Key Points
CD123 CAR T cells specifically target CD123+ AML cells. AML patient-derived T cells can be genetically modified to lyse autologous tumor cells.
The success of adoptive therapy using chimeric antigen receptor (CAR)-expressing T cells partly depends on optimal CAR design. CARs frequently incorporate a spacer/linker region based on the constant region of either IgG1 or IgG4 to connect extracellular ligand-binding with intracellular signaling domains. Here, we evaluated the potential for the IgG4-Fc linker to result in off-target interactions with Fc gamma receptors (FcγRs). As proof-of-principle, we focused on a CD19-specific scFv-IgG4-CD28-zeta CAR and found that, in contrast to CAR-negative cells, CAR+ T cells bound soluble FcγRs in vitro and did not engraft in NSG mice. We hypothesized that mutations to avoid FcγR binding would improve CAR+ T cell engraftment and antitumor efficacy. Thus, we generated CD19-specific CARs with IgG4-Fc spacers that had either been mutated at two sites (L235E; N297Q) within the CH2 region (CD19R(EQ)) or incorporated a CH2 deletion (CD19Rch2Δ). These mutations reduced binding to soluble FcγRs without altering the ability of the CAR to mediate antigen-specific lysis. Importantly, CD19R(EQ) and CD19Rch2Δ T cells exhibited improved persistence and more potent CD19-specific antilymphoma efficacy in NSG mice. Together, these studies suggest that optimal CAR function may require the elimination of cellular FcγR interactions to improve T cell persistence and antitumor responses.
MicroRNAs (miRNAs), such as miR-192, mediate the actions of transforming growth factor β1 (TGF-β) related to the pathogenesis of diabetic kidney diseases. We found that the biphasic induction of miR-192 expression by TGF-β in mouse renal glomerular mesangial cells initially involved the Smad transcription factors, followed by sustained expression that was promoted by acetylation of the transcription factor Ets-1 and histone H3 by the acetyltransferase p300, which was activated by the serine and threonine kinase Akt. In mesangial cells from Ets-1–deficient mice or in cells in which Ets-1 was knocked down, basal amounts of miR-192 were higher than those in control cells, but sustained induction of miR-192 by TGF-β was attenuated. Furthermore, inhibition of Akt or ectopic expression of dominant-negative histone acetyltransferases decreased p300-mediated acetylation and Ets-1 dissociation from the miR-192 promoter, and prevented miR-192 expression in response to TGF-β. Activation of Akt and p300 and acetylation of Ets-1 and histone H3 were increased in glomeruli from diabetic db/db mice compared to non-diabetic db/+ mice, suggesting that this pathway may contribute to diabetic nephropathy. These findings provide insight into the regulation of miRNAs through signaling-mediated changes in transcription factor activity and in epigenetic histone acetylation under normal and disease states.
Purpose
T cells engineered with chimeric antigen receptors (CARs) recognizing CD19 can induce complete remission of B cell malignancies in clinical trials; however, in some disease settings CAR therapy confers only modest clinical benefit due to attenuated persistence of CAR T cells. The purpose of the study was to enhance persistence and augment the antitumor activity of adoptively transferred CD19CAR T cells by re-stimulating CAR+ T cells through an endogenous cytomegalovirus (CMV)-specific T cell receptor.
Experimental design
CMV-specific T cells from CMV seropositive healthy donors were selected after stimulation with pp65 protein and transduced with clincal grade lentivirus expressing the CD19R:CD28:ζ/EGFRt CAR. The resultant bi-specific T cells, targeting CMV and CD19, were expanded via CD19 CAR-mediated signals using CD19-expressing cells.
Results
The bi-specific T cells proliferated vigorously after engagement with either endogenous CMVpp65 T cell receptors or engineered CD19 CARs, exhibiting specific cytolytic activity and IFNγ secretion. Upon adoptive transfer into immunodeficient mice bearing human lymphomas, the bi-specific T cells exhibited proliferative response and enhanced antitumor activity following CMVpp65 peptide vaccine administration.
Conclusions
We have redirected CMV-specific T cells to recognize and lyse tumor cells via CD19CARs, while maintaining their ability to proliferate in response to CMV antigen stimulation. These results illustrate the clinical applications of CMV vaccine to augment the antitumor activity of adoptively transferred CD19CAR T cells in patients with B cell malignancies.
CD62LC precursors enriched for na€ ıve and stem cell memory precursors (T N/SCM ) with that of T CM . We found that cytotoxic T cells (CTLs) derived from T CM transcribed higher levels of CD28, FOS, INFg, Eomesodermin (Eomes), and lower levels of BCL2L11, maintained higher levels of phosphorylated AKT, and displayed enhanced sensitivity to the proliferative and anti-apoptotic effects of g-chain cytokines compared to CTLs derived from T N/SCM . Higher frequencies of CTLs derived from T CM retained CD28 expression and upon activation secreted higher levels of IL-2. In NOD/Scid IL-2RgC null mice, CD8 C T CM derived CTLs engrafted to higher frequencies in response to human IL-15 and mounted robust proliferative responses to an immunostimulatory vaccine. Similarly, CD8C T CM derived CD19CAR C CTLs exhibited superior antitumor potency following adoptive transfer compared to their CD8 C T N/SCM derived counterparts. These studies support the use of T CM enriched cell products for adoptive therapy of cancer.
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