200. Generation of CAR-T Cells Lacking T Cell Receptor and Human Leukocyte Antigen Using Engineered Meganucleases

The manufacture of CAR-T cells depends on peripheral blood donations that contain T cells of sufficient quality and quantity. Currently, many CAR-T programs rely on autologous T cells, but several technical and commercial challenges hinder development. The majority of CAR-T trials have enrolled leukemia or lymphoma patients, many of which are unsuitable donors for CAR-T production due to their disease state or to previous treatments with lymphodepleting agents. In addition, a custom CAR-T production run for each patient is time consuming, lacks standardization and may present regulatory challenges. An alternative strategy is to source T cells from healthy donors and produce large batches of allogeneic CAR-T cells. Allogeneic T cells, however, will display mismatched human leukocyte antigens (HLA) that will be recognized by the recipients’ immune systems, contributing to immune rejection of engrafted CAR-T cells. Additionally, donor T cells will recognize the mismatched HLAs present in the recipient, contributing to graft-versus-host immune pathology. Both undesired immune responses are predicated on interactions between HLA and T cell receptors (TCR), and while the therapeutic effectiveness of CAR-T cells with targeted deletions in TCR genes has been reported by several groups, studies featuring both TCR and HLA deletion are limited. Here, we describe the use of meganucleases engineered to target regions of the TCR α chain constant region and β-2 microglobulin genes to generate TCR and HLA class I knockout primary human T cells. Both nucleases generate knockouts with approximately 75% efficiency and are well-tolerated by primary T cells from at least four separate donors. Purified double knockout cells do not demonstrate functional disadvantages in terms of proliferation or cytokine production, but do exhibit reduced allostimulatory potential toward HLA-mismatched T cells. Together, these findings demonstrate the feasibility of generating therapeutic quantities of CAR-T cells with reduced allo-reactive potential and collateral toxicity to normal tissues in recipients.