IgG.     

Synthesis and biological evaluation of Tc-99m-cyclopentadienyltricarbonyl-technetium-labeled A-85380: An imaging probe for single-photon emission computed tomography investigation of nicotinic acetylcholine receptors in the brain

We have designed (S)-(5-(azetidin-2-ylmethoxy)pyridine-3-yl)methyl cyclopentadienyltricarbonyl technetium carboxylate ([^99mTc]CPTT–A–E) with high affinity for nicotinic acetylcholine receptors (nAChRs) using (2(S)-azetidinylmethoxy)-pyridine (A-85380) as the lead compound to develop a Tc-99m-cyclopentadienyltricarbonyl-technetium (^99mTc)-labeled nAChR imaging probe. Because technetium does not contain a stable isotope, cyclopentadienyltricarbonyl rhenium (CPTR) was synthesized by coordinating rhenium, which is a homologous element having the same coordination structure as technetium. Further, the binding affinity to nAChR was evaluated. CPTR–A–E exhibited a high binding affinity to nAChR (K_i = 0.55 nM). Through the radiosynthesis of [^99mTc]CPTT–A–E, an objective compound could be obtained with a radiochemical yield of 33% and a radiochemical purity of greater than 97%. In vitro autoradiographic study of the brain exhibited that the local nAChR density strongly correlated with the amount of [^99mTc]CPTT–A–E that was accumulated in each region of interest. Further, the in vivo evaluation of biodistribution revealed a higher accumulation of [^99mTc]CPTT–A–E in the thalamus (characterized by the high nAChR density) when compared with that in the cerebellum (characterized by the low nAChR density). Although additional studies will be necessary to improve the uptake of [^99mTc]CPTT–A–E to the brain, [^99mTc]CPTT–A–E met the basic requirements for nAChR imaging.     

Use of liposomes as carriers for radiation synovectomy

Using [^99mTc]pertechnetate as an aqueous space marker, the permeability of liposomes composed of seven different mixtures of distearoylphosphatidylcholine (DSPC) and sphingomyelin (SM) was determined. Liposomes containing 20–33% SM were the least permeable in the presence of rheumatoid synovial fluid. Following injection of ^99mTc-containing liposomes into the knee joints of rabbits, retention of ^99mTc in the knee was more than 200 times greater than following injection of nonencapsulated [^99mTc]pertechnetate. The knee clearance biologic half time of ^99mTe with DSPC/SM (4:1) liposomes was 64 h. Most of the activity that had leaked from the knee was not found in extra-articular tissues, suggesting rapid excretion. When DSPC/SM (4:1) liposomes were labeled with ¹¹¹In(oxine), a knee clearance biologic half time of greater than 1200 h was observed.     

The chemistry of rhenium and technetium as related to the use of isotopes of these elements in therapeutic and diagnostic nuclear medicine

The β emitting isotopes ¹⁸⁶Re and ¹⁸⁸Re are logical choices on which to base therapeutic radiopharmaceuticals that might be expected to be analogous to diagnostic radiopharmaceuticals based on ^99mTc. However, the chemistry of rhenium is sufficiently different from that of technetium so that the development of Re radiopharmaceuticals often cannot be predicated on the known chemistry and biological behavior of ^99mTc radiopharmaceuticals. The relevant chemical differences involve the greater stability of the higher oxidation states of Re (and thus the greater tendency of reduced Re radiopharmaceuticals to undergo re-oxidation to perrhenate), and the greater substitution inertness of reduced Re complexes. These differences are illustrated (1) in the preparation and use of ¹⁸⁶Re (Sn)-HEDP and ^99mTc(Sn)-HEDP diphosphonate radiopharmaceuticals designed, respectively, for palliative therapy and diagnosis of metastatic cancer to bone, and (2) in the preparation and biodistribution of tr-[¹⁸⁶Re(DMPE)₂Cl₂]⁺ and [¹⁸⁶Re(DMPE)₃]⁺, analogs to the potential myocardial perfusion imaging agents tr-[^99mTc(DMPE)₂Cl₂]⁺ and [^99mTc(DMPE)₃]⁺. [HEDP = (1-hydroxyethylidene)diphosphonate; DMPE = 1,2-bis(dimethylphosphino)ethane].     

