Improving the anti-acute myeloid leukemia activity of CD123-specific engager T cells by MyD88 and CD40 costimulation.

The outcome of patients with acute myeloid leukemia remains poor, and immunotherapy has the potential to improve this. T cells expressing chimeric antigen receptors or bispecific T-cell engagers targeting CD123 are actively being explored in preclinical and/or early phase clinical studies. We have shown that T cells expressing CD123-specific bispecific T-cell engagers (CD123.ENG T cells) have anti-acute myeloid leukemia activity. However, like chimeric antigen receptor T cells, their effector function diminishes rapidly once they are repeatedly exposed to antigen-positive target cells. Here we sought to improve the effector function of CD123.ENG T cells by expressing inducible co-stimulatory molecules consisting of MyD88 and CD40 (iMC), MyD88 (iM), or CD40 (iC), which are activated by a chemical inducer of dimerization. CD123.ENG T cells expressing iMC, iM, or iC maintained their antigen specificity in the presence of a chemical inducer of dimerization, as judged by cytokine production (interferon-γ, interleukin-2) and their cytolytic activity. In repeat stimulation assays, activating iMC and iM, in contrast to iC, enabled CD123.ENG T cells to secrete cytokines, expand, and kill CD123-positive target cells repeatedly. Activating iMC in CD123.ENG T cells consistently improved antitumor activity in an acute myeloid leukemia xenograft model. This translated into a significant survival advantage in comparison to that of mice that received CD123.ENG or CD123.ENG.iC T cells. In contrast, activation of only iM in CD123.ENG T cells resulted in donor-dependent antitumor activity. Our work highlights the need for both toll-like receptor pathway activation via MyD88 and provision of co-stimulation via CD40 to consistently enhance the antitumor activity of CD123.ENG T cells.

Targeting glycans for CAR therapy: The advent of sweet CARs.

Chimeric antigen receptor (CAR) T cell therapy has created a paradigm shift in the treatment of hematologic malignancies but has not been as effective toward solid tumors. For such tumors, the primary obstacles facing CAR T cells are scarcity of tumor-specific antigens and the hostile and complex tumor microenvironment. Glycosylation, the process by which sugars are post-translationally added to proteins or lipids, is profoundly dysregulated in cancer. Abnormally glycosylated glycoproteins expressed on cancer cells offer unique targets for CAR T therapy as they are specific to tumor cells. Tumor stromal cells also express abnormal glycoproteins and thus also have the potential to be targeted by glycan-binding CAR T cells. This review will discuss the state of CAR T cells in the therapy of solid tumors, the cancer glycoproteome and its potential for use as a therapeutic target, and the landscape and future of glycan-binding CAR T cell therapy.

Redirecting T cells to hematological malignancies with bispecific antibodies.

There is a need to improve outcomes for patients with recurrent and/or refractory hematological malignancies. Immunotherapy holds the promise to meet this need, because it does not rely on the cytotoxic mechanism of conventional therapies. Among different forms of immunotherapy, redirecting T cells to hematological malignancies with bispecific antibodies (BsAbs) is an attractive strategy. BsAbs are an "off-the-shelf" product that is easily scalable in contrast to adoptive T-cell therapies. Among these, the bispecific T-cell engager blinatumomab has emerged as the most successful BsAb to date. It consists of 2 single-chain variable fragments specific for CD19 present on B-cell malignancies and CD3 expressed on almost all T cells. Blinatumomab has shown potent antitumor activity as a single agent, particularly for acute lymphoblastic leukemia, resulting in its US Food and Drug Administration approval. However, although successful in inducing remissions, these are normally short-lived, with median response durations of <1 year. Nevertheless, the success of blinatumomab has reinvigorated the BsAb field, which is bustling with preclinical and clinical studies for not only B-cell-derived lymphoblastic leukemia and lymphoma but also acute myeloid leukemia and multiple myeloma. Here, we will review the successes and challenges of T-cell-targeted BsAbs for the immunotherapy of hematological malignancies with special focus on conducted clinical studies and strategies to improve their efficacy.

The future of cellular immunotherapy for childhood leukemia.

