Monitoring of tisagenlecleucel transgene DNA using a quantitative polymerase chain reaction assay.

Chimeric antigen receptor (CAR)-T cell therapies reprogram T cells to engage and eliminate cancer cells. Patients' T cells are transduced in vitro using lentiviral or retroviral vectors containing a CAR transgene. Following infusion, CAR-T cells expand in vivo and may persist in the peripheral blood and bone marrow for years. Therefore, monitoring in vivo copies of the CAR transgene requires highly sensitive, validated analytical methods. Herein, we describe the validation of a qPCR assay to detect tisagenlecleucel transgene in patient samples. The limit of detection and lower limit of quantitation were 3.1 and 10 copies/200 ng genomic DNA, respectively, equivalent to ∼50 copies/µg genomic DNA and in alignment with US Food and Drug Administration guidance on bioanalytical method validation. The assay allowed quantitation of the tisagenlecleucel transgene over a wide dynamic range with a high degree of linearity, that is, 101-106 copies/200 ng genomic DNA (R2 ≥ 0.9988). Coefficients of variation of measured transgene copies ranged from 0.2% to 12.8%. A droplet digital PCR assay was performed as a method of validation and showed a strong correlation with the qPCR assay (R2 = 0.9980, p < 0.0001). This qPCR assay is being utilized to monitor tisagenlecleucel expansion and persistence in clinical trials.

Monitoring CAR-T cell kinetics in clinical trials by multiparametric flow cytometry: Benefits and challenges.

Exceptional clinical responses produced by the first chimeric antigen receptor T [CAR-T] cell therapies, and their entry into commercial markets prompted a logarithmic increase in the number of next generation CAR-T clinical trials. As a result, there is a growing interest in understanding the analytical approaches utilized for reliable monitoring of these "living" drugs, and the challenges encountered during their clinical development. Multiparametric flow cytometry (MFC) assays have played a crucial role in understanding the phenotype and function of first approved CAR-T therapies. Herein, three main areas for monitoring CAR-T therapies in clinical trials are discussed: (1) analytical considerations critical for development of MFC assays for the reliable enumeration of CAR-T levels, (2) operational challenges associated with clinical trial sampling and transportation, and (3) differential cellular kinetics observed by MFC and qPCR analyses and their relationship with efficacy (measurable residual disease levels). Initial experiences described here may enable design of fit-for-purpose tools and help to more rapidly advance the development of next generation CAR-T therapies.

Mechanisms of necroptosis in T cells.

Cell populations are regulated in size by at least two forms of apoptosis. More recently, necroptosis, a parallel, nonapoptotic pathway of cell death, has been described, and this pathway is invoked in the absence of caspase 8. In caspase 8-deficient T cells, necroptosis occurs as the result of antigen receptor-mediated activation. Here, through a genetic analysis, we show that necroptosis in caspase 8-deficient T cells is related neither to the programmed necrosis as defined by the requirement for mitochondrial cyclophilin D nor to autophagy as defined by the requirement for autophagy-related protein 7. Rather, survival of caspase 8-defective T cells can be completely rescued by loss of receptor-interacting serine-threonine kinase (Ripk) 3. Additionally, complementation of a T cell-specific caspase 8 deficiency with a loss of Ripk3 gives rise to lymphoproliferative disease reminiscent of lpr or gld mice. In conjunction with previous work, we conclude that necroptosis in antigen-stimulated caspase 8-deficient T cells is the result of a novel Ripk1- and Ripk3-mediated pathway of cell death.

Foxo transcription factors control regulatory T cell development and function.

Foxo transcription factors integrate extrinsic signals to regulate cell division, differentiation and survival, and specific functions of lymphoid and myeloid cells. Here, we showed the absence of Foxo1 severely curtailed the development of Foxp3(+) regulatory T (Treg) cells and those that developed were nonfunctional in vivo. The loss of function included diminished CTLA-4 receptor expression as the Ctla4 gene was a direct target of Foxo1. T cell-specific loss of Foxo1 resulted in exocrine pancreatitis, hind limb paralysis, multiorgan lymphocyte infiltration, anti-nuclear antibodies and expanded germinal centers. Foxo-mediated control over Treg cell specification was further revealed by the inability of TGF-ß cytokine to suppress T-bet transcription factor in the absence of Foxo1, resulting in IFN-γ secretion. In addition, the absence of Foxo3 exacerbated the effects of the loss of Foxo1. Thus, Foxo transcription factors guide the contingencies of T cell differentiation and the specific functions of effector cell populations.

Transcription factor Foxo3 controls the magnitude of T cell immune responses by modulating the function of dendritic cells.

Foxo transcription factors regulate cell cycle progression, cell survival and DNA-repair pathways. Here we demonstrate that deficiency in Foxo3 resulted in greater expansion of T cell populations after viral infection. This exaggerated expansion was not T cell intrinsic. Instead, it was caused by the enhanced capacity of Foxo3-deficient dendritic cells to sustain T cell viability by producing more interleukin 6. Stimulation of dendritic cells mediated by the coinhibitory molecule CTLA-4 induced nuclear localization of Foxo3, which in turn inhibited the production of interleukin 6 and tumor necrosis factor. Thus, Foxo3 acts to constrain the production of key inflammatory cytokines by dendritic cells and to control T cell survival.

A pivotal role for the multifunctional calcium/calmodulin-dependent protein kinase II in T cells: from activation to unresponsiveness.

Stimulation of the TCR leads to an oscillatory release of free calcium that activates members of the calcium/calmodulin-dependent protein kinase II (CaMKII) family. The CaMKII molecules have profound and lasting effects on cellular signaling in several cell types, yet the role of CaMKII in T cells is still poorly characterized. In this report we describe a splice variant of CaMKIIbeta, CaMKIIbeta'e, in mouse T cells. We have determined its function, along with that of CaMKIIgamma, by introducing the active and kinase-dead mutants into activated P14 TCR transgenic T cells using retroviral transduction. Active CaMKII enhanced the proliferation and cytotoxic activity of T cells while reducing their IL-2 production. Furthermore, it induced a profound state of unresponsiveness that could be overcome only by prolonged culture in IL-2. These results indicate that members of the CaMKII family play an important role in regulation of CD8 T cell proliferation, cytotoxic effector function, and the response to restimulation.

Expression of an engrailed-LMO4 fusion protein in mammary epithelial cells inhibits mammary gland development in mice.

LIM domain factors and associated cofactors are important developmental regulators in pattern formation and organogenesis. In addition, overexpression of two LIM-only factors (LMOs) causes acute lymphocytic leukemia. The more recently discovered LMO factor LMO4 is highly expressed in proliferating epithelial cells, and frequently overexpressed in breast carcinoma. Here we show that while LMO4 is expressed throughout mammary gland development, it is dramatically upregulated in mammary epithelial cells during midpregnancy. The LMO coactivator Clim2/Ldb1/NLI showed a similar expression pattern, consistent with the idea that LMO4 and Clim2 act as a complex in mammary epithelial cells. In MCF-7 cells, LMO4 transcripts were upregulated by heregulin, an activator of ErbB receptors that are known to be important in mammary gland development and breast cancer. To test the hypothesis that LMO4 plays roles in mammary gland development, we created an engrailed-LMO4 fusion protein. This fusion protein maintains the ability to interact with Clim2, but acts as a dominant repressor of both basal and activated transcription when recruited to a DNA-regulatory region. When the engrailed-LMO4 fusion protein was expressed under control of the MMTV promoter in transgenic mice, both ductular development in virgin mice and alveolar development in pregnant mice were inhibited. These results suggest that LMO4 plays a role in promoting mammary gland development.