Anti-L1CAM radioimmunotherapy is more effective with the radiolanthanide terbium-161 compared to lutetium-177 in an ovarian cancer model

Purpose: The L1 cell adhesion molecule (L1CAM) is considered a valuable target for therapeutic intervention in different types of cancer. Recent studies have shown that anti-L1CAM radioimmunotherapy (RIT) with 67Cu- and 177Lu-labelled internalising monoclonal antibody (mAb) chCE7 was effective in the treatment of human ovarian cancer xenografts. In this study, we directly compared the therapeutic efficacy of anti-L1CAM RIT against human ovarian cancer under equitoxic conditions with the radiolanthanide 177Lu and the potential alternative 161Tb in an ovarian cancer therapy model. Methods: Tb was produced by neutron bombardment of enriched 160Gd targets. 161Tb and 177Lu were used for radiolabelling of DOTA-conjugated antibodies. The in vivo behaviour of the radioimmunoconjugates (RICs) was assessed in IGROV1 tumour-bearing nude mice using biodistribution experiments and SPECT/CT imaging. After ascertaining the maximal tolerated doses (MTD) the therapeutic impact of 50% MTD of 177Lu- and 161Tb-DOTA-chCE7 was evaluated in groups of ten mice by monitoring the tumour size of subcutaneous IGROV1 tumours. Results: The average number of DOTA ligands per antibody was 2.5 and maximum specific activities of 600MBq/mg were achieved under identical radiolabelling conditions. RICs were stable in human plasma for at least 48h. 177Lu- and 161Tb-DOTA-chCE7 showed high tumour uptake (37.8-39.0 %IA/g, 144h p.i.) with low levels in off-target organs. SPECT/CT images confirmed the biodistribution data. 161Tb-labelled chCE7 revealed a higher radiotoxicity in nude mice (MTD: 10MBq) than the 177Lu-labelled counterpart (MTD: 12MBq). In a comparative therapy study with equitoxic doses, tumour growth inhibition was better by 82.6% for the 161Tb-DOTA-chCE7 than the 177Lu-DOTA-chCE7 RIT. Conclusions: Our study is the first to show that anti-L1CAM 161Tb RIT is more effective compared to 177Lu RIT in ovarian cancer xenografts. These results suggest that 161Tb is a promising candidate for future clinical applications in combination with internalising antibodies

Paclitaxel improved anti-L1CAM lutetium-177 radioimmunotherapy in an ovarian cancer xenograft model

Background Today's standard treatment of advanced-stage ovarian cancer, including surgery followed by a paclitaxel-platinum-based chemotherapy, is limited in efficacy. Recently, we could show that radioimmunotherapy (RIT) with 177Lu-labelled anti-L1 cell adhesion molecule (L1CAM) monoclonal antibody chCE7 is effective in ovarian cancer therapy. We investigated if the efficacy of anti-L1CAM RIT can be further improved by its combination with paclitaxel (PTX). Methods In vitro cell viability and cell cycle arrest of human ovarian cancer cells were assessed upon different treatment conditions. For therapy studies, nude mice (n = 8) were injected subcutaneously with IGROV1 human ovarian carcinoma cells and received a single dose of 6 MBq 177Lu-DOTA-chCE7 alone or in combination with 600 μg PTX (31.6 mg/kg). Tumour growth delay and survival were determined. To investigate whether PTX can influence the tumour uptake of the radioimmunoconjugates (RICs), a biodistribution study (n = 4) and SPECT/CT images were acquired 120 h post injections of 2 MBq 177Lu-DOTA-chCE7 alone or in combination with 600 μg PTX. Results Lu-DOTA-chCE7 in combination with PTX revealed a significantly decreased cell viability of ovarian carcinoma cells in vitro and was effective in a synergistic manner (combination index < 1). PTX increased the RIT efficacy by arresting cells in the radiosensitive G2/M phase of the cell cycle 24 h post treatment start. In vivo combination therapy including 177Lu-DOTA-chCE7 and PTX resulted in a significantly prolonged overall survival (55 days vs. 18 days/PTX and 29 days/RIT), without weight loss and/or signs of toxicity. Biodistribution studies revealed no significant difference in tumour uptakes of 177Lu-DOTA-chCE7 72 h post injection regardless of an additional PTX administration. Conclusions Combination of anti-L1CAM 177Lu-RIT with PTX is a more effective therapy resulting in a prolonged overall survival of human ovarian carcinoma-bearing nude mice compared with either monotherapy. The combination is promising for future clinical applications.ISSN:2191-219

Folate Receptor-Positive Gynecological Cancer Cells: In Vitro and In Vivo Characterization

