Design of an optimized Wilms' tumor 1 (WT1) mRNA construct for enhanced WT1 expression and improved immunogenicity in vitro and in vivo

Tumor antigen-encoding mRNA for dendritic cell (DC)-based vaccination has gained increasing popularity in recent years. Within this context, two main strategies have entered the clinical trial stage: the use of mRNA for ex vivo antigen loading of DCs and the direct application of mRNA as a source of antigen for DCs in vivo. DCs transfected with mRNA-encoding Wilms' tumor 1 (WT1) protein have shown promising clinical results. Using a stepwise approach, we re-engineered a WT1 cDNA-carrying transcription vector to improve the translational characteristics and immunogenicity of the transcribed mRNA. Different modifications were performed: (i) the WT1 sequence was flanked by the lysosomal targeting sequence of dendritic cell lysosomal-associated membrane protein to enhance cytoplasmic expression; (ii) the nuclear localization sequence (NLS) of WT1 was deleted to promote shuttling from the nucleus to the cytoplasm; (iii) the WT1 DNA sequence was optimized in silico to improve translational efficiency; and (iv) this WT1 sequence was cloned into an optimized RNA transcription vector. DCs electroporated with this optimized mRNA showed an improved ability to stimulate WT1-specific T-cell immunity. Furthermore, in a murine model, we were able to show the safety, immunogenicity, and therapeutic activity of this optimized mRNA. This work is relevant for the future development of improved mRNA-based vaccine strategies K

PRIMMO study protocol : a phase II study combining PD-1 blockade, radiation and immunomodulation to tackle cervical and uterine cancer

Background: Immunotherapeutic approaches have revolutionized oncological practice but are less evaluated in gynecological malignancies. PD-1/PD-L1 blockade in gynecological cancers showed objective responses in 13-17% of patients. This could be due to immunosuppressive effects exerted by gynecological tumors on the microenvironment and an altered tumor vasculature.In other malignancies, combining checkpoint blockade with radiation delivers benefit that is believed to be due to the abscopal effect. Addition of immune modulation agents has also shown to enhance immune checkpoint blockade efficacy. Therefore we designed a regimen consisting of PD-1 blockade combined with radiation, and different immune/environmental-targeting compounds: repurposed drugs, metronomic chemotherapy and a food supplement.We hypothesize that these will synergistically modulate the tumor microenvironment and induce and sustain an anti-tumor immune response, resulting in tumor regression. Methods: PRIMMO is a multi-center, open-label, non-randomized, 3-cohort phase 2 study with safety run-in in patients with recurrent/refractory cervical carcinoma, endometrial carcinoma or uterine sarcoma.Treatment consists of daily intake of vitamin D, lansoprazole, aspirin, cyclophosphamide and curcumin, starting 2weeks before the first pembrolizumab dose. Pembrolizumab is administered 3-weekly for a total of 6cycles. Radiation (3x8Gy) is given on days 1, 3 and 5 of the first pembrolizumab dose.The safety run-in consists of 6 patients. In total, 18 and 25 evaluable patients for cervical and endometrial carcinoma respectively are foreseen to enroll. No sample size is determined for uterine sarcoma due to its rarity.The primary objective is objective response rate at week 26 according to immune-related response criteria.Secondary objectives include safety, objective response rate at week 26 according to RECIST v1.1, best overall response, progression-free survival, overall survival and quality of life.Exploratory, translational research aims to evaluate immune biomarkers, extracellular vesicles, cell death biomarkers and the gut microbiome. Discussion: In this study, a combination of PD-1 blockade, radiation and immune/environmental-targeting compounds is tested, aiming to tackle the tumor microenvironment and induce anti-tumor immunity. Translational research is performed to discover biomarkers related to the mode of action of the combination. Trial registration: EU Clinical Trials Register: EudraCT 2016-001569-97, registered on 19-6-2017. Clinicaltrials.gov: NCT03192059, registered on 19-6-2017

Editorial: Approaches to Advance Cancer Vaccines to Clinical Utility

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A Phase 2 Study to Assess the Immunomodulatory Capacity of a Lecithin-based Delivery System of Curcumin in Endometrial Cancer

Curcumin is a botanical with anti-tumor and immunomodulatory properties. We hypothesized that curcumin supplementation might influence inflammatory biomarker levels in endometrial carcinoma (EC). In this open-label, non-randomized phase 2 study (NCT02017353), seven EC patients consumed 2 g/day Curcumin Phytosome (CP) orally for 2 weeks. Blood was taken at baseline, days 1, 7, 14, and 21. The following analytes were measured: curcuminoids and metabolites, 56 inflammatory biomarkers, COX-2, frequencies of myeloid-derived suppressor cells, dendritic cells and NK cells, expression of MHC molecules on leukocytes and monocytes and activation/memory status of T cells. Patients completed quality of life (QoL) questionnaires at baseline and end of treatment. Curcumin metabolites were detectable in plasma upon CP intake. CP downregulated MHC expression levels on leukocytes (P = 0.0313), the frequency of monocytes (P = 0.0114) and ICOS expression by CD8+ T cells (P = 0.0002). However, CP upregulated CD69 levels on CD16- NK cells (P = 0.0313). No differences were observed regarding inflammatory biomarkers, frequencies of other immune cell types, T cell activation and COX-2 expression. A non-significant trend to improved QoL was observed. Overall, CP-induced immunomodulatory effects in EC were modest without significant QoL changes. Given the small population and the observed variability in inter-patient biomarker levels, more research is necessary to explore whether benefits of CP can be obtained in EC by different supplementation regimens. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT02017353; www.clinicaltrialsregister.eu, identifier 2013-001737-40.status: publishe

