Ana Robles

Improved radioiodination of biomolecules using exhaustive Chloramine-T oxidation.

To improve standardization in analytical reagents we investigated Chloramine-T radioiodination (125I) of several biomolecules based on the use of a single amount of the oxidizing agent Chloramine-T as the limiting reagent being exhausted during the course of the reaction. Whenever the labeling yield resulted in less than one atom 125I/molecule, a second amount of the oxidizing agent was added. Thereafter, the integrity of the various biomolecules was assessed using radioimmunoassays, radioreceptor binding assays, or radioimmunometric assays. Purification yields were done by gel permeation (56% +/- 19%, n=230) or by precipitation with trichloroacetic acid (59% +/- 19%, n=230). Specific activity (117 +/- 61 MBq/nmol) and the degree of iodine incorporation (1.4 +/- 0.8 atoms of 125I/molecule) were achieved after 300 sec of incubation. A second addition of Chloramine-T resulted in an increased labeling yield of all biomolecules tested by a mean factor of 1.8 +/- 0.9. After the second addition of Chloramine-T, we observed for some biomolecules a significant (p<0.001) decreased effect in biological performance. In conclusion, the use of Chloramine-T as a limiting reagent resulted in molecules with appropriate immunological and biological performance. In general, tracers were minimally damaged and assessment of the shelf life as well as storing conditions showed the usefulness of the standardization of biomolecule labeling.

Re-HEDP : pharmacokinetic characterization, clinical and dosimetric evaluation in osseous metastatic patients with two levels of radiopharmaceutical dose

BackgroundA study for pain relief therapy with 188Re-HEDP was done in patients with bone metastases secondary to breast and prostate cancer.Materials and MethodsPatients received 1.3 or 2.2 GBq, in single or multiple doses. Platelets, white and red cells were evaluated during 11 weeks. Pharmacokinetic characterization was done from blood and urine samples for 5 patients along 24 hours. Urinary excretion was evaluated in other 16 patients during 6 hours. Bone uptake was estimated as remaining activity in whole body. Scintigraphic images were acquired at 2 and 24 hs post-administration. Absorbed dose in bone marrow was estimated with Mirdose3. Analgesics intake and pain score were daily recorded. Tumour markers (PSA, and Tn-structure) were monitored in 9 patients during 4 to 6 months. Single doses of low activity (1.3 GBq) were given to twelve patients. Nine patients received multiple doses.ResultsAll except one patient had normal levels of platelets, white and red cells. Remaining dose in blood at 2 hours was 9%. Urinary elimination was 58%. Bone uptake at 24 hours was 43% (mean value; n = 5). No changes of the haematological parameters were detected along follow-up period. Pain relief was evidenced by decrease or supression of opioid analgesic and by subjective index. PSA showed a decrease in prostate cancer patients (n = 4). Tn-structure showed a significant increase after 4 to 8 months.ConclusionSingle or multiple dose scheme could be safely used, with administered activity of 188Re-HEDP up to 60 mCi, with low bone marrow absorbed doses.

[177Lu]DOTA-anti-CD20: labeling and pre-clinical studies.

Anti-CD20 (Rituximab®), a specific chimeric monoclonal antibody used in CD20-positive Non-Hodgkins Lymphoma, was conjugated to a bifunctional quelate (DOTA) and radiolabeled with (177)Lu through a simple method. [(177)Lu]-DOTA-anti-CD20 was obtained with a radiochemical purity higher than 97%, and showed good chemical and biological stability, maintaining its biospecificity to CD20 antigens. Monte Carlo simulation showed high doses deposited on a spheroid tumor mass model. This method seems to be an appropriate alternative for the production of [(177)Lu]-DOTA-anti-CD20 as therapeutic radiopharmaceutical.

[125I]IgM (KAU) Human monoclonal cold agglutinin: Labelling and studies on its biological activity

In order to study the interaction between an IgM cold agglutinin and the erythrocyte I antigen, the former antibody was labelled with 125I using the Chloramine-T, IODOGEN and Bolton-Hunter methods. High incorporation and adequate stability of the labelled IgM were obtained with all procedures. However, suitable biological activity was maintained only with the Bolton-Hunter method. Further studies suggest that tyrosine iodination affects antigen recognition by this IgM, whereas iodination of amino groups does not. The reagent thus prepared allowed the determination of the number of I sites per erythrocyte as well as the antibody affinity constant.