The folate receptor (FR) is expressed in a variety of gynecological cancer types. It has been widely used for tumor targeting with folic acid conjugates of diagnostic and therapeutic probes. The cervical KB tumor cells have evolved as the standard model for preclinical investigations of folate-based (radio) conjugates. In this study, a panel of FR-expressing human cancer cell lines—including cervical (HeLa, KB, KB-V1), ovarian (IGROV-1, SKOV-3, SKOV-3.ip), choriocarcinoma (JAR, BeWo) and endometrial (EFE-184) tumor cells—was investigated in vitro and for their ability to grow as xenografts in mice. FR-expression levels were compared in vitro and in vivo and the cell lines were characterized by determination of the sensitivity towards commonly-used chemotherapeutics and the expression of two additional, relevant tumor markers, HER2 and L1-CAM. It was found that, besides KB cells, its multiresistant KB-V1 subclone as well as the ovarian cancer cell lines, IGROV-1 and SKOV-3.ip, could be used as potentially more relevant preclinical models. They would allow addressing specific questions such as the therapeutic efficacy of FR-targeting agents in tumor (mouse) models of multi-resistance and in mouse models of metastases formation

Combining Albumin-Binding Properties and Interaction with Pemetrexed to Improve the Tissue Distribution of Radiofolates

Folic-acid-based radioconjugates have been developed for nuclear imaging of folate receptor (FR)-positive tumors; however, high renal uptake was unfavorable in view of a therapeutic application. Previously, it was shown that pre-injection of pemetrexed (PMX) increased the tumor-to-kidney ratio of radiofolates several-fold. In this study, PMX was combined with the currently best performing radiofolate ([177Lu]cm13), which is outfitted with an albumin-binding entity. Biodistribution studies were carried out in mice bearing KB or IGROV-1 tumor xenografts, both FR-positive tumor types. SPECT/CT was performed with control mice injected with [177Lu]folate only and with mice that received PMX in addition. Control mice showed high uptake of radioactivity in KB and IGROV-1 tumor xenografts, but retention in the kidneys was also high, resulting in tumor-to-kidney ratios of ~0.85 (4 h p.i.) and ~0.60 (24 h p.i.) or ~1.17 (4 h p.i.) and ~1.11 (24 h p.i.) respectively. Pre-injection of PMX improved the tumor-to-kidney ratio to values of ~1.13 (4 h p.i.) and ~0.92 (24 h p.i.) or ~1.79 (4 h p.i.) and ~1.59 (24 h p.i.), respectively, due to reduced uptake in the kidneys. It was found that a second injection of PMX—3 h or 7 h after administration of the radiofolate—improved the tumor-to-kidney ratio further to ~1.03 and ~0.99 or ~1.78 and ~1.62 at 24 h p.i. in KB and IGROV-1 tumor-bearing mice, respectively. SPECT/CT scans readily visualized the tumor xenografts, whereas accumulation of radioactivity in the kidneys was reduced in mice that received PMX. In this study, it was shown that PMX had a positive impact in terms of reducing the kidney uptake of albumin-binding radiofolates; hence, the administration of PMX resulted in ~1.3–1.7-fold higher tumor-to-kidney ratios. This is, however, a rather moderate effect in comparison to the previously shown effect of PMX on conventional radiofolates (without albumin binder), which led to 5–6-fold increased tumor-to-kidney ratios. An explanation for this result may be the different pharmacokinetic profiles of PMX and long-circulating radiofolates, respectively. Despite the promising potential of this concept, it is believed that a clinical translation would be challenging, particularly when PMX had to be injected more than once.ISSN:1420-304

18F-AzaFol for Detection of Folate Receptor-β Positive Macrophages in Experimental Interstitial Lung Disease-A Proof-of-Concept Study

Background: Interstitial lung disease (ILD) is a common and severe complication in rheumatic diseases. Folate receptor-β is expressed on activated, but not resting macrophages which play a key role in dysregulated tissue repair including ILD. We therefore aimed to pre-clinically evaluate the potential of $^{18}$F-AzaFol-based PET/CT (positron emission computed tomography/computed tomography) for the specific detection of macrophage-driven pathophysiologic processes in experimental ILD. Methods: The pulmonary expression of folate receptor-β was analyzed in patients with different subtypes of ILD as well as in bleomycin (BLM)-treated mice and respective controls using immunohistochemistry. PET/CT was performed at days 3, 7, and 14 after BLM instillation using the $^{18}$F-based folate radiotracer $^{18}$F-AzaFol. The specific pulmonary accumulation of the radiotracer was assessed by ex vivo PET/CT scans and quantified by ex vivo biodistribution studies. Results: Folate receptor-β expression was 3- to 4-fold increased in patients with fibrotic ILD, including idiopathic pulmonary fibrosis and connective tissue disease-related ILD, and significantly correlated with the degree of lung remodeling. A similar increase in the expression of folate receptor-β was observed in experimental lung fibrosis, where it also correlated with disease extent. In the mouse model of BLM-induced ILD, pulmonary accumulation of $^{18}$F-AzaFol reflected macrophage-related disease development with good correlation of folate receptor-β positivity with radiotracer uptake. In the ex vivo imaging and biodistribution studies, the maximum lung accumulation was observed at day 7 with a mean accumulation of 1.01 ± 0.30% injected activity/lung in BLM-treated vs. control animals (0.31 ± 0.06% % injected activity/lung; p < 0.01). Conclusion: Our preclinical proof-of-concept study demonstrated the potential of $^{18}$F-AzaFol as a novel imaging tool for the visualization of macrophage-driven fibrotic lung diseases

