Use of Cell and Genome Modification Technologies to Generate Improved "Off-the-Shelf" CAR T and CAR NK Cells

Abstract

The broad success of adoptive immunotherapy to treat human cancer has resulted in a paradigm shift in modern medicine. Modification of autologous and allogenic immune cells with chimeric antigen receptors (CAR) designed to target specific antigens on tumor cells has led to production of CAR T and CAR NK cell therapies, which are ever more commonly introduced into cancer patient treatment protocols. While allogenic T cells may offer advantages such as improved anti-tumor activity, they also carry the risk of adverse reactions like graft-versus-host disease. This risk can be mitigated by use of autologous immune cells, however, the time needed for T and/or NK cell isolation, modification and expansion may be too long for some patients. Thus, there is an urgent need for strategies to robustly produce "off-the-shelf" CAR T and CAR NK cells, which could be used as a bridging therapy between cancer diagnosis or relapse and allogeneic transplantation. Advances in genome modification technologies have accelerated the generation of designer cell therapy products, including development of "off-the-shelf" CAR T cells for cancer immunotherapy. The feasibility and safety of such approaches is currently tested in clinical trials. This review will describe cell sources for CAR-based therapies, provide background of current genome editing techniques and the applicability of these approaches for generation of universal "off-the-shelf" CAR T and NK cell therapeutics.

Keywords: CRISPR-Cas9; T cell; chimeric antigen receptor; genome editing; immunotherapy.

FIGURE 1

Modification of T or NK cells with CAR-encoding retro- and lentiviral vectors. On the left, a lentiviral vector is shown that transfers the genetic cargo into the T or NK cells leading to the expression of a chimeric antigen receptor (CAR) on the cell surface. On the right, the structure of a 3^rd generation CAR is depicted with single chain variable fragment (scFv, including V_H and V_L chains), hinge, transmembrane and signaling domains shown. CARs can be engineered with cell-type specific modules to enhance CAR T or CAR NK cell activity. Examples of cytoplasmic signaling domains that can be engineered into CARs for T and NK cells are shown. Combining such strategies with additional genome modification approaches described in later sections of this review will lead to improved “off-the-shelf” cell products.

FIGURE 2

CRISPR-Cas9-mediated gene editing of CAR T cells. The TCR can be knocked out to lessen the likelihood of graft versus host disease (GVHD). The HLAs can be knocked out to increase persistence of gene-modified cells. Knockout of receptors that can be targeted by other medications, such as antibodies, can be accomplished to allow selective survival of gene-modified cells, e.g., CD52.

FIGURE 3

Reshaping of the tumor microenvironment using T cells redirected for universal cytokine killing (TRUCK). Upon antigen binding, the CAR activates CD3z(eta) signaling, which leads to activation of an NFAT-driven promoter that controls expression of antitumor-cytokine cassette, e.g., IL12 or IL18. The cytokines are then secreted from the CAR T or CAR NK cells into the tumor microenvironment, where they recruit additional immune cells to enhance the antitumor activity.