PURPOSE OF REVIEW: Exciting translational discoveries in recent years have brought realized promise of immunotherapy for children with high-risk leukemias. This review summarizes the current immunotherapeutic landscape with a focus on key clinical trials for patients with acute lymphoblastic leukemia or acute myeloid leukemia. RECENT FINDINGS: Chemotherapy resistance remains a major barrier to cure in children with high-risk leukemias. Immunotherapy approaches have potential to overcome this resistance given alternative mechanisms of action. Based upon preclinical activity and/or success in adult patients, recent clinical trials have demonstrated safety and efficacy of various mAb, antibody-drug conjugate, bispecific T-cell-engaging antibody, natural killer cell, and chimeric antigen receptor-redirected T-cell immunotherapies for children with acute lymphoblastic leukemia or acute myeloid leukemia. Food and Drug Administration approval of several of these immunotherapies has increased the pediatric leukemia therapeutic portfolio and improved clinical outcomes for previously incurable patients. SUMMARY: Several antibody-based or cellular immunotherapy modalities have demonstrated appreciable efficacy in children with relapsed or chemotherapy-refractory leukemia via early-phase clinical trials. Some studies have also identified critical biomarkers of treatment response and resistance that merit further investigation. Continued preclinical and clinical evaluation of novel immunotherapies is imperative to improve cure rates for children with high-risk leukemias.

Programmed cell death protein 1 activation preferentially inhibits CD28.CAR-T cells.

Targeted adoptive immunotherapy with engineered T cells is a promising treatment for refractory hematologic malignancies. However, many patients achieving early complete remissions ultimately relapse. Immunosuppressive ligands are expressed on tumor and supportive cells in the tumor microenvironment (TME). When activated, T cells express associated "checkpoint" receptors. Binding of co-inhibitory ligands and receptors may directly contribute to T-cell functional exhaustion. It is not known whether all T cells engineered to express chimeric antigen receptors (CARs) are subject to checkpoint-mediated regulation. It is also unknown whether distinct CAR signaling moieties modulate T-cell responsiveness to these inhibitory pathways. We have, therefore, directly compared functional co-inhibition in engineered T cells identically targeted to the tumor-associated antigen CD123, but distinct in their mode of T-cell activation: via the endogenous T-cell receptor (ENG), or downstream of CD28 or 41BB-containing CARs. In all cases, we have observed antigen-independent T-cell activation associated with upregulation of the co-inhibitory receptors programmed cell death protein 1 (PD-1, CD279), Tim-3 and Lag-3. Notably, CD28.CAR T cells were uniquely susceptible to PD-1/PD-L1 mediated checkpoint inhibition. Together, our data indicate that PD-1/PD-L1 checkpoint blocking agents may be considered clinically when CD28.CAR T cells do not perform optimally in human trials.

CD123-Engager T Cells as a Novel Immunotherapeutic for Acute Myeloid Leukemia.

Immunotherapy with CD123-specific T-cell engager proteins or with T cells expressing CD123-specific chimeric antigen receptors is actively being pursued for acute myeloid leukemia. T cells secreting bispecific engager molecules (ENG-T cells) may present a promising alternative to these approaches. To evaluate therapeutic potential, we generated T cells to secrete CD123/CD3-bispecific engager molecules. CD123-ENG T cells recognized primary acute myeloid leukemia (AML) cells and cell lines in an antigen-dependent manner as judged by cytokine production and/or tumor killing, and redirected bystander T cells to AML cells. Infusion of CD123-ENG T cells resulted in regression of AML in xenograft models conferring a significant survival advantage of treated mice in comparison to mice that received control T cells. At high effector to target ratios, CD123-ENG T cells recognized normal hematopoietic stem and progenitor cells (HSPCs) with preferential recognition of HSPCs from cord blood compared to bone marrow. We therefore introduced the CD20 suicide gene that can be targeted in vivo with rituximab into CD123-ENG T cells. The expression of CD20 did not diminish the anti-AML activity of CD123-ENG T cells, but allowed for rituximab-mediated ENG-T cell elimination. Thus, ENG-T cells coexpressing CD20 suicide and CD123 engager molecules may present a promising immunotherapeutic approach for AML.

