Development of [225Ac]Ac-DOTA-C595 as radioimmunotherapy of pancreatic cancer: in vitro evaluation, dosimetric assessment and detector calibration.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy which may benefit from radioimmunotherapy. Previously, [177Lu]Lu-DOTA-C595 has been developed as a beta-emitting radioimmunoconjugate to target cancer-specific mucin 1 epitopes (MUC1-CE) overexpressed on PDAC. However, the therapeutic effect may be enhanced by using an alpha-emitting radionuclide such as Actinium-225 (Ac-225). The short range and high linear energy transfer of alpha particles provides dense cellular damage and can overcome typical barriers related to PDAC treatment such as hypoxia. Despite the added cytotoxicity of alpha-emitters, their clinical implementation can be complicated by their complex decay chains, recoil energy and short-range impeding radiation detection. In this study, we developed and evaluated [225Ac]Ac-DOTA-C595 as an alpha-emitting radioimmunotherapy against PDAC using a series of in vitro experiments and conducted a preliminary dosimetric assessment and cross-calibration of detectors for the clinical implementation of Ac-225. RESULTS: Cell binding and internalisation of [225Ac]Ac-DOTA-C595 was rapid and greatest in cells with strong MUC1-CE expression. Over 99% of PDAC cells had positive yH2AX expression within 1 h of [225Ac]Ac-DOTA-C595 exposure, suggesting a high level of DNA damage. Clonogenic assays further illustrated the cytotoxicity of [225Ac]Ac-DOTA-C595 in a concentration-dependent manner. At low concentrations of [225Ac]Ac-DOTA-C595, cells with strong MUC1-CE expression had lower cell survival than cells with weak MUC1-CE expression, yet survival was similar between cell lines at high concentrations irrespective of MUC1-CE expression. A dosimetric assessment was performed to estimate the dose-rate of 1 kBq of [225Ac]Ac-DOTA-C595 with consideration to alpha particles. Total absorption of 1 kBq of Ac-225 was estimated to provide a dose rate of 17.5 mGy/h, confirmed via both detector measurements and calculations. CONCLUSION: [225Ac]Ac-DOTA-C595 was shown to target and induce a therapeutic effect in MUC1-CE expressing PDAC cells.

In vitro characterisation of [177Lu]Lu-DOTA-C595 as a novel radioimmunotherapy for MUC1-CE positive pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) continues to be a malignancy with an unmet clinical demand. Development of radioimmunoconjugates which target cancer-specific receptors provides an opportunity for radioimmunotherapy of both metastatic and primary PDAC. In this study, we characterised the in vitro behaviour of a novel beta-emitting radioimmunoconjugate [177Lu]Lu-DOTA-C595 as a therapeutic agent against PDAC. [177Lu]Lu-DOTA-C595 is designed to target cancer-specific mucin 1 epitopes (MUC1-CE) overexpressed on most epithelial cancers, including PDAC. RESULTS: A series of in vitro experiments were performed on PDAC cell lines (PANC-1, CAPAN-1, BxPC-3 and AsPC-1) exhibiting strong to weak MUC1-CE expression. [177Lu]Lu-DOTA-C595 bound to all cell lines relative to their expression of MUC1-CE. [177Lu]Lu-DOTA-C595 was also rapidly internalised across all cell lines, with a maximum of 75.4% of activity internalised within the PANC-1 cell line at 48 h. The expression of γH2AX foci and clonogenic survival of PANC-1 and AsPC-1 cell lines after exposure to [177Lu]Lu-DOTA-C595 were used to quantify the in vitro cytotoxicity of [177Lu]Lu-DOTA-C595. At 1 h post treatment, the expression of γH2AX foci exceeded 97% in both cell lines. The expression of γH2AX foci continued to increase in PANC-1 cells at 24 h, although expression reduced in AsPC-1. Clonogenic assays showed a high level of cell kill induced by [177Lu]Lu-DOTA-C595. CONCLUSION: [177Lu]Lu-DOTA-C595 has favourable in vitro characteristics to target and treat MUC1-CE positive PDAC. Further investigations to characterise the in vivo effects and potential value of [177Lu]Lu-DOTA-C595 in other MUC1-CE expressing malignancies such as lung, ovarian and colorectal adenocarcinoma are warranted.

Preliminary Development and Testing of C595 Radioimmunoconjugates for Targeting MUC1 Cancer Epitopes in Pancreatic Ductal Adenocarcinoma.

