CD19/22 CAR T cells in children and young adults with B-ALL: phase 1 results and development of a novel bicistronic CAR.

Remission durability following single-antigen targeted chimeric antigen receptor (CAR) T-cells is limited by antigen modulation, which may be overcome with combinatorial targeting. Building upon our experiences targeting CD19 and CD22 in B-cell acute lymphoblastic leukemia (B-ALL), we report on our phase 1 dose-escalation study of a novel murine stem cell virus (MSCV)-CD19/CD22-4-1BB bivalent CAR T-cell (CD19.22.BBζ) for children and young adults (CAYA) with B-cell malignancies. Primary objectives included toxicity and dose finding. Secondary objectives included response rates and relapse-free survival (RFS). Biologic correlatives included laboratory investigations, CAR T-cell expansion and cytokine profiling. Twenty patients, ages 5.4 to 34.6 years, with B-ALL received CD19.22.BBζ. The complete response (CR) rate was 60% (12 of 20) in the full cohort and 71.4% (10 of 14) in CAR-naïve patients. Ten (50%) developed cytokine release syndrome (CRS), with 3 (15%) having ≥ grade 3 CRS and only 1 experiencing neurotoxicity (grade 3). The 6- and 12-month RFS in those achieving CR was 80.8% (95% confidence interval [CI]: 42.4%-94.9%) and 57.7% (95% CI: 22.1%-81.9%), respectively. Limited CAR T-cell expansion and persistence of MSCV-CD19.22.BBζ compared with EF1α-CD22.BBζ prompted laboratory investigations comparing EF1α vs MSCV promoters, which did not reveal major differences. Limited CD22 targeting with CD19.22.BBζ, as evaluated by ex vivo cytokine secretion and leukemia eradication in humanized mice, led to development of a novel bicistronic CD19.28ζ/CD22.BBζ construct with enhanced cytokine production against CD22. With demonstrated safety and efficacy of CD19.22.BBζ in a heavily pretreated CAYA B-ALL cohort, further optimization of combinatorial antigen targeting serves to overcome identified limitations (www.clinicaltrials.gov #NCT03448393).

Intrathecal hydrocortisone for treatment of children and young adults with CAR T-cell immune-effector cell-associated neurotoxicity syndrome.

Immune-effector cell-associated neurotoxicity syndrome (ICANS) is a significant toxicity occurring with chimeric antigen receptor (CAR) T-cell therapy, with first-line treatment options including supportive care and systemic corticosteroids. Sparse data exist on how to approach progressive/refractory cases of ICANS. We present five pediatric and young adult patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL) who had progressively worsening ICANS despite systemic steroids, and received intrathecal hydrocortisone with rapid reversal of ICANS. Therapeutic lumbar punctures are routinely used in upfront ALL therapy in pediatrics, with a demonstrable safety profile, thus use of intrathecal hydrocortisone merits further prospective studies in patients with severe ICANS.

Leaf decomposition and flammability are largely decoupled across species in a tropical swamp forest despite sharing some predictive leaf functional traits.

Decomposition and fire are major carbon pathways in many ecosystems, yet potential linkages between these processes are poorly understood. We test whether variability in decomposability and flammability across species are related to each other and to key plant functional traits in tropical swamp forests, where habitat degradation is elevating decomposition and fire regimes. Using senesced and fresh leaves of 22 swamp tree species in Singapore, we conducted an in situ decomposition experiment and a laboratory flammability experiment. We analysed 16 leaf physical and biochemical traits as predictors of decomposability and components of flammability: combustibility, ignitability and sustainability. Decomposability and flammability were largely decoupled across species, despite some shared predictive traits such as specific leaf area (SLA). Physical traits predicted that thicker leaves with a smaller SLA and volume decomposed faster, while various cation concentrations predicted flammability components, particularly ignitability. We show that flammability and decomposability of swamp forest leaves are decoupled because flammability is mostly driven by biochemical traits, while decomposition is driven by physical traits. Our approach identifies species that are slow to decompose and burn (e.g. Calophyllum tetrapterum and Xanthophyllum flavescens), which could be planted to mitigate carbon losses in tropical swamp reforestation.