Engineering self-propelled tumor-infiltrating CAR T cells using synthetic velocity receptors.

Chimeric antigen receptor (CAR) T cells express antigen-specific synthetic receptors, which upon binding to cancer cells, elicit T cell anti-tumor responses. CAR T cell therapy has enjoyed success in the clinic for hematological cancer indications, giving rise to decade-long remissions in some cases. However, CAR T therapy for patients with solid tumors has not seen similar success. Solid tumors constitute 90% of adult human cancers, representing an enormous unmet clinical need. Current approaches do not solve the central problem of limited ability of therapeutic cells to migrate through the stromal matrix. We discover that T cells at low and high density display low- and high-migration phenotypes, respectively. The highly migratory phenotype is mediated by a paracrine pathway from a group of self-produced cytokines that include IL5, TNFα, IFNγ, and IL8. We exploit this finding to "lock-in" a highly migratory phenotype by developing and expressing receptors, which we call velocity receptors (VRs). VRs target these cytokines and signal through these cytokines' cognate receptors to increase T cell motility and infiltrate lung, ovarian, and pancreatic tumors in large numbers and at doses for which control CAR T cells remain confined to the tumor periphery. In contrast to CAR therapy alone, VR-CAR T cells significantly attenuate tumor growth and extend overall survival. This work suggests that approaches to the design of immune cell receptors that focus on migration signaling will help current and future CAR cellular therapies to infiltrate deep into solid tumors.

The rapid and highly parallel identification of antibodies with defined biological activities by SLISY.

The therapeutic applications of antibodies are manifold and the emergence of SARS-CoV-2 provides a cogent example of the value of rapidly identifying biologically active antibodies. We describe an approach called SLISY (Sequencing-Linked ImmunoSorbent assaY) that in a single experiment can assess the binding specificity of millions of clones, be applied to any screen that links DNA sequence to a potential binding moiety, and requires only a single round of biopanning. We demonstrate this approach using an scFv library applied to cellular and protein targets to identify specific or broadly reacting antibodies. For a cellular target, we use paired HLA knockout cell lines to identify a panel of antibodies specific to HLA-A3. For a protein target, SLISY identifies 1279 clones that bound to the Receptor Binding Domain of the SARS-CoV-2 spike protein, with >40% of tested clones also neutralizing its interaction with ACE2 in in vitro assays. Using a multi-comparison SLISY against the Beta, Gamma, and Delta variants, we recovered clones that exhibited broad-spectrum neutralizing potential in vitro. By evaluating millions of scFvs simultaneously against multiple targets, SLISY allows the rapid identification of candidate scFvs with defined binding profiles facilitating the identification of antibodies with the desired biological activity.

HLH-like toxicities predict poor survival after the use of tisagenlecleucel in children and young adults with B-ALL.

Chimeric antigen receptor-associated hemophagocytic lymphohistiocytosis (HLH)-like toxicities (LTs) involving hyperferritinemia, multiorgan dysfunction, coagulopathy, and/or hemophagocytosis are described as occurring in a subset of patients with cytokine release syndrome (CRS). Case series report poor outcomes for those with B-cell acute lymphoblastic leukemia (B-ALL) who develop HLH-LTs, although larger outcomes analyses of children and young adults (CAYAs) with B-ALL who develop these toxicities after the administration of commercially available tisagenlecleucel are not described. Using a multi-institutional database of 185 CAYAs with B-ALL, we conducted a retrospective cohort study including groups that developed HLH-LTs, high-grade (HG) CRS without HLH-LTs, or no to low-grade (NLG) CRS without HLH-LTs. Primary objectives included characterizing the incidence, outcomes, and preinfusion factors associated with HLH-LTs. Among 185 CAYAs infused with tisagenlecleucel, 26 (14.1%) met the criteria for HLH-LTs. One-year overall survival and relapse-free survival were 25.7% and 4.7%, respectively, in those with HLH-LTs compared with 80.1% and 57.6%, respectively, in those without. In multivariable analysis for death, meeting criteria for HLH-LTs carried a hazard ratio of 4.61 (95% confidence interval, 2.41-8.83), controlling for disease burden, age, and sex. Patients who developed HLH-LTs had higher pretisagenlecleucel disease burden, ferritin, and C-reactive protein levels and lower platelet and absolute neutrophil counts than patients with HG- or NLG-CRS without HLH-LTs. Overall, CAYAs with B-ALL who developed HLH-LTs after tisagenlecleucel experienced high rates of relapse and nonrelapse mortality, indicating the urgent need for further investigations into prevention and optimal management of patients who develop HLH-LTs after tisagenlecleucel.

