Iodination of proteins,glycoproteins, and peptides using a solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen)

A new, commercially available oxidizing agent, 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) was compared with chloramine-T and solid-phase lactoperoxidase in the radioiodination of proteins, glycoproteins, and peptides. A method for performing low-level iodinations is described and was used to determine maximum ¹²⁵I incorporation. Iodinated proteins were purified on analytical gel filtration columns and peptides by reverse-phase high-performance liquid chromatography. Both methods were designed to analyze the tracers for the presence of aggregate and breakdown products caused by the iodination. All tracers prepared were tested in antibody dilution and dose-response curves in their respective radioimmunoassays. Results indicate that Iodogen can be used for a wide range of proteins and peptides, can permit theoretical iodine incorporation with minimal oxidation damage, and can produce tracer stable for up to 3 months.     

Biodistribution of p-Iodobenzoyl (PIP) labeled antibodies in a murine lymphoma model

A monoclonal antibody against the murine T-cell antigen Thy 1.1 was radioiodinated using N-succin-imidyl p-iodobenzoate (PIP) in an attempt to decrease deiodination of the labeled antibody. The biodistribution of the PIP labeled antibody was compared to Iodogen labeled antibody in Thy 1.1⁺ lymphoma bearing AKR/Cum mice, where the antibody was tumor specific, and AKR/J mice where the antibody reacted with both tumor and normal T-cells. PIP labeling resulted in decreased iodine concentrations in stomach and salivary gland as compared to Iodogen labeling. There was little difference in radioiodine concentrations between the two preparations in tumor, lymphoid tissues or other organs. These results suggest deiodination of intact antibody plays little role in the clearance of radioiodinated anti-Thy 1.1 antibody from tissues.     

Advantage of residualizing radiolabels for an internalizing antibody against the B-cell lymphoma antigen, CD22

 LL2 is an anti-CD22 pan-B-cell monoclonal antibody which, when radiolabeled, has a high sensitivity for detecting B-cell, non-Hodgkin’s lymphoma (NHL), as well as an antitumor efficacy in therapeutic applications. The aim of this study was to determine whether intracellularly retained radiolabels have an advantage in the diagnosis and therapy of lymphoma with LL2. In vitro studies showed that iodinated LL2 is intracellularly catabolized, with a rapid release of the radioiodine from the cell. In contrast, residualizing radiolabels, such as radioactive metals, are retained intracellularly for substantially longer. In vivo studies were performed using LL2-labeled with radioiodine by a non-residualizing (chloramine-T) or a residualizing method (dilactitol-tyramine, DLT), or with a radioactive metal (¹¹¹In). The biodistribution of a mixture of ¹²⁵I (non-residualizing chloramine-T compared to residualizing DLT), ¹¹¹In-labeled LL2 murine IgG2a or its fragments [F(ab′)₂, Fab′], as well as its humanized, CDR-grafted form, was studied in nude mice bearing the RL human B-cell NHL cell line. Radiation doses were calculated from the biodistribution data according to the Medical International Radiation Dose scheme to assess the potential advantage for therapeutic applications. At all assay times, tumor uptake was higher with the residualizing labels (i.e., ¹¹¹In and DLT-¹²⁵I) than with the non-residualizing iodine label. For example, tumor/blood ratios of ¹¹¹In-labeled IgG were 3.2-, 3.5- and 2.8-fold higher than for non-residualizing iodinated IgG on days 3, 7 and 14, respectively. Similar results were obtained for DLT-labeled IgG and fragments with residualized radiolabels. Tumor/organ ratios also were higher with residualizing labels. No significant differences in tumor, blood and organ uptake were observed between murine and humanized LL2. The conventionally iodinated anti-CD20 antibody, 1F5, had tumor uptake values comparable to those of iodinated LL2, the uptake of both antibodies being strongly dependent on tumor size. These data suggest that, with internalizing antibodies such as LL2, labeling with intracellularly retained isotopes has an advantage over released ones, which justifies further clinical trials with residualizing ¹¹¹In-labeled LL2 for diagnosis, and residualizing ¹³¹I and ⁹⁰Y labels for therapy. Received: 23 August 1996 / Accepted: 7 February 1997     

