Abozeid, Mohamed (2018) Imaging and Radio-immunotherapy of pancreatic cancer. [Tesi di dottorato]
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The aims of this study are imaging and Radio-immunotherapy (RIT) of pancreatic cancer through the development of new target-specific radiopharmaceuticals based on mAb directed to prostate stem cell antigen (PSCA) and Mesothelin antigen, which are heavily overexpressed in this tumor histotype.
Flow cytometry studies were carried out in order to confirm the ability of both anti-APSCA (APSCA) and anti-mesothelin (AM) mAbs to recognize the specific antigen receptors PSCA and mesothelin on the surface of the malignant cells. Two tumor cell lines were used, the human pancreatic adenocarcinoma T3M4 cell transduced or not to express PSCA receptors (T3M4-PSCA) and the embryonic human kidney 293-cell transduced or not to express mesothelin receptors (293-Meso).
Cytometry studies were done to identify the expression of PSCA and Mesothelin antigen on the cell surface. First, both mAbs conjugated to Alexa 680 using the SAIVI rapid antibody labelling kit, the labelled products was purified and the labelling degree and protein concentration were determined by absorbance. Then, 3x105 cells were stained with 1 µg of Alexa 680-APSCA or Alexa 680-AM mAbs to be analysed using a FACS Calibur flow cytometry.
Optical imaging studies were performed in mice to confirm the ability of both mAbs to identify specifically the antigens in vivo.
Direct labelling of APSCA and AM mAbs was performed by reduction of mAb disulphide bridges with 2-mercaptoehanol (2-ME). Reduced mAbs were radiolabeled by the addition of eluted 99mTc-pertechnetate to mAb, followed by the reducing agent SnCl2. The preparation was incubated at room temperature and gently mixed. Radiochemical purity (RCP) analyses were performed using radio-HPLC and stability was tested after dilution in different solutions at different ratios.
Conjugations of DOTA were done by incubation of p-SCN-Bz-DOTA to APSCA and AM mAbs, then characterization studies of DOTA conjugates were done using size exclusion HPLC. Conjugated mAbs were indirectly radiolabelled with Lutetium-177 177(Lu), incubated at 37oC and labelling efficiency (LE) was evaluated. Stability against dilution and transchelation were also assessed at different time points by size exclusion HPLC and ITLC-SG.
Results and conclusions
AM and APSCA mAbs that efficiently detected pancreatic cancer cells in vitro and in vivo were successfully labeled using direct and indirect methods with 99mTc and 177Lu, respectively. All the resulting compounds were prepared by rapid simple methods and demonstrated high RCP, high in vitro stability and high in vitro binding specificity. Therefore, our results are highly encouraging and open additional opportunities for further in vivo studies to assess such mAbs as novel imaging diagnostic and effective therapeutic tools in pancreatic cancer.
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Gli obiettivi di questo studio sono l'imaging e la radioimmunoterapia (RIT) del cancro del pancreas attraverso lo sviluppo di nuovi radiofarmaci target-specifici basati su mAb diretti all'antigene della prostata staminale prostatica (PSCA) e all'antigene della mesotelina, che sono fortemente sovraespressi in questo istotipo tumorale .
Gli mAb AM e APSCA che hanno rilevato in modo efficiente le cellule di cancro al pancreas in vitro e in vivo sono stati etichettati con successo usando metodi diretti e indiretti con 99mTc e 177Lu, rispettivamente. Tutti i composti risultanti sono stati preparati con metodi semplici rapidi e hanno dimostrato un alto RCP, elevata stabilità in vitro ed elevata specificità di legame in vitro. Pertanto, i nostri risultati sono altamente incoraggianti e aprono ulteriori opportunità per ulteriori studi in vivo per valutare tali mAb come nuovi strumenti diagnostici per immagini e strumenti terapeutici efficaci nel cancro del pancreas.
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