T Cell Reprogramming Against Cancer.

Advances in academic and clinical studies during the last several years have resulted in practical outcomes in adoptive immune therapy of cancer. Immune cells can be programmed with molecular modules that increase their therapeutic potency and specificity. It has become obvious that successful immunotherapy must take into account the full complexity of the immune system and, when possible, include the use of multifactor cell reprogramming that allows fast adjustment during the treatment. Today, practically all immune cells can be stably or transiently reprogrammed against cancer. Here, we review works related to T cell reprogramming, as the most developed field in immunotherapy. We discuss factors that determine the specific roles of αß and γδ T cells in the immune system and the structure and function of T cell receptors in relation to other structures involved in T cell target recognition and immune response. We also discuss the aspects of T cell engineering, specifically the construction of synthetic T cell receptors (synTCRs) and chimeric antigen receptors (CARs) and the use of engineered T cells in integrative multifactor therapy of cancer.

A versatile flow-based assay for immunocyte-mediated cytotoxicity.

Cell-mediated cytotoxicity is a critical function of the immune system in mounting defense against pathogens and cancers. Current methods that allow direct evaluation of cell-mediated cytotoxicity suffer from a wide-range of drawbacks. Here, we present a novel strategy to measure cytotoxicity that is direct, sensitive, rapid, and highly adaptable. Moreover, it allows accurate measurement of viability of both target and effector cells. Target cells are fluorescently labeled with a non-toxic, cell-permeable dye that covalently binds to cell proteins, including nuclear proteins. The labeled target cells are incubated with effector cells to begin killing. Following the killing reaction, the cell mixture is incubated with another dye that specifically stains proteins of dead cells, including nuclear proteins. In the final step, cell nuclei are released by Triton X-100, and analyzed by flow cytometry. This results in four nuclear staining patterns that separate target and effector nuclei as well as nuclei of live and dead cells. Analyzing nuclei, instead of cells, greatly reduces flow cytometry errors caused by the presence of target-effector cell aggregates. Target killing time can often be reduced to 2 h and the assay can be done in a high throughput format. We have successfully validated this assay in a variety of cytotoxicity scenarios including those mediated by NK-92 cells, Chimeric Antigen Receptor (CAR)-T cells, and Tumor Infiltrating Lymphocytes (TIL). Therefore, this technique is broadly applicable, highly sensitive and easily administered, making it a powerful tool to assess immunotherapy-based, cell-mediated cytotoxicity.

Cell engineering with synthetic messenger RNA.

mRNA has become an important alternative to DNA as a tool for cell reprogramming. To be expressed, exogenous DNA must be transmitted through the cell cytoplasm and placed into the nucleus. In contrast, exogenous mRNA simply has to be delivered into the cytoplasm. This can result in a highly uniform transfection of the whole population of cells, an advantage that has not been observed with DNA transfer. The use of mRNA, instead of DNA, in medical applications increases protocol safety by abolishing the risk of transgene insertion into host genomes. In this chapter, we review the aspects of mRNA structure and function that are important for its "transgenic" behavior, such as the composition of mRNA molecules and complexes with RNA binding proteins, localization of mRNA in cytoplasmic compartments, translation, and the duration of mRNA expression. In immunotherapy, mRNA is employed in reprogramming of antigen presenting cells (vaccination) and cytolytic lymphocytes. Other applications include generation of induced pluripotent stem (iPS) cells, and genome engineering with modularly assembled nucleases. The most investigated applications of mRNA technology are also reviewed here.

Chimeric receptor mRNA transfection as a tool to generate antineoplastic lymphocytes.

mRNA transfection is a useful approach for temporal cell reprogramming with minimal risk of transgene-mediated mutagenesis. We applied this to redirect lymphocyte cytotoxicity toward malignant cells. Using the chimeric immune receptor (CIR) constructs anti-CD19 CIR and 8H9 CIR, we achieved uniform expression of CIRs on virtually the entire population of lymphocytes. We reprogrammed CD3+ CD8+, CD3+ CD4+, and natural killer (NK ) cells toward autologous and allogeneic targets such as B cells, Daudi lymphoma, primary melanoma, breast ductal carcinoma, breast adenocarcinoma, and rhabdomyosarcoma. The reprogramming procedure is fast. Although most of the experiments were performed on lymphocytes obtained after 7-day activation, only 1-day activation of T cells with anti-CD3, anti-CD28 antibodies, and interleukin-2 is sufficient to develop both lymphocyte cytotoxicity and competence for mRNA transfer. The entire procedure, which includes lymphocyte activation and reprogramming, can be completed in 2 days. The efficiency of mRNA-modified human T cells was tested in a murine xenograft model. Human CD3+CD8+ lymphocytes expressing anti-CD19 CIR mRNA inhibited Daudi lymphoma growth in NOD=SCID mice. These results demonstrate that a mixed population of cytotoxic lymphocytes, including T cells together with NK cells, can be quickly and simultaneously reprogrammed by mRNA against autologous malignancies. With relatively minor modifications the described method of lymphocyte reprogramming can be scaled up for cancer therapy.

Synthetic messenger RNA as a tool for gene therapy.

Transfection of human cells with DNA in biomedical applications carries the risk of insertional mutagenesis. Transfection with mRNA avoids this problem; however, in vitro production of mRNA, based on preliminary DNA template cloning in special vectors, is a laborious and time-consuming procedure. We report an efficient vectorfree method of mRNA production from polymerase chain reaction-generated DNA templates. For all cell types tested mRNA was transfected more readily than DNA, and its expression was highly uniform in cell populations. Even cell types relatively resistant to transfection with DNA could express transfected mRNA well. The level of mRNA expression could be controlled over a wide range by changing the amount of input RNA. Cells could be efficiently and simultaneously loaded with several different transcripts. To test a potential clinical application of this method, we transfected human T lymphocytes with mRNA encoding a chimeric immune receptor directed against CD19, a surface antigen widely expressed in leukemia and lymphoma. The transfected mRNA conferred powerful cytotoxicity to T cells against CD19+ targets from the same donor. These results demonstrate that this method can be applied to generate autologous T lymphocytes directed toward malignant cells.