A study of some factors that influence the iodination of ox insulin

1. The influence of carrier iodide, iodine monochloride and pH on the labelling of ox insulin with ¹³¹I by the iodine monochloride method have been studied. 2. The quantitative effect of the iodide in the radioactive iodine preparation was that predicted from a calculation of its specific activity. No other interfering factors were detected in the [¹³¹I]iodide solutions used. 3. Increasing the molar ratio of iodine monochloride to insulin resulted in an increase followed progressively by a decrease in the proportion of ¹³¹I bound, while the total iodine bound increased to an amount characteristic of pH and thereafter remained constant. 4. The influence of pH on the iodination of insulin with iodine monochloride was complex and the pH curve showed two maxima, at pH2·8 and 6·4. At pH2·8 it was not possible to exceed 8 atoms of iodine bound per molecule by increasing the molar ratio of iodine monochloride. Similarly, at pH6·4 the substitution value of 11·5 atoms of iodine per molecule could not be exceeded. 5. Iodinated insulins containing an average of 1·96, 2·74, 6·0 and 7·0 atoms of iodine per molecule fully retained the ability to bind guinea-pig anti-(ox insulin) serum, and the ability to compete with unlabelled insulin for antibody sites only became significantly changed in the most highly substituted preparations and in the presence of large concentrations of unlabelled insulin. 6. The method for the iodination of insulin with 98% incorporation of ¹³¹I by using chloramine-t is described. 7. ¹³¹I-iodinated insulin prepared with graded quantities of chloramine-t in excess of that required for efficient labelling was less efficiently bound by guinea-pig anti-(ox insulin) serum than insulin labelled by the iodine monochloride method.     

Receptor-Purified,Bolton-Hunter Radioiodinated,Recombinant, Human Epidermal Growth Factor: An Improved Radioligand for Receptor Studies

Abstract

We report an assessment of the applicability of the Bolton-Hunter method to the radioiodination of epidermal growth factor (EGF). Recombinant human EGF (hEGF) could be radioiodinated successfully by this method, whereas murine EGF could not. Bolton-Hunter ¹²⁵I-labeled hEGF was compared with commercial ¹²⁵I-labeled hEGF prepared by the chloramine-T radioiodination method. Neither radioligand was sufficiently pure for a detailed characterization of the purportedly heterogeneous pattern of binding of EGF to its receptors. A procedure based on receptor adsorption was thus developed for repurification of the Bolton-Hunter ¹²⁵I-labeled hEGF. This provided a much purer radioligand suitable for detailed studies of receptor-binding heterogeneity.     

A procedure for the preferential radioiodination of small amounts of protein in the presence of excess lipids.

The chloramine-T, iodine monochloride, and lactoperoxidase radioiodination procedures were evaluated for their ability to label proteins in the presence of large amounts of reactive lipid. Mouse serum very low density lipoproteins (VLDL) (10% protein, 90% lipid) and detergent-disrupted Friend murine leukemia virus (FLV) (60% protein, 33% lipid) were used as model systems. Based on the distribution of label between protein and lipid, as well as the total amount of label incorporated by each procedure, only a modification of the iodine monochloride procedure preferentially labeled proteins to a high specific activity in both VLDL and FLV. The technique, which is described in detail, is technically simple, rapid, and causes no detectable degradation of proteins.     

Allosteric effects of monoclonal antibodies on human growth hormone

We have previously shown that a monoclonal antibody (MAb) recognizing the human growth hormone (hGH) antigenic domain left exposed after binding to lactogenic receptors enhanced hGH binding probably through allosteric effects on the hormone binding site. Since receptors displaying different specificities would not recognize exactly the same hGH region, we explored whether some of our MAb could affect hGH binding to somatogenic receptors from rabbit liver and to human liver hGH-specific receptors.The effect of MAbAE5, AC8 and F11 on hGH binding was measured by determining the formation of¹²⁵I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding¹²⁵I-MAb, indicated that the hGH domain defined by epitopes AE5, AC8 and F11 is uncovered in the various hormone:receptor complexes.Procedure B was devised to reveal any alteration in the hGH molecule induced by the MAb. In this case preformed¹²⁵I-MAb:hGH complexes were added to microsomes. Data showed that¹²⁵I-MAb AE5:hGH complexes bound better to the various receptors than¹²⁵I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to¹²⁵I-MAb AC8 or¹²⁵I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding of MAb AE5 or MAb F11 to hGH 20 K (a natural hGH variant lacking residues 32–46) also enhanced its affinity to the various receptors whereas MAb AC8 did not inhibit hGH 20 K binding.Results indicated that MAb recognizing the hGH antigenic area that remains unmasked after binding to different membrane-bound receptors are able to affect hormone binding site. MAb would induce either positive or negative allosteric changes in the hormone region involved in its binding to lactogenic, somatogenic and hGH-specific receptors.     

