Artificial Targets: a versatile cell-free platform to characterize CAR T cell function in vitro.

Cancer immunotherapies using chimeric antigen receptor (CAR) T cells have tremendous potential and proven clinical efficacy against a number of malignancies. Research and development are emerging to deepen the knowledge of CAR T cell efficacy and extend the therapeutic potential of this novel therapy. To this end, functional characterization of CAR T cells plays a central role in consecutive phases across fundamental research and therapeutic development, with increasing needs for standardization. The functional characterization of CAR T cells is typically achieved by assessing critical effector functions, following co-culture with cell lines expressing the target antigen. However, the use of target cell lines poses several limitations, including alterations in cell fitness, metabolic state or genetic drift due to handling and culturing of the cells, which would increase variabilities and could lead to inconsistent results. Moreover, the use of target cell lines can be work and time intensive, and introduce significant background due to the allogenic responses of T cells. To overcome these limitations, we developed a synthetic bead-based platform ("Artificial Targets") to characterize CAR T cell function in vitro. These synthetic microparticles could specifically induce CAR T cell activation, as measured by CD69 and CD137 (4-1BB) upregulation. In addition, engagement with Artificial Targets resulted in induction of multiple effector functions of CAR T cells mimicking the response triggered by target cell lines including cytotoxic activity, as assessed by exposure of CD107a (LAMP-1), expression and secretion of cytokines, as well as cell proliferation. Importantly, in contrast to target cells, stimulation with Artificial Targets showed limited unspecific CAR T cell proliferation. Finally, Artificial Targets demonstrated flexibility to engage multiple costimulatory molecules that can synergistically enhance the CAR T cell function and represented a powerful tool for modulating CAR T cell responses. Collectively, our results show that Artificial Targets can specifically activate CAR T cells for essential effector functions that could significantly advance standardization of functional assessment of CAR T cells, from early development to clinical applications.

Aptamers: novelty tools for cancer biology.

Although the term 'cancer' was still over two thousand years away of being coined, the first known cases of the disease date back to about 3000BC, in ancient Egypt. Five thousand years later, still lacking a cure, it has become one of the leading causes of death, killing over half a dozen million people yearly. So far, monoclonal antibodies are the most successful immune-therapy tools when it comes to fighting cancer. The number of clinical trials that use them has been increasing steadily during the past few years, especially since the Food and Drug Administration greenlit the use of the first immune-checkpoint blockade antibodies. However, albeit successful, this approach does come with the cost of auto-inflammatory toxicity. Taking this into account, the development of new therapeutic reagents with low toxicity becomes evident, particularly ones acting in tandem with the tools currently at our disposal. Ever since its discovery in the early nineties, aptamer technology has been used for a wide range of diagnostic and therapeutic applications. With similar properties to those of monoclonal antibodies, such as high-specificity of recognition and high-affinity binding, and the advantages of being developed using in vitro selection procedures, aptamers quickly became convenient building blocks for the generation of multifunctional constructs. In this review, we discuss the steps involved in the in vitro selection process that leads to functional aptamers - known as Systematic Evolution of Ligands by Exponential Enrichment - as well as the most recent applications of this technology in diagnostic and treatment of oncological illnesses. Moreover, we also suggest ways to improve such use.