99mTc(Cu)—mannitol complex: a new agent for dynamic renal function studies

Mannitol has been labelled with ^99mTc by using cuprous chloride as a reducing agent. Blood and kidney clearance of ^99mTc(Cu)-mannitol was slightly faster than that of ^99mTc(Sn)-DTPA in rat and maximum radioactivity ratio of kidneys to blood was 84.6 at 5 min. A comparative study of ^99mTc(Cu)-mannitol, ^99mTc(Sn)-DTPA was made in rabbits by taking serial images of kidneys and bladder with a γ camera. Results show superiority of ^99mTc(Cu)-mannitol over other agents for dynamic renal function studies.     

Transchelation of 99mTc from low affinity sites to high affinity sites of antibody

An exclusive labeling of high affinity sites of IgG and its F(ab′)₂ fragments with ^99mTc was accomplished. Antibody was first labeled in 0.1 M acetate buffer at pH 4.5, using stannous chloride as a reducing agent. Thus, high capacity, low affinity sites and low capacity, high affinity sites were both labeled. These ^99mTc complexes were stable at pH 4.5 and 7.0; however, they became destabilized at pH 8.2 and 9.0. Transchelation of ^99mTc to DTPA took place at the higher pH values and leveled off at 54% ^99mTc-F(ab′)₂ and 73% ^99mTc-IgG. These results indicate that the majority of ^99mTc bound to the low affinity sites was transchelated to the high affinity sites rather than to DTPA since low affinity sites account for 84% of total F(ab′)₂ sites and 76% of IgG sites. Biodistribution data in mice at 2.5 h postinjection were consistent with this hypothesis in that tissue concentrations of ¹¹¹In-DTPA-F(ab′)₂ were similar to the reequilibrated ^99mTc-F(ab′)₂ but were much higher than that of the unequilibrated ^99mTc-F(ab′)₂.     

Water-soluble phosphines for direct labeling of peptides with technetium and rhenium: insights from electrospray mass spectrometry

Direct labeling of salmon calcitonin (sCT) is possible in one step using water-soluble phosphines (sulfonated triphenylphosphines) as the reducing agent both for disulfide and for pertechnetate. Phosphines were the most efficient reducing agent for disulfide bonds among those examined. The phosphines both reduced the pertechnetate to Tc(III), and contributed to the technetium coordination sphere in the labeled product. In contrast, the phosphines did not reduce rhenium below oxidation state V, nor did they participate in the rhenium coordination sphere in the labeled peptide. Instead, the expected oxorhenium(V) moiety was incorporated. Both Tc and Re labeling processes gave rise to dimers with two peptides linked by the metal center, as well as simple monomeric species. Positive mode electrospray mass spectrometry not only revealed the presence of phosphine bound to technetium and oxygen bound to rhenium in the metallopeptides but also revealed the oxidation states of the metals. Electrospray mass spectrometry is proving to be an exceptionally valuable technique for characterizing radiopharmaceuticals. Although the one-step direct labeling method described gives mixed products and poor receptor affinity when applied to the small peptide sCT, it might be readily adapted to monoclonal antibodies.     

A 99mTc generator based on the adsorption of [99Mo]molybdophosphate on hydrous manganese dioxide

The adsorption behaviour on hydrous manganese dioxide of ⁹⁹Mo in the form of molybdate and molybdophosphate and of ^99mTc in the form of pertechnetate was investigated at different solution acidities. The adsorption capacity of molybdenum is much higher for the molybdophosphate form. A ^99mTc-generator is suggested. This generator is based on the adsorption of [⁹⁹Mo]molybdophosphate on MnO₂, at an acidity of 0.5 N. ^99mTc is then eluted either by 0.9% NaCl or by methyl ethyl ketone. Radionuclidic, radiochemical and chemical purity tests of the eluates were performed and the present generator seems to offer some advantages over the traditional alumina generator.