Sandmeyer reaction repurposed for the site-selective,non-oxidizing radioiodination of fully-deprotected peptides: Studies on the endogenous opioid peptide α-neoendorphin

Standard radioiodination methods lack site-selectivity and either mask charges (Bolton-Hunter) or involve oxidative reaction conditions (chloramine-T). Opioid peptides are very sensitive to certain structural modifications, making these labeling methods untenable. In our model opioid peptide, α-neoendorphin, we replaced a tyrosyl hydroxyl with an iodine, and in cell lines stably expressing mu, delta, or kappa opioid receptors, we saw no negative effects on binding. We then optimized a repurposed Sandmeyer reaction using copper(I) catalysts with non-redoxing/non-nucleophilic ligands, bringing the radiochemical yield up to around 30%, and site-selectively incorporated radioactive iodine into this position under non-oxidizing reaction conditions, which should be broadly compatible with most peptides. The ¹²⁵I- and ¹³¹I-labeled versions of the compound bound with high affinity to opioid receptors in mouse brain homogenates, thus demonstrating the general utility of the labeling strategy and of the peptide for exploring opioid binding sites.     

Imaging of systemic Candida albicans infections with a radioiodinated monoclonal antibody: Experimental study in the guinea pig

Guinea pigs intravenously infected with Candida albicans were scanned to evaluate the use of radioiodinated monoclonal antibodies (MAb) to fungal antigens for detecting tissue infection sites. A total of 18 infected and 8 uninfected animals were used. MAb and F(ab′)₂ fragments directed against cell wall glycoproteins of C. albicans were labeled with ¹³¹I. Another MAb directed against a Schistosoma mansoni glycoprotein was labeled with ¹²⁵I and used as a nonspecific control. Radiolabeled MAbs were injected at a dose of 12.5 μg (500 kBq) per animal. Images were acquired 24 h later. Animals were then killed and the dissected organs were separately gamma-counted. The number of C. albicans colony forming units (cfu) per gram was determined in each organ. A clear relationship was found between the anatomic distributions of C. albicans and ¹³¹I. The biodistribution of ¹³¹I radioactivity associated with anti-Candida MAb was greater in infected animals than in healthy animals and increased with the number of cfu per g in each organ. The distribution was highly specific in animals with Candida endophthalmitis, a pathognomic feature of organ involvement during hematogenous dissemination. In contrast, the distribution of ¹²⁵I radioactivity associated with the nonspecific MAb was similar in healthy and infected animals. In infected animals, it was totally independent of the intensity of fungal infection.     

Radioimmunodetection of human tumor xenografts by monoclonal antibody F(ab′)2 fragments

Experimental procedures are described for the radiolocalization of human tumors by murine monoclonal antibodies (MAb) in animal model systems. Visualization of tumor xenografts was clearer in nude mice as compared to experimentally immunosuppressed mice due to the higher viability of the tumors in nude mice. MAb localization in tumor tissue was greatly enhanced when F(ab′)₂ fragments rather than intact antibody molecules were used. Although tumors could be visualized with either ¹³¹I-, ¹²³I- or ¹¹¹In-labeled MAb fragments without using background subtraction, tumor-to-background ratios of radioactivity were highest for ¹³¹I-labeled fragments. ¹³¹I-labeled F(ab′)₂ fragments of eight MAb against human colorectal carcinoma, melanoma or lung carcinoma localized specifically only in those tumors that bound the MAb in vitro and not in unrelated tumors. Radiolabeled fragments of MAb with other specificities (anti-hepatitis virus MAb) did not localize in tumors. All MAb that inhibited tumor growth in nude mice effectively localized these tumors by γ-scintigraphy. On the other hand, some MAb were effective in localizing tumors but ineffective in inhibiting their growth. The ability of the specific radiolabeled F(ab′)₂ fragments to localize in tumor grafts correlated significantly with MAb binding affinity and density of antigenic sites on tumor cells together, but not with either in vitro binding parameter alone. Thus, Scatchard analysis of MAb binding to tumor cells may be an effective means to screen for MAb with tumor radiolocalization potential.     

Some quantitative aspects of the labelling of proteins with 125I by the iodine monochloride method

The labelling of proteins by the iodine monochloride method was studied by using a mathematical model. The equations used were primarily derived from the mass law equation of the isotopic exchange reaction between [¹²⁵I]iodide and iodine monochloride. For convenient application, all equations were programmed into a computing desk-top calculator. To support the validity of the theoretical model, a series of iodinations of insulin were performed under various labelling conditions. The results of these experiments compare well with the theoretically derived values. Deviations from the theoretical values occurring at molar ratios of [¹²⁵I]iodide to iodine monochloride < 0.1 and > 4.0 are explained and suggestions made about how to prevent them. The mathematical model was used to simulate the isotopic exchange, and the iodination reaction under various conditions, to study (a) the influence of the amount of [¹²⁵I]iodide on the amount of [¹²⁵I]iodine monochloride formed, (b) the influence of the specific radioactivity of [¹²⁵I]iodide on the amount of [¹²⁵I]iodine monochloride formed, and (c) the influence of the specific radioactivity of [¹²⁵I]iodide on the number of millicuries needed for labelling to a desired extent.     