Repurposing Drugs in Oncology (ReDO)—clarithromycin as an anti-cancer agent

Clarithromycin (CAM) is a well-known macrolide antibiotic available as a generic drug. CAM is traditionally used for many types of bacterial infections, treatment of Lyme disease and eradication of gastric infection with Helicobacter pylori. Extensive preclinical and clinical data demonstrate a potential role for CAM to treat various tumours in combination with conventional treatment. The mechanisms of action underlying the anti-tumour activity of CAM are multiple and include prolonged reduction of pro-inflammatory cytokines, autophagy inhibition, and anti-angiogenesis. Here, we present an overview of the current preclinical (in vitro and in vivo) and clinical evidence supporting the role of CAM in cancer. Overall these findings justify further research with CAM in many tumour types, with multiple myeloma, lymphoma, chronic myeloid leukaemia (CML), and lung cancer having the highest level of evidence. Finally, a series of proposals are being made to further investigate the use of CAM in clinical trials which offer the greatest prospect of clinical benefit to patients

Priming of cytotoxic T lymphocyte responses by dendritic cells: induction of potent anti-tumor immune responses

It is well established that CD8+ cytotoxic T lymphocytes (CTLs) play a major role in eradicating tumor cells. The path from naive CD8+ T cell to effector CD8+ T cell is guided by antigen presenting cells, such as dendritic cells (DCs) that start a developmental program in the CD8+ T cells through the delivery of MHC/peptide complexes, co-stimulatory and pro-inflammatory signals. These signals determine the magnitude of the ensuing response and the ability of the CD8+ CTLs to successfully complete their quest, eradicating tumor cells whilst conquering enemies, such as immunosuppressive regulatory T cells (Tregs). In this chapter, we discuss the role of DCs and of the signals critical for effector CD8+ T cell differentiation, and how the differences in the nature of these signals contribute to the diversity of CD8+ T cell responses

ReDO_DB: the repurposing drugs in oncology database

Repurposing is a drug development strategy that seeks to use existing medications for new indications. In oncology, there is an increased level of activity looking at the use of non-cancer drugs as possible cancer treatments. The Repurposing Drugs in Oncology (ReDO) project has used a literature-based approach to identify licensed non-cancer drugs with published evidence of anticancer activity. Data from 268 drugs have been included in a database (ReDO_DB) developed by the ReDO project. Summary results are outlined and an assessment of clinical trial activity also described. The database has been made available as an online open-access resource (http://www.redo-project.org/db/).status: publishe

PRIMMO study protocol: a phase II study combining PD-1 blockade, radiation and immunomodulation to tackle cervical and uterine cancer

BACKGROUND: Immunotherapeutic approaches have revolutionized oncological practice but are less evaluated in gynecological malignancies. PD-1/PD-L1 blockade in gynecological cancers showed objective responses in 13-17% of patients. This could be due to immunosuppressive effects exerted by gynecological tumors on the microenvironment and an altered tumor vasculature. In other malignancies, combining checkpoint blockade with radiation delivers benefit that is believed to be due to the abscopal effect. Addition of immune modulation agents has also shown to enhance immune checkpoint blockade efficacy. Therefore we designed a regimen consisting of PD-1 blockade combined with radiation, and different immune/environmental-targeting compounds: repurposed drugs, metronomic chemotherapy and a food supplement. We hypothesize that these will synergistically modulate the tumor microenvironment and induce and sustain an anti-tumor immune response, resulting in tumor regression. METHODS: PRIMMO is a multi-center, open-label, non-randomized, 3-cohort phase 2 study with safety run-in in patients with recurrent/refractory cervical carcinoma, endometrial carcinoma or uterine sarcoma. Treatment consists of daily intake of vitamin D, lansoprazole, aspirin, cyclophosphamide and curcumin, starting 2 weeks before the first pembrolizumab dose. Pembrolizumab is administered 3-weekly for a total of 6 cycles. Radiation (3 × 8 Gy) is given on days 1, 3 and 5 of the first pembrolizumab dose. The safety run-in consists of 6 patients. In total, 18 and 25 evaluable patients for cervical and endometrial carcinoma respectively are foreseen to enroll. No sample size is determined for uterine sarcoma due to its rarity. The primary objective is objective response rate at week 26 according to immune-related response criteria. Secondary objectives include safety, objective response rate at week 26 according to RECIST v1.1, best overall response, progression-free survival, overall survival and quality of life. Exploratory, translational research aims to evaluate immune biomarkers, extracellular vesicles, cell death biomarkers and the gut microbiome. DISCUSSION: In this study, a combination of PD-1 blockade, radiation and immune/environmental-targeting compounds is tested, aiming to tackle the tumor microenvironment and induce anti-tumor immunity. Translational research is performed to discover biomarkers related to the mode of action of the combination. TRIAL REGISTRATION: EU Clinical Trials Register: EudraCT 2016-001569-97 , registered on 19-6-2017. Clinicaltrials.gov: NCT03192059 , registered on 19-6-2017.status: Published onlin