Promising potential of [Lu-177]Lu-DOTA-folate to enhance tumor response to immunotherapy-a preclinical study using a syngeneic breast cancer model

Purpose It was previously demonstrated that radiation effects can enhance the therapy outcome of immune checkpoint inhibitors. In this study, a syngeneic breast tumor mouse model was used to investigate the effect of [Lu-177]Lu-DOTA-folate as an immune stimulus to enhance anti-CTLA-4 immunotherapy. Methods In vitro and in vivo studies were performed to characterize NF9006 breast tumor cells with regard to folate receptor (FR) expression and the possibility of tumor targeting using [Lu-177]Lu-DOTA-folate. A preclinical therapy study was performed over 70 days with NF9006 tumor-bearing mice that received vehicle only (group A); [Lu-177]Lu-DOTA-folate (5 MBq; 3.5 Gy absorbed tumor dose; group B); anti-CTLA-4 antibody (3 x 200 mu g; group C), or both agents (group D). The mice were monitored regarding tumor growth over time and signs indicating adverse events of the treatment. Results [Lu-177]Lu-DOTA-folate bound specifically to NF9006 tumor cells and tissue in vitro and accumulated in NF9006 tumors in vivo. The treatment with [Lu-177]Lu-DOTA-folate or an anti-CTLA-4 antibody had only a minor effect on NF9006 tumor growth and did not substantially increase the median survival time of mice (23 day and 19 days, respectively) as compared with untreated controls (12 days). [Lu-177]Lu-DOTA-folate sensitized, however, the tumors to anti-CTLA-4 immunotherapy, which became obvious by reduced tumor growth and, hence, a significantly improved median survival time of mice (> 70 days). No obvious signs of adverse effects were observed in treated mice as compared with untreated controls. Conclusion Application of [Lu-177]Lu-DOTA-folate had a positive effect on the therapy outcome of anti-CTLA-4 immunotherapy. The results of this study may open new perspectives for future clinical translation of folate radioconjugates.ISSN:1619-7070ISSN:1619-708

Downregulation of Varicella-Zoster Virus (VZV) Immediate-Early ORF62 Transcription by VZV ORF63 Correlates with Virus Replication In Vitro and with Latency

Varicella-zoster virus (VZV) open reading frame 63 (ORF63) protein is expressed during latency in human sensory ganglia. Deletion of ORF63 impairs virus replication in cell culture and establishment of latency in cotton rats. We found that cells infected with a VZV ORF63 deletion mutant yielded low titers of cell-free virus and produced very few enveloped virions detectable by electron microscopy compared with those infected with parental virus. Microarray analysis of cells infected with a recombinant adenovirus expressing ORF63 showed that transcription of few human genes was affected by ORF63; a heat shock 70-kDa protein gene was downregulated, and several histone genes were upregulated. In experiments using VZV transcription arrays, deletion of ORF63 from VZV resulted in a fourfold increase in expression of ORF62, the major viral transcriptional activator. A threefold increase in ORF62 protein was observed in cells infected with the ORF63 deletion mutant compared with those infected with parental virus. Cells infected with ORF63 mutants impaired for replication and latency (J. I. Cohen, T. Krogmann, S. Bontems, C. Sadzot-Delvaux, and L. Pesnicak, J. Virol. 79:5069-5077, 2005) showed an increase in ORF62 transcription compared with those infected with parental virus. In contrast, cells infected with an ORF63 mutant that is not impaired for replication or latency showed ORF62 RNA levels equivalent to those in cells infected with parental virus. The ability of ORF63 to downregulate ORF62 transcription may play an important role in virus replication and latency

Anti-L1CAM radioimmunotherapy is more effective with the radiolanthanide terbium-161 compared to lutetium-177 in an ovarian cancer model

