Engineering T-Cells Beyond Chimeric Antigen Receptor

Adoptive T cell therapy exploits the ability of T lymphocytes to recognize and destroy specific targets, on microbes or tumors, through their T cell receptors (TCR), leading to efficient killing and long-term protection against diseases. Unfortunately, tumor antigens are often overexpressed, unmodified self-antigens, subject to tolerance mechanisms; so tumor-specific T lymphocytes are rare cells. Conversely, neoantigens derive from oncogenic mutations can elicit productive T cell responses, but for tumors with a low mutational load, such as the majority of hematological malignancies, such tumor-specific T cells are rarely identified. These limitations can be overcome by genetic engineering of T lymphocyte specificity. Recently, unprecedented clinical results were obtained with chimeric antigen receptor (CAR) engineered T cells in patients affected by B-cell malignancies, raising high expectations among the scientific community, patient associations, biotech companies and general public.

While clearly proving the ability of redirected T cells to recognize and efficiently kill cancer cells, CAR therapy has also shown some limitations: the nature of CAR-mediated recognition imposes to restrict the array of targeted antigens to those expressed on the surface of cancer cells. As a consequence, antigens involved in the oncogenic process, that are often expressed as intracellular molecules, cannot be targeted by current CARs. Furthermore, when the natural counterpart of cancer cells cannot be spared, the identification of a proper CAR target on cancer cell surface might become a real challenge.

TCR genetic engineering represents a suitable alternative to CAR T cell therapy for several tumors. The core of this approach is the transfer in patients' T cells of genes encoding for rare tumor-specific TCR. TCRs recognize antigen-derived peptides processed and presented on HLA molecules, thus allowing to largely increasing the array of potential targets. However, the simple transfer of tumor specific TCR genes into T cells is affected by other limitations: genetically modified T cells shall express four different TCR chains, that might mispair, leading to unpredictable toxicity and to an overall dilution of the tumor specific TCR on lymphocyte surface, thus limiting the efficacy of therapeutic cellular product.

To overcome these issues, we developed a TCR gene editing procedure, based on the knockout of the endogenous TCR genes by transient exposure to alfa and beta chain specific Zinc Finger Nucleases (ZFNs), followed by the introduction of tumor-specific TCR genes by lentiviral vectors (Provasi et al, Nature Medicine 2012). The TCR gene editing technology, proved safer and more effective than conventional TCR gene transfer in vitro and in animal studies. Early differentiated T cells, such as memory stem T cells and central memory T cells, cells endowed with long term persistence capacity, can be genetically engineered by TCR gene transfer and TCR gene editing, thus allowing to produce long-lasting living drugs, with the aim of eliminating cancer cells and patrol the organism for tumor recurrence

To enter the phase of clinical practice adoptive T cell therapy needs today to face several challenges: compliance to the dynamic and heterogeneous regulatory framework, susceptibility to automated processes, reproducibility, and sustainability shall be relevant variables in determining the fate of these innovative cellular products.