Abstract
The efficacy of T cells expressing chimeric antigen receptors (CARs) for solid tumors has been limited by insufficient CAR T cell expansion and persistence. The use of virus-specific T cells (VSTs) as carriers for CARs may overcome this limitation since CAR-VSTs can be boosted by viral vaccines or oncolytic viruses. However, there is limited understanding of the optimal combination of endodomains and their influence on the native T cell receptor (TCR) in VSTs. We therefore compared the function of GD2.CARs expressing the TCR zeta chain (ζ) alone or combined with endodomains from CD28 and 4-1BB in varicella zoster virus-specific (VZV) T cells. VZVSTs expressing GD2-CARs recognized VZV-derived peptides and killed GD2-expressing tumor cells. However, after repeated stimulation through their native TCR, the expansion of GD2-CAR.CD28ζ-VZVSTs was 3.3-fold greater (p < 0.001) than non-transduced VZVSTs, whereas GD2-CARζ- and GD2-CAR.41BBζ inhibited VZVST expansion (p < 0.01). Compared to control VZVSTs, GD2-CAR.ζ VZVSTs showed a greater frequency of apoptotic (p < 0.01) T cells, whereas prolonged downregulation of the native αβ TCR was observed in GD2-CAR.41BBζ VZVSTs (p < 0.001). We confirmed that CD28ζ can best maintain TCR function by expressing GD2.CARs in Epstein-Barr virus-specific T cells and CD19-CARs in VZVSTs. In response to CAR stimulation VSTs with CD28ζ endodomains also showed the greatest expansion (6 fold > GD2-CAR.41BBζ VZVSTs (p < 0.001), however anti-tumor efficacy was superior in GD2-CAR.41BBζ-VZVSTs. These findings demonstrate that CAR signaling domains can enhance or diminish the function of the native TCR and indicate that only CD28ζ may preserve the function of the native TCR in tonically signaling CAR-VSTs.
Highlights
Chimeric antigen receptors (CARs) redirected to the B-cell antigen CD19 have yielded impressive clinical results, associated with exponential CAR-T cell expansion in patients with B cell acute lymphoblastic leukemia (B-ALL) and lymphoma [1,2,3,4]
VZVSTs were transduced with retroviral vectors encoding the GD2.CAR constructs GD2.ζ, GD2.CD28ζ and GD2.41BBζ, which differed only in their intracellular costimulatory endodomains (Supplementary Figure S1) [17]
virus-specific T cells (VSTs) transduced with GD2.CD28ζ and GD2.41BBζ CARs contained a trend toward higher frequencies of CD45RO+CCR7+ central memory T cells than the non-transduced (NT) and GD2.ζ-transduced cells, in both the CD4 and CD8 subsets
Summary
Chimeric antigen receptors (CARs) redirected to the B-cell antigen CD19 have yielded impressive clinical results, associated with exponential CAR-T cell expansion in patients with B cell acute lymphoblastic leukemia (B-ALL) and lymphoma [1,2,3,4]. CAR-T cells targeting solid tumors have been less effective, as they fail to expand sufficiently after infusion despite the presence of costimulatory endodomains. This failure is likely in part because solid tumors lack the costimulatory molecules expressed by B-cells and instead express inhibitory cytokines and ligands, which prevents adequate T cell expansion at the tumor site [5]. The successful treatment of solid tumors might require additional strategies to preserve and enhance the in vivo function of CAR T cells. One means to enhance the in vivo proliferation and function of CAR-T cells is via their native T cell receptor (TCR). In a clinical trial of patients receiving VSTs transduced with a CD19.CAR possessing a CD28ζ signaling domain, we observed that patients with Epstein-Barr virus (EBV) reactivation exhibited a concomitant increase in both EBVspecific T cells and the CD19.CAR signal, suggesting virusinduced expansion of CD19.CAR-VSTs [6]
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