Effect of radiochemical modification on biodistribution of scFvD2B antibody fragment recognising prostate specific membrane antigen.

Antibody-based reagents represent a promising strategy as clinical diagnostic tools. Prostate cancer (PCa) is the second-leading cause of death in males in the Western population. There is a presently unmet need for accurate diagnostic tool to localize and define the extent of both primary PCa and occult recurrent disease. One of the most suitable targets for PCa is the prostate-specific membrane antigen (PSMA) recognised by the monoclonal antibody D2B that we re-shaped into the single chain Fv (scFv format). Aim of this study was to evaluate in preclinical in vivo models the target specificity of scFvD2B after labelling with different radionuclides. (111)In radiolabelling was performed via the chelator Bz-NOTA, and (131)I radioiodination was performed using iodogen. The potential for molecular imaging and the biological behaviour of the radiolabelled scFvD2B were evaluated in mice bearing two subcutaneous PCa isogenic cell lines that differed only in PSMA expression. Biodistribution studies were performed at 3, 9, 15 and 24h after injection to determine the optimal imaging time point. A significant kidney accumulation, as percentage of injected dose of tissue (%ID/g), was observed for (111)In-scFvD2B at 3h after injection (45%ID/g) and it was maintained up to 24h (26%ID/g). By contrast, kidney accumulation of (131)I-scFvD2B was only marginally (0.3%ID/g at 24h). At the optimal time point defined between 15h and 24h, regardless of the radionuclide used, the scFvD2B was able to localize significantly better in the PSMA expressing tumours compared to the negative control; with (131)I-scFvD2B yielding a significantly better target/background ratio compared to (111)In-scFvD2B. These data suggest that, besides antigen specificity, chemical modification may affect antibody fragment biodistribution.

Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using an IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex.

Bispecific antibodies (BsAb) have proven to be useful targeting vectors for pretargeted radioimmunotherapy (PRIT). We sought to overcome key PRIT limitations such as high renal radiation exposure and immunogenicity (e.g., of streptavidin-antibody fusions), to advance clinical translation of this PRIT strategy for diasialoganglioside GD2-positive [GD2(+)] tumors. For this purpose, an IgG-scFv BsAb was engineered using the sequences for the anti-GD2 humanized monoclonal antibody hu3F8 and C825, a murine scFv antibody with high affinity for the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexed with ß-particle-emitting radiometals such as (177)Lu and (90)Y. A three-step regimen, including hu3F8-C825, a dextran-based clearing agent, and p-aminobenzyl-DOTA radiolabeled with (177)Lu (as (177)Lu-DOTA-Bn; t1/2 = 6.71 days), was optimized in immunocompromised mice carrying subcutaneous human GD2(+) neuroblastoma (NB) xenografts. Absorbed doses for tumor and normal tissues were approximately 85 cGy/MBq and ≤3.7 cGy/MBq, respectively, with therapeutic indices (TI) of 142 for blood and 23 for kidney. A therapy study (n = 5/group; tumor volume, 240 ± 160 mm(3)) with three successive PRIT cycles (total (177)Lu: ∼33 MBq; tumor dose ∼3,400 cGy), revealed complete tumor response in 5 of 5 animals, with no recurrence up to 28 days after treatment. Tumor ablation was confirmed histologically in 4 of 5 mice, and normal organs showed minimal overall toxicities. All nontreated mice required sacrifice within 12 days (>1.0-cm(3) tumor volume). We conclude that this novel anti-GD2 PRIT approach has sufficient TI to successfully ablate subcutaneous GD2(+)-NB in mice while sparing kidney and bone marrow.

Preclinical pharmacology and safety of a novel avidin derivative for tissue-targeted delivery of radiolabelled biotin.