Three-year results from phase I of ZUMA-4: KTE-X19 in pediatric relapsed/refractory acute lymphoblastic leukemia.

Here we present the 3-year results of ZUMA-4, a phase I/II multicenter study evaluating the safety and efficacy of KTEX19, an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, in pediatric/adolescent patients with relapsed/refractory B-cell acute lymphoblastic leukemia. Phase I explored two dose levels and formulations. The primary endpoint was the incidence of dose-limiting toxicities. Thirty-one patients were enrolled; KTE-X19 was administered to 24 patients (median age 13.5 years, range 3-20; median follow-up 36.1 months). No dose-limiting toxicities were observed. All treated patients had grade ≥3 adverse events, commonly hypotension (50%) and anemia (42%). Grade 3 cytokine release syndrome rates were 33% in all treated patients, 75% in patients given the dose of 2×106 CAR T cells/kg, 27% in patients given the dose of 1×106 cells/kg in the 68 mL formulation, and 22% in patients given the dose of 1×106 cells/kg in the 40 mL formulation; the percentages of patients experiencing grade ≥3 neurologic events were 21%, 25%, 27%, and 11% respectively. Overall complete remission rates (including complete remission with incomplete hematologic recovery) were 67% in all treated patients, 75% in patients given 2×106 CAR T cells/kg, 64% in patients given 1×106 cells/kg in the 68 mL formulation, and 67% in patients given 1×106 cells/kg in the 40 mL formulation. Overall minimal residual diseasenegativity rates were 100% among responders; 88% of responders underwent subsequent allogeneic stem-cell transplantation. In the 1×106 (40 mL) group (recommended phase II dose), the median duration of remission censored at allogeneic stem-cell transplantation and median overall survival were not reached. Pediatric/adolescent patients with relapsed/refractory B-cell acute lymphoblastic leukemia achieved high minimal residual disease-negative remission rates with a manageable safety profile after a single dose of KTE-X19. Phase II of the study is ongoing at the dose of 1×106 CAR T cells/kg in the 40 mL formulation. ClinicalTrials.gov: NCT02625480.

Disease detection methodologies in relapsed B-cell acute lymphoblastic leukemia: Opportunities for improvement.

BACKGROUND: Accurate disease detection is integral to risk stratification in B-cell acute lymphoblastic leukemia (ALL). The gold standard used to evaluate response in the United States includes morphologic evaluation and minimal residual disease (MRD) testing of aspirated bone marrow (BM) by flow cytometry (FC). This MRD assessment is usually made on a single aspirate sample that is subject to variability in collection techniques and sampling error. Additionally, central nervous system (CNS) assessments for ALL include evaluations of cytopathology and cell counts, which can miss subclinical involvement. PROCEDURE: We retrospectively compared BM biopsy, aspirate, and FC samples obtained from children and young adults with relapsed/refractory ALL to identify the frequency and degree of disease discrepancies in this population. We also compared CNS FC and cytopathology techniques. RESULTS: Sixty of 410 (14.6%) BM samples had discrepant results, 41 (10%) of which were clinically relevant as they resulted in a change in the assignment of marrow status. Discrepant BM results were found in 28 of 89 (31.5%) patients evaluated. Additionally, cerebrospinal fluid (CSF) FC identified disease in 9.7% of cases where cytopathology was negative. CONCLUSIONS: These results support further investigation of the role of concurrent BM biopsy, with aspirate and FC evaluations, and the addition of FC to CSF evaluations, to fully assess disease status and response, particularly in patients with relapsed/refractory ALL. Prospective studies incorporating more comprehensive analysis to evaluate the impact on clinical outcomes are warranted.

ASTCT Consensus Grading for Cytokine Release Syndrome and Neurologic Toxicity Associated with Immune Effector Cells.

