Bispecific T-cells Expressing Polyclonal Repertoire of Endogenous γδ T-cell Receptors and Introduced CD19-specific Chimeric Antigen Receptor

Abstract

Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR(+)γδ T cells were expanded on CD19(+) artificial antigen-presenting cells (aAPC), which resulted in >10(9) CAR(+)γδ T cells from <10(6) total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR(+)γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19(+) tumor cell lines compared with CAR(neg)γδ T cells. CD19(+) leukemia xenografts in mice were reduced with CAR(+)γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells.

Avoidance of On-Target Off-Tumor Activation Using a Co-stimulation-Only Chimeric Antigen Receptor

“…Autologous gdT cells can be expanded to sufficient numbers from the blood of cancer patients to have therapeutic efficacy against a range of tumor targets, 39,40 and CAR-expressing gdT cells are readily expanded. 41 We also demonstrate this ( Figure 2D); extrapolation of these figures indicates that a cell dose of 10 7 -10 8 cells would be achievable from a 100-mL blood draw.…”

T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia

“…48 The antileukemic effect observed in our studies is comparable with what is often seen in xenogeneic models of acute leukemia void of tumor-derived costimulation, using either single or multiple infusions of CAR T cells. 9,49 The in vivo potency of our CD123 CAR T cells may be further augmented by using a less differentiated memory T-cell subset or by altering several features of the CAR including scFv affinity, linker length, and intracellular costimulatory domain (s), all of which have been shown to play key roles in CAR T-cell potency. 8,10,43,50 Recently, Tettamanti, Marin, et al have demonstrated that cytokine-induced killer cells expressing a first-generation CD123 CAR exhibit potent cytolytic activity against AML cell lines and primary patient samples while sparing normal hematopoietic progenitors in vitro.…”

Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor

“…13 Specificity for EGFR was based on using EGFR neg control cell lines, including tCD19 + EL4 40 and the malignant B-cell tumor NALM-6. To reduce variability, only donors in which DNA- and mRNA-modified CAR + T cells contained a similar proportion of CD8 + T cells and expressed similar levels of CAR were analyzed (see data, Supplemental Digital Content 10).…”