Mediport use as an acceptable standard for CAR T cell infusion.

Introduction: Mediport use as a clinical option for the administration of chimeric antigen receptor T cell (CAR T cell) therapy in patients with B-cell malignancies has yet to be standardized. Concern for mediport dislodgement, cell infiltration, and ineffective therapy delivery to systemic circulation has resulted in variable practice with intravenous administration of CAR T cell therapy. With CAR T cell commercialization, it is important to establish practice standards for CAR T cell delivery. We conducted a study to establish usage patterns of mediports in the clinical setting and provide a standard of care recommendation for mediport use as an acceptable form of access for CAR T cell infusions. Methods: In this retrospective cohort study, data on mediport use and infiltration rate was collected from a survey across 34 medical centers in the Pediatric Real-World CAR Consortium, capturing 504 CAR T cell infusion routes across 489 patients. Data represents the largest, and to our knowledge sole, report on clinical CAR T cell infusion practice patterns since FDA approval and CAR T cell commercialization in 2017. Results: Across 34 sites, all reported tunneled central venous catheters, including Broviac® and Hickman® catheters, as accepted standard venous options for CAR T cell infusion. Use of mediports as a standard clinical practice was reported in 29 of 34 sites (85%). Of 489 evaluable patients with reported route of CAR T cell infusion, 184 patients were infused using mediports, with no reported incidences of CAR T cell infiltration. Discussion/Conclusion: Based on current clinical practice, mediports are a commonly utilized form of access for CAR T cell therapy administration. These findings support the safe practice of mediport usage as an accepted standard line option for CAR T cell infusion.

Gene-Based Natural Killer Cell Therapies for the Treatment of Pediatric Hematologic Malignancies.

Pediatric blood cancers are among the most common malignancies that afflict children. Intensive chemotherapy is not curative in many cases, and novel therapies are urgently needed. NK cells hold promise for use as immunotherapeutic effectors due to their favorable safety profile, intrinsic cytotoxic properties, and potential for genetic modification that can enhance specificity and killing potential. NK cells can be engineered to express CARs targeting tumor-specific antigens, to downregulate inhibitory and regulatory signals, to secrete cytokine, and to optimize interaction with small molecule engagers. Understanding NK cell biology is key to designing immunotherapy for clinical translation.

Transgenic Expression of IL15 Retains CD123-Redirected T Cells in a Less Differentiated State Resulting in Improved Anti-AML Activity in Autologous AML PDX Models.

