TCR Gene Editing to Treat Hematological Malignancies

Tumor-specific T lymphocytes are rare cells, able to recognize cancer cells through their T cell receptors (TCR), and kill them. To exploit tumor-specific T cells for cancer treatment, and overcome their paucity, it is today possible to transfer in patients’ T cells genes encoding for rare tumor-specific TCR. However, such genetically modified T cells express four different TCR chains, that might mispair, leading to unpredictable toxicity and to an overall dilution of the tumor specific TCR on cell 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 2102). The TCR gene editing technology, proved safer and more effective than conventional TCR gene transfer in vitro and in animal studies. While successful, complete editing requires multiple manipulation steps and four transduction procedures. To reduce the duration and complexity of the procedure, we recently developed and tested the ‘single TCR editing’ (SE) approach, that allows to redirect T cell specificity in clinically suitable conditions.

We validated SE using an HLA-A2 restricted TCR specific for NY-ESO-1_157-165, a cancer testis antigen expressed by several tumor types, including multiple myeloma. Conventional TCR gene transfer (TR) and SE cells were compared in terms of efficacy and safety in vitro and in vivo.
 SE rapidly produced high numbers of tumor specific T cells, with an early differentiation phenotype, including memory stem T cells, associated to long term survival in adoptive immunotherapy clinical trials (Oliveira et al., STM 2015). SE proved highly efficient, and similar to TR T cells, in killing multiple myeloma in vitro and in immunodeficient NSG mice. However, while TR lymphocytes proved highly alloreactive, SE cells showed a favorable safety profile. Indeed, in contrast to the TR cells, SE cells mediated tumor rejection without inducing xenogeneic graft versus host disease in NSG mice, thus promoting a significantly higher survival rate. The detrimental alloreactive effect mediated by TR cells led to a xenogeneic GvHD rate comparable to that of unmodified allogenic T cells.

Overall, the single TCR gene editing procedure provides a rapid and efficient method for generating primary T cells that highly express a tumor specific TCR and are devoid of their endogenous TCR repertoire, and thus represents a protean platform for the effective and safe adoptive transfer of allogeneic and autologous T lymphocytes redirected towards any desired tumor associated antigen.