Chimeric antigen receptor (CAR) T cell therapy is rapidly emerging as one of the most promising therapies for hematologic malignancies. Two CAR T products were recently approved in the United States and Europe for the treatment ofpatients up to age 25years with relapsed or refractory B cell acute lymphoblastic leukemia and/or adults with large B cell lymphoma. Many more CAR T products, as well as other immunotherapies, including various immune cell- and bi-specific antibody-based approaches that function by activation of immune effector cells, are in clinical development for both hematologic and solid tumor malignancies. These therapies are associated with unique toxicities of cytokine release syndrome (CRS) and neurologic toxicity. The assessment and grading of these toxicities vary considerably across clinical trials and across institutions, making it difficult to compare the safety of different products and hindering the ability to develop optimal strategies for management of these toxicities. Moreover, some aspects of these grading systems can be challenging to implement across centers. Therefore, in an effort to harmonize the definitions and grading systems for CRS and neurotoxicity, experts from all aspects of the field met on June 20 and 21, 2018, at a meeting supported by the American Society for Transplantation and Cellular Therapy (ASTCT; formerly American Society for Blood and Marrow Transplantation, ASBMT) in Arlington, VA. Here we report the consensus recommendations of that group and propose new definitions and grading for CRS and neurotoxicity that are objective, easy to apply, and ultimately more accurately categorize the severity of these toxicities. The goal is to provide a uniform consensus grading system for CRS and neurotoxicity associated with immune effector cell therapies, for use across clinical trials and in the postapproval clinical setting.

Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells.

BACKGROUND: Clinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes. METHODS: Counter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We investigated the use of PBMC concentrates enriched for lymphocytes using elutriation for manufacturing 8 CD19- and 5 GD2-CAR T cell products. RESULTS: When compared to PBMC concentrates, lymphocyte-enriched elutriation fractions contained greater proportions of CD3+ and CD56+ cells and reduced proportions of CD14+ and CD15+ cells. All 13 CAR T cell products manufactured using the elutriated lymphocytes yielded sufficient quantities of transduced CAR T cells to meet clinical dose criteria. The GD2-CAR T cell products contained significantly more T cells and transduced T cells than the CD19-CAR T cell products. A comparison of the yields of CAR T cells produced from elutriated lymphocytes with the yields of CAR T cells previous produced from cells isolated from PBMC concentrates by anti-CD3/CD28 bead selection or by anti-CD3/CD28 bead selection plus plastic adherence found that greater quantities of GD2-CAR T cells were produced from elutriated lymphocytes, but not CD19-CAR T cells. CONCLUSIONS: Enrichment of PBMC concentrates for lymphocytes using elutriation increased the quantity of GD2-CAR T cells produced. These results provide further evidence that CAR T cell expansion is inhibited by monocytes and granulocytes.

Autologous lymphapheresis for the production of chimeric antigen receptor T cells.

BACKGROUND: The first step in manufacturing chimeric antigen receptor (CAR) T cells is to collect autologous CD3+ lymphocytes by apheresis. Patients, however, often have leukopenia or have other disease-related complications. We evaluated the feasibility of collecting adequate numbers of CD3+ cells, risk factors for inadequate collections, and the rate of adverse events. STUDY DESIGN AND METHODS: Apheresis lymphocyte collections from patients participating in three CAR T-cell clinical trials were reviewed. Collections were performed on the COBE Spectra by experienced nurses, with the goal of obtaining a minimum of 0.6 × 109 and a target of 2 × 109 CD3+ cells. Preapheresis peripheral blood counts, apheresis parameters, and product cell counts were analyzed. RESULTS: Of the 71 collections, 69 (97%) achieved the minimum and 55 (77%) achieved the target. Before apheresis, the 16 patients with yields below the target had significantly lower proportions and absolute numbers of circulating lymphocytes and CD3+ lymphocytes and higher proportions of circulating blasts and NK cells than those who achieved the target (470 × 106 lymphocytes/L vs. 1340 × 106 lymphocytes/L, p = 0.008; 349 × 106 CD3+ cells/L vs. 914 × 106 CD3+ cells/L, p = 0.001; 17.6% blasts vs. 4.55% blasts, p = 0.029). Enrichment of blasts in the product compared to the peripheral blood occurred in four patients, including the two patients whose collections did not yield the minimum number of CD3+ cells. Apheresis complications occurred in 11 patients (15%) and, with one exception, were easily managed in the apheresis clinic. CONCLUSIONS: In most patients undergoing CAR T-cell therapy, leukapheresis is well tolerated, and adequate numbers of CD3+ lymphocytes are collected.