Interaction of a monoclonal antibody against hEGF with a receptor site for EGF

Epidermal growth factor (EGF) has been detected by radioimmunoassay (RIA) in different body fluids such as serum, amniotic fluid, and urine. Human tumor tissues with EGF receptors (EGF-Rc) may be saturated with EGF, which may be of prognostic value. An RIA was envisaged to measure human epidermal growth factor (hEGF) levels using EGF-Rc as capture agent and a monoclonal antibody anti-hEGF (MAb anti-hEGF) labeled with 125Iodine as a marker for this binding. The purpose of this work was to study the feasibility of MAb anti-hEGF to detect the receptor binding sites and to investigate the interaction between MAb anti-hEGF and the EGF-Rc. Various binding experiments were performed to study possible interference and interactions in the complex MAb anti-hEGF and the receptor. Affinity constants were determined by means of Scatchard plot analysis to interpret the complex stability challenged with other compounds for a better understanding of the interaction process. Binding constants were of the same order for all the ligands tested separately involving the EGF-Rc, but were significantly higher (t = 15.7, p < 0.05) for hEGF in its binding to MAb anti-hEGF. It was possible with equilibrium studies and competition experiments to evaluate the interaction of EGF and MAb anti-hEGF with the EGF receptor. This observation makes the MAb anti-hEGF a potential tracer for the quantitation of receptors in vitro, and possibly for the detection of membrane receptors on tumor cells in vivo.

Monoclonal Antibodies against the Tn-Specific Isolectin B4 from Vicia villosa Seeds: Characterization of the Epitope of the Blocking Antibody VV34

Vicia villosa isolectin B4 (VVLB4) recognizes the Tn antigen (GalNAc-O-Ser/Thr) exposed in certain human carcinomas. We have produced anti-VVLB4 monoclonal antibodies (MAbs), and their lectin recognition selectivity was assessed by ELISA and Western blot against the purified Gal/GalNAc-specific lectins from Vicia villosa, Salvia sclarea, Helix pomatia, Arachis hypogaea, Glycine max, and Dolichos biflorus. The antibodies were also tested for their ability to block the binding of VVLB4 to the Tn antigen expressed on immobilized asialo ovine submaxillary mucin. Two MAbs, VV34 and VV2, specifically recognized VVLB4 and impaired the binding of the lectin to the Tn antigen by 98% and 21%, respectively. On the other hand, MAbs VV1 and VV22 cross-reacted with other purified lectins. The four antibodies recognized native and periodate-oxidized nonreduced as well as reduced VVLB4 after SDS-PAGE and Western blot, suggesting that they were recognizing continuous polypeptide epitopes. The VV34 antibody recognized two tryptic peptides (7-29 and 96-106) from VVLB4, which are contiguous in the three-dimensional structure of the lectin. The minimum structural determinant of the epitope was mapped to the polypeptide stretch (18)LILQED(23) using a set of overlapping synthetic peptides. This region of the molecule encompasses the C-terminal part of the loop joining strands beta1 and beta2 and the N-terminal part of beta2, and is located about 20-25 A away from the center of the Tn-combining site.

In-site interaction evaluation of Tn density by inhibition/competition assays.

The tumor-associated structure N-acetyl-galactosamine-O-Ser/Thr (Tn antigen), which is overexpressed in various tumor cell types, notably of the breast, ovary and colon, is an interesting determinant that is useful for cancer diagnosis and follow-up. The aim of this research was to study different assay strategies in order to determine the most sensitive system for further application in epitope characterization and binding assessment. The tetrameric isolectin obtained from Vicia villosa seeds (VVLB(4)) shows high affinity for the tumor-associated structure. A monoclonal antibody against VVLB(4), MabVV(34), was generated, and the interaction between MabVV(34) and VVLB(4) was studied by means of binding and inhibition assays. Several synthetic peptides (10 amino acid sequences) designed from the amino acid sequence of VVLB(4) and obtained from trypsin digestion were tested to determine which amino acids were involved in the interaction between MabVV(34) and VVLB(4). The further unraveling of this epitope was investigated by inhibition using designed synthetic peptides as well as mixtures mimicking variable density effect. Under the experimental circumstances, MabVV(34) was able to inhibit the binding of VVLB(4) to Tn. Two of the four peptide sequences assayed showed better inhibition properties. Finally, mixtures containing these selected sequences allowed the evaluation of binding and inhibition as a function of Tn density. We conclude that the present study facilitates the further development of a specific Tn marker and may contribute to the development of Tn-like radiolabelled peptides or Tn-specific radiolabelled fragments providing a highly selective tool for cancer diagnosis and treatment. This strategy may contribute to characterize the new generation of radiopharmaceuticals for diagnosis and therapy based on biomolecules like antibodies, fragments or peptides, whose application is directly guided by their specific molecular recognition.