Radioiodine D amino acids labeling of rituximab, a new method for enhancing the radiopharmaceutical targeting and biostability

Radioimmunotherapy [RIT] is a very promising new therapy for the treatment of recurrent B-Cell non-Hodgkin's lymphoma [NHL]. Iodine-131 is the most frequently used nuclide in clinical RIT, but its usefulness has been limited by dehalogenation of monoclonal antibodies labeled via conventional methods. To circumvent this problem, we have synthesized a tri-peptide consisting of non-metabolizable D amino acids attached to N-Hydroxysuccinimide [NHS]. Tri-peptide was synthesized by standard Fmoc solid phase synthesis on tritylchloride resin. Labeling of tri-peptide was performed using the chloramine-T method and the conventional extraction. Radioiodination of tri-peptide was followed by conjugation to anti-CD20 antibody. In vitro stability of labeled antibody in serum and phosphate buffered saline [PBS] was measured for 48hr by [thin layer chromatography] TLC. Raji cell line was used to test cell binding of the labeled anti-CD20. The chemical purity of synthesized peptide as assessed by analytical [high performance liquid chromatography] HPLC was 95%. Labeling of tri-peptide resulted in a radiochemical yield of 50-71% with radiochemical purity of > 95%. At Rituximab concentration of 10mg/ml, coupling efficiencies of 65-80% was obtained with radiochemical purity of 95% and Specific activity [SA] of 185MBq/mg [5mCi/mg]. This study showed that labeling monoclonal antibodies with radioiodine by non-metabolizable D amino acids will improve bio-stability of the product

Evaluation of tumor targeting with radiolabeled F [AB'] 2 fragment of a humanized monoclonal antibody

Humanized monoclonal antibody U36 and its F[ab']2 fragment, radio labeled with 125I, were tested for tumor localization in nude mice bearing a squamous cell carcinoma xenograft line derived from a head and neck carcinoma. Monoclonal antibody IgG or F[ab']2 fragment were injected in parallel and at days 1, 2 and 3, mice were dissected for determination of isotope biodistribution. IgG as well as F[ab']2 showed highly specific localization in tumor tissue. The mean tumor uptake [n=3] is expressed as the percentage of the injected dose per gram of tumor tissue [%ID/g].%ID/g of IgG was 11.7% at day 1 and decreased to 10.9% at day 3 whereas%ID/g of F[ab']2 was 2.9% at day 1 and decreased on following days. Tumor to blood ratios [T/B] at day 1 were 0.86 for IgG and 1.32 for F[ab']2 and reached a maximum at day 3 with values of 4.41 and 1.84 respectively. These findings suggest that the superior tumor to non-tumor ratios in the day of 1 render the F[ab']2 fragment more qualified for specific targeting radioisotopes to tumor xenografts in this exprimental setting