Cancer is rapidly becoming the most frequent cause of morbidity and mortality in the EU, accounting for a quarter of all deaths in EU. Without breakthroughs in treatment, cancer is likely to remain one of the biggest killers in the 21st century. Immunotherapy of cancer by checkpoint inhibitors, vaccines or adoptive T cell therapy is coming of age and has the potential to cure cancer, but is still hampered by some major limitations. For instance, Adoptive Cell Therapy (ACT) with unmanipulated or engineered T cells (TCR-transgenic and CAR-T cells) has indeed demonstrated success in the treatment of patients affected by leukemias, but is much less effective against lymphomas and solid tumors. One likely explanation is that we do not educate the right type of anti-tumor T cells. The T cells considered to be the gold standard for tumor therapy have stem cell memory features, but the proper and safe way to generate these fit T cells for clinical purposes is still an unresolved matter. Here we propose an advanced transformative technology termed INCITE, utilizing a novel high-resolution 3D microfabrication technology to engineer a specially tailored microenvironment that will be inhabited by cells central for T cells education in order to generate the fittest anti-tumor T cells for advanced adoptive T cell therapy. INCITE will bring together a transdisciplinary consortium capable of developing this innovative platform by combining state-of-the-art 3D printing, computer modeling, bioengineering, bioinformatics, immunology, developmental and cancer biology approaches, toward the development of a functional immune niche for selection and expansion of tumor-rejecting T cells. The INCITE platform will revolutionize the treatment of cancer patients with ACT, with a profound impact on the quality of life and well-being of millions of people.

A Nanovaccine Approach For The Treatment of Pancreatic Cancer By Multicomponent Immuno-Modulation: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in men and women and still fatal in over 90% of patients. It is characterised by its extremely aggressive nature where it is also responsible for the highest mortality rate compared to other major cancers, resulting in excess of 250,000 deaths worldwide per annum. Current state-of-art therapies for advanced PDAC including chemo- and/or radiotherapy, despite extensive efforts, have met with only limited success. Surgery is only applicable for those with early stages of the disease, or to relieve symptoms, if the cancer is blocking the bile duct or the bowel. There are two major reasons for the resistance of PDAC to conventional therapy. Firstly, PDAC has a very defining hallmark, where an abundance of stromal content is present in the tumour microenvironment (TME) to form a physical and biochemical barrier. Secondly, during progression of the disease, the body's immune system is hijacked to support the proliferation of the cancer. New approaches, such as immunotherapy, are therefore needed where it has already shown promise in overcoming many aspects of this resistance. Immunotherapy has the potential to treat minimal residual disease after pancreatic resection (surgery) as well as for metastatic and non-resectable PDAC. Our objective for this project is to bring together a multidisciplinary and intersectoral group to develop novel vaccine approaches, including use of multiple immunomodulating components.

Although immunotherapy of select hematological malignancies using Chimeric Antigen Receptor (CAR) redirected T lymphocytes has recently gained regulatory approval, successful treatment of solid tumors using CAR T cells remains elusive. One salient problem is the limited efficacy and untimely exhaustion of CAR T cells in the tumor microenvironment (TME). Combining innovative methods of genome editing, chemistry and immunology, CAR T-REX proposes to explore a novel concept of building auto-regulated genetic circuits into CAR T cells to selectively circumvent their exhaustion upon activation in the TME. Genetic rewiring will be achieved by precisely inserting artificial miRNAs under endogenous exhaustion-related “Driver” promoters to downregulate “Target” genes that cause exhaustion. Proprietary technology enables specific replacement of the “Driver” gene without risking off-target mutations. Further advantages of combined insertion and silencing are (i) the ability to regulate when a gene is turned on/off by biologically and clinically relevant cellular cues, and (ii) multiple gene-knockdown with a single dsDNA cleavage and RNA-silencing of both alleles. These genetic modifications will be implemented using a novel high-performance peptide-based gene delivery platform with unlimited loading capacity, allowing combination of several types of cargo, as well as economical large scale GMP production. Rewired HER2/Neu (ErbB2) redirected CAR T cells will be tested on preclinical breast and gastric carcinomas, and variants that eliminate tumors resistant to conventional 2nd and 3rd generation peers (without adverse events) will be developed/manufactured following quality-by-design principles under GMP-like conditions, thus accelerating the pathway towards clinical translation. These approaches will also constitute a proof-of-concept for modifying therapeutic cell products, with the potential to considerably improve their safety, specificity, efficacy, scalability and cost.

Cell and gene therapies offer a massive paradigm shift from current treatment options and hold the potential to cure previously untreatable diseases. Naturally-occurring and genetically modified T cells with chimeric antigen (CAR) or T cell receptors (TCR) have demonstrated remarkable curative capacities against advanced hematologic malignancies but have shown limited efficacy in treating solid tumors. Major barriers hindering the full antitumor potential of T cells are the immunosuppressive signals and persisting antigenic stimuli within the tumor microenvironment that inexorably push T cells into a highly dysfunctional state called “exhaustion”. Herein, we propose a groundbreaking technology, T-FITNESS, which will enable antitumor T cells to become refractory to exhaustion. At the core of the platform are microRNA (miRNA)-based synthetic logic circuits capable of rewiring the transcriptional networks orchestrating T cell exhaustion. By harnessing the power of CRISPR/Cas genome editing, we will integrate sensors of miRNAs upregulated in exhausted cells into untranslated regions of one or more transcription factors driving T cell exhaustion, to enable their fine-tuned downregulation. We will validate the reprogramming efficacy of T-FITNESS by performing extensive functional analyses in vitro and in vivo and advance the best circuits towards the clinic by developing an automated cGMP-compliant manufacturing process for point-of-care production of T-FITNESS-edited CAR-T cells. To develop this innovative platform, we will bring together a multidisciplinary consortium of academic and industry partners that combine their unique expertise in T cell therapy and immunology, synthetic biology, genome editing, cGMP manufacturing, bioinformatics, and communication. Easily integrable within CAR-T, TCR-T, and tumor-infiltrating lymphocyte (TIL) platforms, T-FITNESS will unleash the curative potential of T cell therapy for the benefit of an ever-growing number of cancer patients.

Cancer immunotherapy based on chimeric antigen receptor (CAR)-engineered T-cells represents a highly efficient therapy and a paradigm shift as living drugs. This platform allows further improvement of therapeutic cells and circuits that guide them. T-FITNESS project addressed the problem of T cell exhaustion aiming to enable antitumor T cells to become refractory to exhaustion. T-FITNESS proposed an innovative sensing of exhaustion-specific miRNAs (ex-miRNA) and transcription factors (exTF) to introduce downregulation of exTF by CRISPR-mediated genomic integration of miRNA target sites and also included the development of a cGMP-compliant manufacturing process. T-FITNESS-HOP-ON aims to achieve the same goal by introducing additional strategies based on protein design and synthetic biology tools. Here inhibitory TFs will be designed to prevent T cell exhaustion where they will be regulated either by external signals or through an autonomous ex-miRNA regulation. An innovative, single-transcript sensing of ex-miRNA and TFinh upregulation will be implemented. Additionally, CAR constructs will be designed to enable the rest of T-cells upon the onset of exhaustion and tested in T-cells and in a preclinical model. The proposed designs have a small genetic footprint and will not require genome editing, which could enable robust clinical translation and could be useful as a technological platform for therapy of other types of disease. The introduction of an additional widening partner, who brings new expertise and ideas will be beneficial both for the existing consortium as well as for the new partner, to improve dissemination, visibility, scientific excellence, facilitate clinical and industrial translation, strengthen the links with international partners and improve career prospects.