Periodic Reporting for period 1 - CARiPSCTcells (Generation of safe and efficient, off-the-shelf, chimeric antigen receptor (CAR)-engineered T cells for broad application)
Reporting period: 2015-07-01 to 2017-06-30
We have previously reported in a proof-of-concept study (Themeli et al. Nat Biotechnol 2013) that genetic engineering of T-cell derived induced pluripotent stem cells (TiPSC) with Chimeric Antigen Receptors (CARs) can be an efficient strategy to concomitantly harness the unlimited availability of induced pluripotent stem cells and direct the specificity and functional potential of TiPSC-derived T cells. Based on this technology, we aimed in this study to further investigate novel stem cell genetic engineering strategies in order to obtain in vitro, unlimited, safe and broadly applicable T cells. We proposed to target MM with a novel inducible CD38-targeting CAR (CD38CAR). In addition, we aimed to simultaneously knockout the expression the endogenous T cell receptor (TCR) and the HLA antigens on the CAR-engineered TiPSCs (CARTiPSC) in order to extend the applicability of CARTiPSC-derived T cells across HLA-barriers.
Objective 1: We generated CAR constructs from human anti-CD38 antibodies. CD38CART cells effectively eradicate human MM cell lines and MM tumors in vitro and in vivo (Drent et al. Mol Ther 2017). We attempted to improve the safety of TiPSC-derived CD38CART cells by using a doxycycline (Dox)-controlled expression of the CAR with a Tet-on system, thus, the expression of the CAR could be induced by the administration of Dox. Using the CRISPR/Cas9-mediated gRNA-guided genome editing technology, we targeted both TRAC (TCRa chain) alleles of TiPSCs in order to generate TCR knockout TiPSC lines. 3 TCR-knockout clonal lines were selected, which expressed the CD38CAR upon Dox treatment. Inducible CD38-TiPSC were further successfully differentiated to mature CD8ab T cells.
Objective 2: For this obective we modified the HLA class I expression of inducible CD38-TiPSCs which were generated in objective 1. We generated b2m-/- inducible (ind)CD38-TiPSCs using the CRISPR/Cas9 system. The generated b2m-/- indCD38-TiPSCs could be efficiently differentiated towards CD8 CD38CAR-T cells in vitro.
Overall results: We have succeeded in generating an unlimited source of non-alloreactive, low-immunogenic inducible CD38CAR-T cells. These cells can recognize and kill MM cells in vitro.