Immunotherapy with T-cells expressing bispecific T-cell engagers (ENG T-cells) is a promising approach to improve the outcomes for patients with recurrent/refractory acute myeloid leukemia (AML). However, similar to T-cells expressing chimeric antigen receptors (CARs), their antitumor activity is limited in the setting of chronic antigen stimulation. We therefore set out to explore whether transgenic expression of IL15 improves the effector function of ENG T-cells targeting CD123-positive AML. T-cells expressing CD123-specific ENG (CD123-ENG) ± IL15 were generated by retroviral transduction from peripheral blood T cells from healthy donors or patients with AML. In this study, we characterized in detail the phenotype and effector functions of ENG T-cell populations in vitro and in vivo. IL15-expressing CD123-ENG (CD123-ENG.IL15) T-cells retained their antigen-specificity and effector function in the setting of chronic antigen exposure for more 30 days of coculture with AML blasts in contrast to CD123-ENG T-cells, whose effector function rapidly eroded. Furthermore, CD123-ENG.IL15 T-cells remained in a less differentiated state as judged by a high frequency of naïve/memory stem T-cell-like cells (CD45RA+CCR7+/CD45RO-CD62L+ cells) without evidence of T-cell exhaustion. Single cell cytokine profiling using IsoPlexis revealed enhanced T-cell polyfunctionality of CD123-ENG.IL15 T-cells as judged by effector cytokine production, including, granzyme B, IFN-γ, MIP-1α, perforin, TNF-α, and TNF-ß. In vivo, CD123-ENG.IL15 T-cells exhibited superior antigen-specific anti-AML activity and T-cell persistence in both peripheral blood and tissues (BM, spleens, and livers), resulting in a significant survival advantage in one AML xenograft model and two autologous AML PDX models. In conclusion, we demonstrate here that the expansion, persistence, and anti-AML activity of CD123-ENG T-cells can be significantly improved by transgenic expression of IL15, which promotes a naïve/TSCM-like phenotype. However, we also highlight that targeting a single tumor antigen (CD123) can lead to immune escape, reinforcing the need to develop approaches to target multiple antigens. Likewise, our study demonstrates that it is feasible to evaluate autologous T cells in AML PDX models, which will be critical for future preclinical evaluations of next generation AML-redirected T-cell therapies.

Impact of High Disease Burden on Survival in Pediatric Patients with B-ALL Treated with Tisagenlecleucel.

CD19-specific chimeric antigen receptor (CAR) T-cell therapies, including the FDA-approved tisagenlecleucel, induce high rates of remission in pediatric patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, post-treatment relapse remains an issue. Optimal management of B-ALL after tisagenlecleucel treatment remains elusive, and continued tracking of outcomes is necessary to establish a standard of care for this population. We sought to evaluate outcomes on the real-world use of tisagenlecleucel in a contemporary pediatric patient population and to identify risk factors influencing event-free survival (EFS) and overall survival (OS). Additionally, we aimed to describe post-tisagenlecleucel management strategies, including use of allogeneic hematopoietic cell transplantation (AlloHCT) or repeat CAR T-cell infusions. We report on 31 pediatric and adolescent and young adult patients (AYA) with B-ALL, treated with lymphodepleting chemotherapy followed by tisagenlecleucel. Patients were treated at Johns Hopkins Hospital and St. Jude Children's Research Hospital between March 2018 and November 2020. Data on patient, disease, and treatment characteristics were collected retrospectively from medical records and described. EFS and OS were estimated by the Kaplan-Meier method and compared by the log-rank test. Single-factor and multiple-factor analysis of EFS and OS were performed by fitting Cox regression models. Of the 30 evaluable patients, 25 (83.3%) experienced a complete response, with 21 having negative minimal residual disease. Treatment was well tolerated, with expected rates of cytokine release syndrome (61.3%) and immune effector cell-associated neurotoxicity (29%). After initial complete response, 12 patients (48%) had subsequent disease recurrence, with CD19-negative relapse (n = 6) occurring sooner than CD19-positive relapse (P = .0125). With a median follow-up time of 386 days (range 11-1187 days), the EFS for the entire cohort (n = 31) at 6 and 12 months after infusion was 47% (95% confidence interval [CI], 28.4%-63.4%) and 35.2% (95% CI, 18.4%-52.5%), respectively. In multivariate analysis, high pretreatment leukemic burden (≥5% bone marrow blasts) was an independent risk factor for inferior EFS (HR 5.98 [95% CI, 1.1-32.4], P = .0380) and OS (HR 4.2 [95% CI, 1.33-13.39], P = .0148). Tisagenlecleucel induced high initial response rates in a contemporary cohort of pediatric and AYA patients with B-ALL. However, 48% of patients experienced subsequent disease relapse, including 6 with antigen-escape variants. This highlights a considerable limitation of single-agent autologous CD19-CAR T-cell therapy. Pretreatment leukemic disease burden of ≥5% blasts was significantly associated with worse outcomes in this study, including lower EFS and OS. Our findings suggest that reducing preinfusion leukemic burden is a viable treatment strategy to improve outcomes of CAR T-cell therapy.