Purpose The L1 cell adhesion molecule (L1CAM) is considered a valuable target for therapeutic intervention in different types of cancer. Recent studies have shown that anti-L1CAM radioimmunotherapy (RIT) with 67Cu- and 177Lu-labelled internalising monoclonal antibody (mAb) chCE7 was effective in the treatment of human ovarian cancer xenografts. In this study, we directly compared the therapeutic efficacy of anti-L1CAM RIT against human ovarian cancer under equitoxic conditions with the radiolanthanide 177Lu and the potential alternative 161Tb in an ovarian cancer therapy model. Methods Tb was produced by neutron bombardment of enriched 160Gd targets. 161Tb and 177Lu were used for radiolabelling of DOTA-conjugated antibodies. The in vivo behaviour of the radioimmunoconjugates (RICs) was assessed in IGROV1 tumour-bearing nude mice using biodistribution experiments and SPECT/CT imaging. After ascertaining the maximal tolerated doses (MTD) the therapeutic impact of 50 % MTD of 177Lu- and 161Tb-DOTA-chCE7 was evaluated in groups of ten mice by monitoring the tumour size of subcutaneous IGROV1 tumours. Results The average number of DOTA ligands per antibody was 2.5 and maximum specific activities of 600 MBq/mg were achieved under identical radiolabelling conditions. RICs were stable in human plasma for at least 48 h. 177Lu- and 161Tb-DOTA-chCE7 showed high tumour uptake (37.8–39.0 %IA/g, 144 h p.i.) with low levels in off-target organs. SPECT/CT images confirmed the biodistribution data. 161Tb-labelled chCE7 revealed a higher radiotoxicity in nude mice (MTD: 10 MBq) than the 177Lu-labelled counterpart (MTD: 12 MBq). In a comparative therapy study with equitoxic doses, tumour growth inhibition was better by 82.6 % for the 161Tb-DOTA-chCE7 than the 177Lu-DOTA-chCE7 RIT. Conclusions Our study is the first to show that anti-L1CAM 161Tb RIT is more effective compared to 177Lu RIT in ovarian cancer xenografts. These results suggest that 161Tb is a promising candidate for future clinical applications in combination with internalising antibodies.ISSN:1619-7070ISSN:1619-708

Combination of lutetium-177 labelled anti-L1CAM antibody chCE7 with the clinically relevant protein kinase inhibitor MK1775: a novel combination against human ovarian carcinoma

Abstract Background Protein kinase inhibitors (PKIs) are currently tested in clinical studies (phase I-III) as an alternative strategy against (recurrent) ovarian cancer. Besides their anti-tumour efficacy, several PKIs have also shown radiosensitizing effects when combined with external beam radiation. Based on these results we asked if the addition of PKIs offers a therapeutic opportunity to improve radioimmunotherapy (RIT) against ovarian cancer. Five PKIs (alisertib, MK1775, MK2206, saracatinib, temsirolimus) were chosen for cytotoxicity screenings based on their current clinical trials in the treatment of ovarian cancer and their influence on cell cycle regulation and DNA damage repair pathways. We combined selected PKIs with 177Lu-labelled anti-L1CAM monoclonal antibody chCE7 for our investigations. Methods PKIs cytotoxicity was determined via cell colony-forming assays. Biomarker of DNA double-strand breaks (DSBs, γH2A.X) was analysed by western blot and fluorescence microscopy. Flow cytometric measurements were performed to evaluate levels of apoptosis based on mono- or combination treatments. The best combination was used for in vivo combination therapy studies in nude mice with SKOV3ip and IGROV1 human ovarian cancer xenografts. Bonferroni correction was used to determine statistical significance for multiple comparisons. Results The highest cytotoxicity against both cell lines was observed for MK1775 and alisertib. Combinations including 177Lu-labelled mAb chCE7 and MK1775 decreased 177Lu-DOTA-chCE7 IC60-values 14-fold, compared to 6-fold, when the radioimmunoconjugate was combined with alisertib. The most effective PKI MK1775 was further evaluated and demonstrated synergistic effects in combination with 177Lu-DOTA-chCE7 against IGROV1 cells. Significantly higher amounts of DSBs were detected in IGROV1 cells after combination (91%) compared to either treatment alone (MK1775: 52%; radioimmunoconjugate: 72%; p < 0.0125). Early-apoptosis was significantly enhanced in IGROV1 cells correlating with induced DSBs (177Lu-DOTA-chCE7: 8%, MK1775: 28%, 177Lu-DOTA-chCE7 + MK1775: 40%, p < 0.0125). Immunohistochemistry analysis of γH2A.X expression levels after therapy in SKOV3ip xenografts revealed a high sensitivity of the tumour cells to MK1775 and a high radioresistance. A prominent effect of tumour growth inhibition of the RIT and of the combination therapy was observed in vivo in a late stage IGROV1 xenograft model. Conclusions Our results warrant further evaluation of combination of MK1775 and radioimmunotherapy