Mammary tumor immunoscintigraphy in rats: the use of 131I-anti-estriol 3-sulfate antibody

The scintigraphic imaging of mammary tumors with anti-estriol 3-sulfate (E₃ 3-S) antibody was studied in rats. A chemical carcinogen, 7,12-dimethylbenz(a)anthracine (DMBA), induced mammary tumors in Sprague-Dawley female rats. Highly specific anti-E₃ 3-S antibody was prepared and radioiodinated by [¹³¹I]NaI using the chloramine-T method. At 24 h after administration of ¹³¹ I-anti-E₃ 3-S antibody, goat anti-guinea pig immunogloblin G (IgG) was injected as the second antibody (SA) and nuclear scintigraphy was performed. Mammary tumors were clearly visualized following SA injection.     

Radioiodination of proteins using prosthetic group: a convenient way to produce labelled proteins with in vivo stability.

Radiolabelled peptides can provide new approaches for radiopharmaceutical development. Several prosthetic groups have been developed for radioiodination of proteins in order to minimize in vivo dehalogenation. In this work, the prosthetic group N-succinimidyl 4-[131I]iodobenzoate ([131I]SIB) was obtained by an alternative procedure that employs Cu(I) assisted radioiododebromination to produce p-[131I]iodobenzoic acid with a radiochemical yield of 92.73 +/- 1.51% (N = 6), followed by the reaction with TSTU (O-(N-succinimidyl)-N,N,N'N'-tetramethyluronium) in alkaline medium. The HPLC profile of the final product, revealed that [131I]SIB was obtained with a radiochemical purity of 98.19 +/- 1.14% (N = 6 Swiss mices (normal group) and animals with inflammation focus developed on the right thigh by tupertine injection) were injected with human immunoglobulin (IgG) radioiodinated with [131I]SIB and by direct method (Iodogen). The comparison of results showed a fast blood clearance, better target organ/background relation and low uptake in thyroid and stomach (p < 0.01) for the protein labelled with [131I]SIB, what suggests a greater in vivo stability.     

Preparation and properties of biologically active radioiodinated parathyroid hormone

A constant-current microelectrolytic radioiodination method was used to label bovine parathyroid hormone (BPTH) with ¹²⁵I to an overall iodination ratio of 1:1 iodide atoms per PTH molecule. Such iodinated preparations were shown to be fully active in several bioassay systems: in vitro adenylate cyclase activation in rat renal and skeletal membranes, in vitro calcium release from rat calvaria, and the in vivo hypercalcemic response in chickens. Analysis by Sephadex G-15 chromatography after enzymatic digestion showed the radioiodine to be incorporated predominantly as monoiodotyrosine. Bioassay of iodinated preparations from which uniodinated hormone had been removed by isoelectric focusing showed essentially full hormonal activity. Such methods can be used to consistently produce radioiodinated biologically active preparations of BPTH 1–84 with high specific activity (2000 Ci/mmol).     

Comparative biodistribution studies of DTPA-derivative bifunctional chelates for radiometal labeled monoclonal antibodies

Biodistribution of five different backbone-substituted derivatives of SCN-Bz-DTPA (1B4M-DTPA, 1M3B-DTPA, 1B3M-DTPA, GEM-DTPA and 2B-DTPA) linked to MAb B72.3 were compared to that of the parent molecule after labeling with ¹¹¹indium. Athymic mice, bearing human colon carcinoma xenografts (LS-174T) were injected i.v. to determine the biodistribution of the MAb chelate conjugates. Three of the MAb metal chelate conjugates (1B4M-DTPA, 1M3B-DTPA, and 1B3M-DTPA), labeled with ¹¹¹In showed efficient and stable tumor localization as well as a slower blood clearance rate than SCN-Bz-DTPA, GEM-DTPA or 2B-DTPA MAb chelate conjugates. Major differences were also seen in normal organ uptake, especially liver and spleen. Tumor-to-liver ratios rose as a function of time for 1B4M-DTPA, 1M3B-DTPA and 1B3M-DTPA MAb chelate conjugates with virtually no accumulation of the radiometal into this organ, as revealed by no increase in the liver-to-blood values. Small accretion in normal liver was noted for SCN-Bz-DTPA, GEM-DTPA or 2B-DTPA MAb chelate conjugates. The results reviewed here, and described previously (Roselli et al., 1991) demonstrate that the use in vivo of backbone-substituted forms of the SCN-Bz-DTPA, such as 1B4M-DTPA, 1M3B-DTPA, and 1B3M-DTPA bound to MAbs, can reduce uptake of indium to normal organs while maximizing the dose to tumor.     