We recently described an oxidized avidin variant, named AvidinOX(®) , which is a product that chemically links to tissue proteins while maintaining the capacity to uptake intravenously administered biotin. Such product proved to be successful in targeting radionuclide therapy in a mouse model of inoperable breast cancer. Here, we show that the uptake of a single or multiple doses of biotin (up to five times), by the tissue-bound AvidinOX(®) , is stable for 2 weeks. Taking into account that oxidized avidin is the first chemically reactive protein to be proposed for clinical use, we evaluated its tolerability, immunogenicity and mutagenicity. Present in vitro data indicate that AvidinOX(®) (up to 10 µg/5 × 10(5) cells) does not affect cell viability or proliferation of PC3 human prostate cancer or 3T3 mouse fibroblast cell lines as well as primary mouse spleen cells. Safety pharmacology and toxicology studies were conducted using AvidinOX(®) up to the highest concentration compatible with its solubility (about 12 mg/mL), representing four times the product concentration intended for human use, and in the maximum administrable volume compatible with each study system. The intramuscular administration in rat and monkey induced a moderate to strong inflammatory response particularly after a second administration and consistently with the induction of an immune response. Interestingly, the intramuscular administration of AvidinOX(®) to rodents and monkeys exhibiting very high anti-avidin antibody titres was well tolerated with no systemic symptoms of any kind. Intravenous administration of AvidinOX(®) , performed to mimic an accidental injection of the dose intended for a local administration (15 µL of 3.3 mg/mL solution), showed significant localization of the product into the spleen not associated with uptake of the radiolabelled biotin intravenously injected after 24 hr, thus suggesting rapid inactivation. No mutagenic activity was induced by oxidized avidin in prokaryotic and eukaryotic cells. Overall, the present data indicate that AvidinOX(®) is well tolerated in rodents and non-human primates, thus supporting its clinical use within protocols of radionuclide therapy of inoperable tumour lesions.

Imunoterapia específica - avaliação dinâmica e cinética in vivo do extrato terapêutico em doentes alérgicos / Specific immunotherapy - in vivo dinamic and kinetic evaluation of a therapeutic extract in allergic patients

Introdução: A imunoterapia específica (IT) é uma forma comum de tratamento da doença alérgica. O mecanismo preciso desta terapêutica não é conhecido, embora a eficácia clínica esteja plenamente documentada. O objetivo deste estudo foi avaliar a cinética da IT , aplicando uma técnica de Medicina Nuclear, marcação de leucócitos com 99mTc-HMPAO, em doentes em fase de manutenção e com excelente eficácia clínica à terapêutica.Pacientes e métodos: Foram estudados 14 doentes alérgicos agrupados de acordo com o tipo de extrato e via de administração: extratos aquosos subcutâneos (látex= 4 doentes; veneno de abelha= 2 doentes), extrato depot subcutâneo (ácaros= 2 doentes; gramíneas= 2 doentes); extrato modificado subcutâneo (gramíneas= 1 doente; Parietária= 1 doente) extrato sublingual (ácaros= 2 doentes). O grupo controle foi constituído por dois doentes alérgicos a ácaros, submetidos respectivamente a: injecção subcutânea de soluto salino e injecção de extrato bacteriano por via subcutânea (controle positivo).Simultaneamente à administração do extrato alergênico terapêutico, procedeu-se à re-injecção de leucócitos marcados com 99mTc-HMPAO, em veia periférica contralateral. A aquisição dinâmica decorreu durante 60 minutos, com matriz de 64x64, 2 imagens/ minuto em projeção torácica anterior. As aquisições estáticas, com matrizes 256x256, durante 5 minutos cada foram adquiridas aos 60, 90, 120, 180, 240, 300 e 360 minutos, em projeção torácica (anterior e posterior) e abdominal (anterior).Resultados: A atividade inflamatória no ocal de administração da IT para os extratos subcutâneos aquosos e depot iniciou-se na primeira hora e manteve um aumento ao longo do tempo de estudo. Para os extratos sublinguais a atividade inflamatória foi observada logo nos primeiros minutos. Todos os estratos subcutâneos condicionaram dreanagem linfática ascendente para áreas axilares homolaterais nos primeiros minutos após a adminstração do extrato e, posteriormente, para tecido linfóide do mediastino superior e anterior, e áreas cervicais. As focalizações torácicas estiveram presentes em todos os doentes estudados, o mesmo não acontecendo para a atividade de intestinal. A via sublingual não induziu focalizações axilares ou instetinais, mesmo tendo havido deglutição do alérgeno. Foram calculados coeficientes corrigidos de captação em áreas individualizadas (ROIs- region of interest) em relação aos coeficientes de captação das áreas de background.