Chimeric antigen receptor (CAR) T cells targeting CD19 or CD22 have shown remarkable activity in B cell acute lymphoblastic leukemia (B-ALL). The major cause of treatment failure is antigen downregulation or loss. Dual antigen targeting could potentially prevent this, but the clinical safety and efficacy of CAR T cells targeting both CD19 and CD22 remain unclear. We conducted a phase 1 trial in pediatric and young adult patients with relapsed or refractory B-ALL (n = 15) to test AUTO3, autologous transduced T cells expressing both anti-CD19 and anti-CD22 CARs (AMELIA trial, EUDRA CT 2016-004680-39). The primary endpoints were the incidence of grade 3–5 toxicity in the dose-limiting toxicity period and the frequency of dose-limiting toxicities. Secondary endpoints included the rate of morphological remission (complete response or complete response with incomplete bone marrow recovery) with minimal residual disease-negative response, as well as the frequency and severity of adverse events, expansion and persistence of AUTO3, duration of B cell aplasia, and overall and event-free survival. The study endpoints were met. AUTO3 showed a favorable safety profile, with no dose-limiting toxicities or cases of AUTO3-related severe cytokine release syndrome or neurotoxicity reported. At 1 month after treatment the remission rate (that is, complete response or complete response with incomplete bone marrow recovery) was 86% (13 of 15 patients). The 1 year overall and event-free survival rates were 60% and 32%, respectively. Relapses were probably due to limited long-term AUTO3 persistence. Strategies to improve CAR T cell persistence are needed to fully realize the potential of dual targeting CAR T cell therapy in B-ALL.

Natural killer (NK) cell responses are regulated by a dynamic equilibrium between activating and inhibitory receptor signals at the immune synapse (or interface) with target cells. Although the organization of receptors at the immune synapse is important for appropriate integration of these signals, there is little understanding of this in detail, because research has been hampered by the limited resolution of light microscopy. Through the use of superresolution single-molecule fluorescence microscopy to reveal the organization of the NK cell surface at the single-protein level, we report that the inhibitory receptor KIR2DL1 is organized in nanometer-scale clusters at the surface of human resting NK cells. Nanoclusters of KIR2DL1 became smaller and denser upon engagement of the activating receptor NKG2D, establishing an unexpected crosstalk between activating receptor signals and the positioning of inhibitory receptors. These rearrangements in the nanoscale organization of surface NK cell receptors were dependent on the actin cytoskeleton. Together, these data establish that NK cell activation involves a nanometer-scale reorganization of surface receptors, which in turn affects models for signal integration and thresholds that control NK cell effector functions and NK cell development.

• The large extracellular domains of the tyrosine phosphatases CD45 and CD148 prevent them from inhibiting T-cell receptor triggering.• These domains are required for optimal segregation from the engaged T-cell receptor, supporting the kineticsegregation model of triggering.T-cell receptor (TCR) triggering results in a cascade of intracellular tyrosine phosphorylation events that ultimately leads to T-cell activation. It is dependent on changes in the relative activities of membrane-associated tyrosine kinases and phosphatases near the engaged TCR. CD45 and CD148 are transmembrane tyrosine phosphatases with large ectodomains that have activatory and inhibitory effects on TCR triggering. This study investigates whether and how the ectodomains of CD45 and CD148 modulate their inhibitory effect on TCR signaling. Expression in T cells of forms of these phosphatases with truncated ectodomains inhibited TCR triggering. In contrast, when these phosphatases were expressed with large ectodomains, they had no inhibitory effect. Imaging studies revealed that truncation of the ectodomains enhanced colocalization of these phosphatases with ligated TCR at the immunological synapse. Our results suggest that the large ectodomains of CD45 and CD148 modulate their inhibitory effect by enabling their passive, size-based segregation from ligated TCR, supporting the kinetic-segregation model of TCR triggering. (Blood. 2013;121(21):4295-4302) Introduction T cells are stimulated through the T-cell receptor (TCR) when it binds cognate peptide presented by a major histocompatibility complex molecule (pMHC) on another cell. As a consequence of ligation, immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of the TCR/CD3 complex are phosphorylated by lymphocyte-specific protein tyrosine kinase (Lck). These phosphorylated ITAMs recruit z-chain-associated protein tyrosine kinase 70 to the membrane, and Zap-70 phosphorylates substrates such as linker of activated T cells (LAT). 1Despite the extensive research in this field, the mechanism by which the binding of TCR to pMHC leads to phosphorylation of TCR/CD3 ITAMs is still contested, and several models have been proposed. 2One common feature of some of these models is that TCR triggering is initiated by changes in the relative concentrations of membrane tyrosine kinases and phosphatases in the vicinity of ligated TCR. 2The principal membrane tyrosine phosphatases involved in regulating TCR-induced tyrosine phosphorylation are CD45 and CD148. 3 The importance of this dynamic equilibrium between kinase and phosphatase activity in TCR triggering was highlighted in studies that use phosphatase inhibitors such as pervanadate. [4][5][6] Treatment of T cells with these inhibitors alone, in the absence of any TCR ligand, was sufficient to induce full activation of TCR signaling pathways, ranging from early events such as phosphorylation of TCR ITAMs, Zap-70, and LAT to late events such as interleukin 2 (IL-2) production. [4][5][6] Several mechanisms have been propos...

Engineered T cell therapies show considerable promise in the treatment of refractory malignancies. Given the ability of engineered T cells to engraft and persist for prolonged periods along with unpredicted toxicities, incorporation of a suicide gene to allow selective depletion after administration is desirable. Rapamycin is a safe and widely available immunosuppressive pharmaceutical that acts by heterodimerization of FKBP12 with the FRB fragment of mTOR. The apical caspase caspase 9 is activated by homodimerization through its CARD domain. We developed a rapamycin-induced caspase 9 suicide gene. First, we showed that caspase 9 could be activated by a two-protein format with replacement of the CARD domain with both FRB and FKBP12. We next identified an optimal compact single-protein rapamycin caspase 9 (rapaCasp9) by fusing both FRB and FKBP12 with the catalytic domain of caspase 9. Functionality of rapaCasp9 when co-expressed with a CD19 CAR was demonstrated in vitro and in vivo.

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