CD3ζ-based chimeric antigen receptors mediate T cell activation via cis- and trans-signalling mechanisms: implications for optimization of receptor structure for adoptive cell therapy.

Chimeric antigen receptors (CARs) can mediate redirected lysis of tumour cells in a major histocompatibility complex (MHC)-independent manner, thereby enabling autologous adoptive T cell therapy for a variety of malignant neoplasms. Currently, most CARs incorporate the T cell receptor (TCR) CD3ζ signalling chain; however, the precise mechanisms responsible for CAR-mediated T cell activation are unclear. In this study, we used a series of immunoreceptor tyrosine-based activation motif (ITAM)-mutant and transmembrane-modified receptors to demonstrate that CARs activate T cells both directly via the antigen-ligated signalling chain and indirectly via associated chains within the TCR complex. These observations allowed us to generate new receptors capable of eliciting polyfunctional responses in primary human T cells. This work increases our understanding of CAR function and identifies new avenues for the optimization of CAR-based therapeutic interventions.

Ligation of the CD2 co-stimulatory receptor enhances IL-2 production from first-generation chimeric antigen receptor T cells.

T cells bearing chimeric antigen receptors (CARs) are broadly categorised into first- and second-generation receptors. Second-generation CARs contain a co-stimulatory signalling molecule and have been shown to secrete IL-2, undergo greater proliferation and to have enhanced persistence in vivo. However, we have shown that T cells bearing a first-generation CAR containing a CD19-targeting scFv (single-chain variable fragment) and the CD3ζ-signalling domain are able to produce IL-2 upon co-culture with CD19(+) B-cell lymphomas independent of CD28 activity. Here, we report that signalling through endogenous CD2 following ligation with its ligands, CD48 in mouse and CD58 in humans, drives IL-2 production by first-generation CD19-specific CAR. Moreover, the high levels of IL-2 produced by human T cells engrafted with a second-generation CD28-containing CAR during target-cell recognition are dependent to a degree upon CD2 receptor activity. These observations highlight the fact that the functional activity induced by T-cell-expressed CARs is dependent upon endogenous 'natural' receptor interactions. A deeper understanding of the role of these activities will serve to further refine the design of future CARs to either exploit or avoid these interactions.

Lentiviral transduction of the murine lung provides efficient pseudotype and developmental stage-dependent cell-specific transgene expression.

Gene transfer for cystic fibrosis (CF) airway disease has been hampered by the lung's innate refractivity to pathogen infection. We hypothesized that early intervention with an integrating gene transfer vector capable of transducing the lung via the lumen may be a successful therapeutic approach. An HIV-based lentiviral vector pseudotyped with the baculovirus gp64 envelope was applied to the fetal, neonatal or adult airways. Fetal intra-amniotic administration resulted in transduction of approximately 14% of airway epithelial cells, including both ciliated and non-ciliated epithelia of the upper, mid and lower airways; there was negligible alveolar or nasal transduction. Following neonatal intra-nasal administration we observed significant transduction of the airway epithelium (approximately 11%), although mainly in the distal lung, and substantial alveolar transduction. This expression was still detectable at 1 year after application. In the adult, the majority of transduction was restricted to the alveoli. In contrast, vesicular stomatitis virus glycoprotein pseudotyped virus transduced only alveoli after adult and neonatal application and no transduction was observed after fetal administration. Repeat administration did not increase transduction levels of the conducting airway epithelia. These data demonstrate that application at early developmental stages in conjunction with an appropriately pseudotyped virus provides efficient, high-level transgene expression in the murine lung. This may provide a modality for treatment for lung disease in CF.