Glycoprotein Targeted CAR-NK Cells for the Treatment of SARS-CoV-2 Infection.

H84T-Banana Lectin (BanLec) CAR-NK cells bind high mannose glycosites that decorate the SARS-CoV-2 envelope, thereby decreasing cellular infection in a model of SARS-CoV-2. H84T-BanLec CAR-NK cells are innate effector cells, activated by virus. This novel cellular agent is a promising therapeutic, capable of clearing circulating SARS-CoV-2 virus and infected cells. Banana Lectin (BanLec) binds high mannose glycans on viral envelopes, exerting an anti-viral effect. A point mutation (H84T) divorces BanLec mitogenicity from antiviral activity. SARS-CoV-2 contains high mannose glycosites in proximity to the receptor binding domain of the envelope Spike (S) protein. We designed a chimeric antigen receptor (CAR) that incorporates H84T-BanLec as the extracellular moiety. Our H84T-BanLec CAR was devised to specifically direct NK cell binding of SARS-CoV-2 envelope glycosites to promote viral clearance. The H84T-BanLec CAR was stably expressed at high density on primary human NK cells during two weeks of ex vivo expansion. H84T-BanLec CAR-NK cells reduced S-protein pseudotyped lentiviral infection of 293T cells expressing ACE2, the receptor for SARS-CoV-2. NK cells were activated to secrete inflammatory cytokines when in culture with virally infected cells. H84T-BanLec CAR-NK cells are a promising cell therapy for further testing against wild-type SARS-CoV-2 virus in models of SARS-CoV-2 infection. They may represent a viable off-the-shelf immunotherapy for patients suffering from COVID-19.

Engineering CAR-NK cells to secrete IL-15 sustains their anti-AML functionality but is associated with systemic toxicities.

BACKGROUND: The prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. Natural Killer (NK) cells can elicit an antileukemic response via a repertoire of activating receptors that bind AML surface ligands. NK-cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity and interleukin (IL)-15 to enhance their persistence. METHODS: We characterized in detail NK-cell populations expressing a panel of AML (CD123)-specific CARs and/or IL-15 in vitro and in AML xenograft models. RESULTS: CARs with 2B4.ζ or 4-1BB.ζ signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.ζ Chimeric Antigen Receptor (CAR)-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.ζ CAR-NK cells. However, the benefit was transient due to limited CAR-NK-cell persistence. Transgenic expression of secretory interleukin (sIL)-15 in 2B4.ζ CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK-cell subsets. 2B4.ζ/sIL-15 CAR and sIL-15 NK cells maintained an overall activated NK-cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK-cell groups. In vivo, 2B4.ζ/sIL-15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.ζ/sIL-15 CAR and sIL-15 NK cells induced lethal toxicity in a second model. CONCLUSION: Transgenic expression of CD123-CARs and sIL-15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early-phase clinical testing.

Converging genetic and epigenetic drivers of paediatric acute lymphoblastic leukaemia identified by an information-theoretic analysis.

In cancer, linking epigenetic alterations to drivers of transformation has been difficult, in part because DNA methylation analyses must capture epigenetic variability, which is central to tumour heterogeneity and tumour plasticity. Here, by conducting a comprehensive analysis, based on information theory, of differences in methylation stochasticity in samples from patients with paediatric acute lymphoblastic leukaemia (ALL), we show that ALL epigenomes are stochastic and marked by increased methylation entropy at specific regulatory regions and genes. By integrating DNA methylation and single-cell gene-expression data, we arrived at a relationship between methylation entropy and gene-expression variability, and found that epigenetic changes in ALL converge on a shared set of genes that overlap with genetic drivers involved in chromosomal translocations across the disease spectrum. Our findings suggest that an epigenetically driven gene-regulation network, with UHRF1 (ubiquitin-like with PHD and RING finger domains 1) as a central node, links genetic drivers and epigenetic mediators in ALL.

Activation of p53-dependent growth suppression in human cells by mutations in PTEN or PIK3CA.

