Multiplex Genome-Edited T-cell Manufacturing Platform for “Off-the-Shelf” Adoptive T-cell Immunotherapies

Abstract

Adoptive immunotherapy using autologous T cells endowed with chimeric antigen receptors (CAR) has emerged as a powerful means of treating cancer. However, a limitation of this approach is that autologous CAR T cells must be generated on a custom-made basis. Here we show that electroporation of transcription activator-like effector nuclease (TALEN) mRNA allows highly efficient multiplex gene editing in primary human T cells. We use this TALEN-mediated editing approach to develop a process for the large-scale manufacturing of T cells deficient in expression of both their ab T-cell receptor (TCR) and CD52, a protein targeted by alemtuzumab, a chemotherapeutic agent. Functionally, T cells manufactured with this process do not mediate graft-versus-host reactions and are rendered resistant to destruction by alemtuzumab. These characteristics enable the administration of alemtuzumab concurrently or prior to engineered T cells, supporting their engraftment. Furthermore, endowing the TALEN-engineered cells with a CD19 CAR led to efficient destruction of CD19 þ tumor targets even in the presence of the chemotherapeutic agent. These results demonstrate the applicability of TALEN-mediated genome editing to a scalable process, which enables the manufacturing of third-party CAR T-cell immunotherapies against arbitrary targets. As such, CAR T-cell immunotherapies can therefore be used in an "off-the-shelf" manner akin to other biologic immunopharmaceuticals. Cancer Res; 75(18); 3853-64. Ó2015 AACR.

Genome-Edited T Cell Therapies

“…While codon optimization, murinization of human TCRs, and the addition of cysteine residues have been found to reduce mispairing [54], complete knockout of the endogenous TCR would be advantageous. Further work exploring genome editing to knockout endogenous TCRα/β chains has been achieved using ZFNs [55, 56], TALENs [57, 58], megaTAL, and CRISPR/Cas [59] nucleases. Bonini et al developed α- and β-chain specific ZFNs against endogenous TCR genes and subsequently introduced the Wilms tumor-1-specific TCR via lentiviral gene transfer.…”

“…The development of a “universal” CAR T cell platform has recently harnessed the gene editing capabilities of TALENs in order to create an “off-the-shelf” adoptive T cell immunotherapy which was validated in vitro [58] and successfully applied to B-ALL [33••]. In order to overcome immune barriers intrinsic to allogeneic transplantations, targeted knockout of the endogenous αβ TCR within donor-derived T cells was performed [91].…”

Recent advances in T‐cell immunotherapy for haematological malignancies

“…(Poirot et al 2015, Torikai and Cooper 2016). Poirot et al (2015) developed an “off the shelf” approach to CAR19 T cells (lentiviral vector, CD19 scFv-41BB-CD3ζ, linked to an abbreviated suicide gene encoding both CD34 and CD20 epitopes). Following manufacture, these GMP-grade CAR19 T cells were electroporated with two pairs of TALEN mRNA, targeting both the TCR and CD52 loci.…”

“…Following manufacture, these GMP-grade CAR19 T cells were electroporated with two pairs of TALEN mRNA, targeting both the TCR and CD52 loci. (Poirot et al 2015) This simultaneous targeting rendered the HLA-mismatched CAR T cells insensitive to alemtuzumab, which can be administered to prevent rejection of allogeneic CAR19 T cells. The resultant product (UCART19) expressed <1% TCR, though some required CliniMacs TCR depletion after TALEN due to residual TCR expression.…”

Current Status of Engineered T-Cell Therapy for Synovial Sarcoma

“…Preliminary data on dose reductions in pre-conditioning cyclophosphamide regimens in ovarian cancer show decreased response to ACT, although whether a similar effect would be seen in synovial sarcoma remains unclear. Lympho-depletion methods that avoid the use of chemotherapy are under development and include monoclonal antibodies that target CD45 and CD52 [13,28,39]. Other strategies to enhance T-cell expansion also being investigated involve expression of IL15 and IL7Rα in genetically modified T cells [13,[29][30].…”