Specific Etiologies Associated With the Multiple Organ Dysfunction Syndrome in Children: Part 1.

OBJECTIVE: To describe a number of the conditions associated with multiple organ dysfunction syndrome presented as part of the Eunice Kennedy Shriver National Institute of Child Health and Human Development multiple organ dysfunction syndrome workshop (March 26-27, 2015). DATA SOURCES: Literature review, research data, and expert opinion. STUDY SELECTION: Not applicable. DATA EXTRACTION: Moderated by an expert from the field, issues relevant to the association of multiple organ dysfunction syndrome with a variety of conditions were presented, discussed, and debated with a focus on identifying knowledge gaps and research priorities. DATA SYNTHESIS: Summary of presentations and discussion supported and supplemented by the relevant literature. CONCLUSIONS: There is a wide range of medical conditions associated with multiple organ dysfunction syndrome in children. Traditionally, sepsis and trauma are the two conditions most commonly associated with multiple organ dysfunction syndrome both in children and adults. However, there are a number of other pathophysiologic processes that may result in multiple organ dysfunction syndrome. In this article, we discuss conditions such as cancer, congenital heart disease, and acute respiratory distress syndrome. In addition, the relationship between multiple organ dysfunction syndrome and clinical therapies such as hematopoietic stem cell transplantation and cardiopulmonary bypass is also considered. The purpose of this article is to describe the association of multiple organ dysfunction syndrome with a variety of conditions in an attempt to identify similarities, differences, and opportunities for therapeutic intervention.

T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial.

BACKGROUND: Chimeric antigen receptor (CAR) modified T cells targeting CD19 have shown activity in case series of patients with acute and chronic lymphocytic leukaemia and B-cell lymphomas, but feasibility, toxicity, and response rates of consecutively enrolled patients treated with a consistent regimen and assessed on an intention-to-treat basis have not been reported. We aimed to define feasibility, toxicity, maximum tolerated dose, response rate, and biological correlates of response in children and young adults with refractory B-cell malignancies treated with CD19-CAR T cells. METHODS: This phase 1, dose-escalation trial consecutively enrolled children and young adults (aged 1-30 years) with relapsed or refractory acute lymphoblastic leukaemia or non-Hodgkin lymphoma. Autologous T cells were engineered via an 11-day manufacturing process to express a CD19-CAR incorporating an anti-CD19 single-chain variable fragment plus TCR zeta and CD28 signalling domains. All patients received fludarabine and cyclophosphamide before a single infusion of CD19-CAR T cells. Using a standard 3 + 3 design to establish the maximum tolerated dose, patients received either 1 × 10(6) CAR-transduced T cells per kg (dose 1), 3 × 10(6) CAR-transduced T cells per kg (dose 2), or the entire CAR T-cell product if sufficient numbers of cells to meet the assigned dose were not generated. After the dose-escalation phase, an expansion cohort was treated at the maximum tolerated dose. The trial is registered with ClinicalTrials.gov, number NCT01593696. FINDINGS: Between July 2, 2012, and June 20, 2014, 21 patients (including eight who had previously undergone allogeneic haematopoietic stem-cell transplantation) were enrolled and infused with CD19-CAR T cells. 19 received the prescribed dose of CD19-CAR T cells, whereas the assigned dose concentration could not be generated for two patients (90% feasible). All patients enrolled were assessed for response. The maximum tolerated dose was defined as 1 × 10(6) CD19-CAR T cells per kg. All toxicities were fully reversible, with the most severe being grade 4 cytokine release syndrome that occurred in three (14%) of 21 patients (95% CI 3·0-36·3). The most common non-haematological grade 3 adverse events were fever (nine [43%] of 21 patients), hypokalaemia (nine [43%] of 21 patients), fever and neutropenia (eight [38%] of 21 patients), and cytokine release syndrome (three [14%) of 21 patients). INTERPRETATION: CD19-CAR T cell therapy is feasible, safe, and mediates potent anti-leukaemic activity in children and young adults with chemotherapy-resistant B-precursor acute lymphoblastic leukaemia. All toxicities were reversible and prolonged B-cell aplasia did not occur. FUNDING: National Institutes of Health Intramural funds and St Baldrick's Foundation.

