Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors

Abstract

Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR’s in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies.

Chimeric antigen receptor (CAR)-transduced natural killer cells in tumor immunotherapy

“…The SB transposon system transfers genes via a bi-component vector system that consists of a transposon containing a gene of interest (eg, CAR) flanked by inverted terminal repeats and a transposase (eg, SB100X) that binds to the inverted terminal repeats and "cutsand-pastes" the transposon for integration into the genome. Several groups have successfully applied the SB transposon system to CAR transgenes in T cells using electroporation [66][67][68] . Phase I trials have been sponsored that use SB to generate CD19-specific CAR-T cells for the treatment of patients with advanced non-Hodgkin's lymphoma and acute lymphoblastic leukemia who underwent HSCT and CAR-T cell infusion as adjuvant therapy in the autologous or allogeneic settings.…”

Adoptive CD8+ T cell therapy against cancer:Challenges and opportunities

“…Upon binding to AP1903, iCasp9 activates and leads to quick apoptotic cell death of CTLs [18,94]. Introducing these suicide genes made the CAR-T and TCR-T more controllable, but remains to be challenging in clinic.…”

Non-viral therapeutic cell engineering with the Sleeping Beauty transposon system

“…Several mechanisms may contribute to the development of this phenotype including lymphoid-to-myeloid transdifferentiation, selection of pre-existing CD19-low/negative leukemia cell clones and emergence of leukemia cell clones that have lost the epitope that is targeted by the CD19 CAR due to alternative splicing [54][55][56]. In ALL, CD19low/negative leukemia cells may still express CD20, CD22 and/or CD123 that are being pursued as rescue antigens [57][58][59]. A recent study has highlighted the potential to re-induce remissions in patients that had relapsed with CD19-low/negative leukemia and received a subsequent infusion of CD22 CAR-T cells [60 ].…”