In an effort to identify genes whose expression is regulated by activated phosphatidylinositol 3-kinase (PI3K) signaling, we performed microarray analysis and subsequent quantitative reverse transcription-PCR on an isogenic set of PTEN gene-targeted human cancer cells. Numerous p53 effectors were upregulated following PTEN deletion, including p21, GDF15, PIG3, NOXA, and PLK2. Stable depletion of p53 led to reversion of the gene expression program. Western blots revealed that p53 was stabilized in HCT116 PTEN(-/-) cells via an Akt1-dependent and p14(ARF)-independent mechanism. Stable depletion of PTEN in untransformed human fibroblasts and epithelial cells also led to upregulation of p53 and senescence-like growth arrest. Simultaneous depletion of p53 rescued this phenotype, enabling PTEN-depleted cells to continue proliferating. Next, we tested whether oncogenic PIK3CA, like inactivated PTEN, could activate p53. Retroviral expression of oncogenic human PIK3CA in MCF10A cells led to activation of p53 and upregulation of p53-regulated genes. Stable depletion of p53 reversed these PIK3CA-induced expression changes and synergized with oncogenic PIK3CA in inducing anchorage-independent growth. Finally, targeted deletion of an endogenous allele of oncogenic, but not wild-type, PIK3CA in a human cancer cell line led to a reduction in p53 levels and a decrease in the expression of p53-regulated genes. These studies demonstrate that activation of PI3K signaling by mutations in PTEN or PIK3CA can lead to activation of p53-mediated growth suppression in human cells, indicating that p53 can function as a brake on phosphatidylinositol (3,4,5)-triphosphate-induced mitogenesis during human cancer pathogenesis.

Improving CAR T-cells: The next generation.

The introduction of chimeric antigen receptor (CAR) T-cell therapy in acute lymphoblastic leukemia (ALL) has dramatically altered the landscape of treatment options available to children and adults with ALL. With complete remission induction rates exceeding 70% in most trials and FDA approval of one CD19 CAR T-cell construct in ALL, CAR T-cell therapy has become a mainstay in the ALL treatment algorithm for those with relapsed/refractory disease. Despite the high remission induction rate, with growing experience using CAR T-cell therapy in ALL, a host of barriers to maintaining long-term durable remissions have been identified. Specifically, relapse after, resistance to, or loss of long-term CAR T-cell persistence may all hinder CAR T-cell efficacy. In this review, we provide an overview of the current limitations which inform the design of the next generation of CAR T-cells and discuss advances in CAR T-cell engineering aimed to improve upon outcomes with CAR T-cell-based therapy in ALL.

The Art and Science of Selecting a CD123-Specific Chimeric Antigen Receptor for Clinical Testing.

Chimeric antigen receptor (CAR) T cells targeting CD123, an acute myeloid leukemia (AML) antigen, hold the promise of improving outcomes for patients with refractory/recurrent disease. We generated five lentiviral vectors encoding CD20, which may serve as a target for CAR T cell depletion, and 2nd or 3rd generation CD123-CARs since the benefit of two costimulatory domains is model dependent. Four CARs were based on the CD123-specific single-chain variable fragment (scFv) 26292 (292) and one CAR on the CD123-specific scFv 26716 (716), respectively. We designed CARs with different hinge/transmembrane (H/TM) domains and costimulatory domains, in combination with the zeta (z) signaling domain: 292.CD8aH/TM.41BBz (8.41BBz), 292.CD8aH/TM.CD28z (8.28z), 716.CD8aH/TM.CD28z (716.8.28z), 292.CD28H/TM. CD28z (28.28z), and 292.CD28H/TM.CD28.41BBz (28.28.41BBz). Transduction efficiency, expansion, phenotype, and target cell recognition of the generated CD123-CAR T cells did not significantly differ. CAR constructs were eliminated for the following reasons: (1) 8.41BBz CARs induced significant baseline signaling, (2) 716.8.28z CAR T cells had decreased anti-AML activity, and (3) CD28.41BBz CAR T cells had no improved effector function in comparison to CD28z CAR T cells. We selected the 28.28z CAR since CAR expression on the cell surface of transduced T cells was higher in comparison to 8.28z CARs. The clinical study (NCT04318678) evaluating 28.28z CAR T cells is now open for patient accrual.