3-([3-125I]iodo-4-hydroxyphenyl)propionyl carbohydrazide,a new radioiodination reagent for ultrasensitive detection and determination of periodate-oxidized nucleoside derivatives and other carbonyl compounds

A new radioiodination reagent for the identification and quantitation of periodate-oxidized ribonucleosides was developed. The reagent, 3-([3-¹²⁵I]iodo-4-hydroxyphenyl)propionyl carbohydrazide, was prepared by radioiodination of 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester in the presence of chloramine T, followed by reduction of the latter with sodium arsenite and treatment of the radioiodinated ester with an excess of carbohydrazide. The reagent reacted quantitatively with periodate-oxidized nucleosides to form ¹²⁵I-labeled morpholine derivatives which were separated by thin-layer chromatography and quantitated by liquid scintillation counting. The reagent was found to react also with other carbonyl compounds and thus may find more general application in the qualitative and quantitative ultramicroanalysis of aldehydes and ketones.     

Transfer of Immunity to Tumour Isografts by the Systemic Administration of Xenogeneic “Immune” RNA

SPECIFIC immunoreactivity can be conferred on lymphoid cells by incubation with RNA-rich extracts prepared from lymphoid tissues exposed to specific antigens in vivo¹ and in vitro^2,3. We have shown transfer of immunity to tumour specific antigens in vivo⁴ and in vitro⁵ by incubation of syngeneic spleen cells in vitro with RNA extracted from the lymphoid tissues of xenogeneic or syngeneic animals immunized with the tumour to be treated. Administration of these spleen cells to normal animals decreased the development and growth of isografts of the same tumour.     

Analysis of polypeptide disposition in human erythrocyte membranes employing membrane inversion

High resolution segregation of erythrocyte membrane polypeptides achieved by isoelectric focusing in 8 M urea was employed in conjunction with surface-restricted radioiodination to analyze the disposition of polypeptides within the human erythrocyte membrane. Several membrane polypeptides showed significant uptake of radioiodine, with the principal labeled component migrating between pH values of 3.0 and 3.5. Two approaches were taken in examining membrane polypeptide disposition on both faces of the erythrocyte membrane. Saturation labeling of the outer face of the membrane with one iodine isotope followed by cell lysis and re-iodination with a second iodine isotope did not prove feasible and another procedure based on surface iodination with ¹²⁵I, formation of sealed inside-out vesicles and re-iodination with ¹³¹I was adopted. Studies of sialic acid release from the membrane surface and trypsin cleavage of radioiodinated peptides indicated that selectively labeled, sealed inside-out vesicles had been formed. The ratio of ¹²⁵I to ¹³¹I in membrane polypeptides separated by isoelectric focusing confirmed the existence of externally disposed, internally disposed and spanning proteins.     

Non-dehalogenation mechanisms for excretion of radioiodine after administration of labeled antibodies

In patients or mice with cancer the pharmacokinetic behavior of radioiodinated and radiometal chelated antibodies has been observed to be different. Rapid clearance from the tissues and excretion into the urine can occur after injection of radioiodinated antibodies. These observations have been interpreted to reflect in vivo dehalogenation of the antibody. This publication describes a variety of other mechanisms that can underlie these phenomena. These mechanisms include receptor uptake and catabolism of antibody and instability of the labeled antibody due to the labeling conditions. Specifically, the relative masses of chloramine-T and antibody in the iodination reaction mixture, the level of iodination of the antibody, and the amount of antibody administered to the recipient are all factors which can influence the clearance of radioiodinated antibody from the recipient. The final determinant for the different behavior of radioiodinated and In-111 metal chelated antibody relate to the different biologic pathways of indium when compared to iodine.     

Radioiodination of sulfhydryl-sensitive proteins.

A new procedure is described for the radioiodination of proteins with sulfhydryl groups essential for their biological activity. Aniline is iodinated with ¹²⁵I-labeled sodium iodide in the presence of chloramine-T, the product separated by solvent extraction, diazotized and coupled to protein.