Mucin 1 is a transmembrane glycoprotein which overexpresses cancer-specific epitopes (MUC1-CE) on pancreatic ductal adenocarcinoma (PDAC) cells. As PDAC is a low survival and highly aggressive malignancy, developing radioimmunoconjugates capable of targeting MUC1-CE could lead to improvements in PDAC outcomes. The aim of this study was to develop and perform preliminary testing of diagnostic and therapeutic radioimmunoconjugates for PDAC using an anti-MUC1 antibody, C595. Firstly, p-SCN-Bn-DOTA was conjugated to the C595 antibody to form a DOTA-C595 immunoconjugate. The stability and binding affinity of the DOTA-C595 conjugate was evaluated using mass spectrometry and ELISA. DOTA-C595 was radiolabelled to Copper-64, Lutetium-177, Gallium-68 and Technetium-99m to form novel radioimmunoconjugates. Cell binding assays were performed in PANC-1 (strong MUC1-CE expression) and AsPC-1 (weak MUC1-CE expression) cell lines using 64Cu-DOTA-C595 and 177Lu-DOTA-C595. An optimal molar ratio of 4:1 DOTA groups per C595 molecule was obtained from the conjugation process. DOTA-C595 labelled to Copper-64, Lutetium-177, and Technetium-99m with high efficiency, although the Gallium-68 labelling was low. 177Lu-DOTA-C595 demonstrated high cellular binding to the PANC-1 cell lines which was significantly greater than AsPC-1 binding at concentrations exceeding 100 nM (p < 0.05). 64Cu-DOTA-C595 showed similar binding to the PANC-1 and AsPC-1 cells with no significant differences observed between cell lines (p > 0.05). The high cellular binding of 177Lu-DOTA-C595 to MUC1-CE positive cell lines suggests promise as a therapeutic radioimmunoconjugate against PDAC while further work is required to harness the potential of 64Cu-DOTA-C595 as a diagnostic radioimmunoconjugate.

Radioimmunotherapy of glioblastoma multiforme - Current status and future prospects.

Glioblastoma multiforme (GBM) or grade IV astrocytoma is the most diagnosed form of primary brain tumours in adults. Radioimmunotherapy (RIT), mostly in combination with conventional therapies, is presented in the current review as a therapeutic strategy of high potential in the management of GBM. A systematic literature search was performed following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) to identify clinical studies that employed a form of radioimmunotherapy using alpha- or beta-emitting radioisotopes. The available literature on RIT in GBM and high-grade gliomas is presented and discussed. The results suggest that this promising treatment approach merits further investigation in future clinical studies.

The Expression Profile and Textural Characteristics of C595-Reactive MUC1 in Pancreatic Ductal Adenocarcinoma for Targeted Radionuclide Therapy.

Improvements in the prognosis of pancreatic ductal adenocarcinoma (PDAC) rely on the development of effective treatments to target advanced disease. Mucin 1 (MUC1) is a transmembrane glycoprotein which is involved in the metastatic progression of PDAC and is a receptor-of-interest for targeted radionuclide therapy. The aim of this study was to determine the feasibility of MUC1-based targeted radionuclide therapy for PDAC, by evaluating the expression profile of MUC1 in different pancreatic cells and tissues using the C595 antibody. MUC1 expression was evaluated in four PDAC cell lines (PANC-1, BxPC-3, CAPAN-1 and AsPC-1) using flow cytometry and immunocytochemistry. Immunohistochemistry was performed on primary and metastatic PDAC, pancreatitis, pancreatic intra-epithelial neoplasia and normal pancreatic tissue samples to identify potential changes in C595-reactive MUC1 expression across different disease groups. C595-reactive MUC1 expression was found to varying degrees in the cell lines (11.5-93.1%). A pixel analysis of the immunohistochemical staining demonstrated highest MUC1 expression in primary PDAC tissue (mean pixel value of 205.4), followed by other pancreatic cancer types (204.9), pancreatic intra-epithelial neoplasia (203.8), metastatic PDAC (201.5), chronic pancreatitis (198.1) and normal pancreatic tissue (191.4). The increased expression in malignant tissues and reduced expression in benign tissues indicate that C595-reactive MUC1 is a potential target for targeted radionuclide therapy of PDAC.

Radioimmunotherapy of Pancreatic Ductal Adenocarcinoma: A Review of the Current Status of Literature.

Pancreatic ductal adenocarcinoma (PDAC) has long been associated with low survival rates. A lack of accurate diagnostic tests and limited treatment options contribute to the poor prognosis of PDAC. Radioimmunotherapy using α- or ß-emitting radionuclides has been identified as a potential treatment for PDAC. By harnessing the cytotoxicity of α or ß particles, radioimmunotherapy may overcome the anatomic and physiological factors which traditionally make PDAC resistant to most conventional treatments. Appropriate selection of target receptors and the development of selective and cytotoxic radioimmunoconjugates are needed to achieve the desired results of radioimmunotherapy. The aim of this review is to examine the growing preclinical and clinical trial evidence regarding the application of α and ß radioimmunotherapy for the treatment of PDAC. A systematic search of MEDLINE® and Scopus databases was performed to identify 34 relevant studies conducted on α or ß radioimmunotherapy of PDAC. Preclinical results demonstrated α and ß radioimmunotherapy provided effective tumour control. Clinical studies were limited to investigating ß radioimmunotherapy only. Phase I and II trials observed disease control rates of 11.2%-57.9%, with synergistic effects noted for combination therapies. Further developments and optimisation of treatment regimens are needed to improve the clinical relevance of α and ß radioimmunotherapy in PDAC.