Current concepts in the diagnosis and management of cytokine release syndrome.

As immune-based therapies for cancer become potent, more effective, and more widely available, optimal management of their unique toxicities becomes increasingly important. Cytokine release syndrome (CRS) is a potentially life-threatening toxicity that has been observed following administration of natural and bispecific antibodies and, more recently, following adoptive T-cell therapies for cancer. CRS is associated with elevated circulating levels of several cytokines including interleukin (IL)-6 and interferon γ, and uncontrolled studies demonstrate that immunosuppression using tocilizumab, an anti-IL-6 receptor antibody, with or without corticosteroids, can reverse the syndrome. However, because early and aggressive immunosuppression could limit the efficacy of the immunotherapy, current approaches seek to limit administration of immunosuppressive therapy to patients at risk for life-threatening consequences of the syndrome. This report presents a novel system to grade the severity of CRS in individual patients and a treatment algorithm for management of CRS based on severity. The goal of our approach is to maximize the chance for therapeutic benefit from the immunotherapy while minimizing the risk for life threatening complications of CRS.

Myeloid cells in peripheral blood mononuclear cell concentrates inhibit the expansion of chimeric antigen receptor T cells.

BACKGROUND AIMS: Autologous chimeric antigen receptor (CAR) T-cell therapies have shown promising clinical outcomes, but T-cell yields have been variable. CD19- and GD2-CAR T-cell manufacturing records were reviewed to identify sources of variability. METHODS: CD19-CAR T cells were used to treat 43 patients with acute lymphocytic leukemia or lymphoma and GD2-CAR T cells to treat eight patients with osteosarcoma and three with neuroblastoma. Both types of CAR T cells were manufactured using autologous peripheral blood mononuclear cells (PBMC) concentrates and anti-CD3/CD28 beads for T-cell enrichment and simulation. RESULTS: A comparison of the first 6 GD2- and the first 22 CD19-CAR T-cell products manufactured revealed that GD2-CAR T-cell products contained fewer transduced cells than CD19-CAR T-cell products (147 ± 102 × 10(6) vs 1502 ± 1066 × 10(6); P = 0.0059), and their PBMC concentrates contained more monocytes (31.4 ± 12.4% vs 18.5 ± 13.7%; P = 0.019). Among the first 28 CD19-CAR T-cell products manufactured, four had poor expansion yielding less than 1 × 10(6) transduced T cells per kilogram. When PBMC concentrates from these four patients were compared with the 24 others, PBMC concentrates of poorly expanding products contained greater quantities of monocytes (39.8 ± 12.9% vs. 15.3 ± 10.8%, P = 0.0014). Among the patients whose CD19-CAR T cells expanded poorly, manufacturing for two patients was repeated using cryopreserved PBMC concentrates but incorporating a monocyte depleting plastic adherence step, and an adequate dose of CAR T cells was produced for both patients. CONCLUSIONS: Variability in CAR T-cell expansion is due, at least in part, to the contamination of the starting PBMC concentrates with monocytes.

Paving the road ahead for CD19 CAR T-cell therapy.

PURPOSE OF REVIEW: Modern immunotherapies, most notably in the form of anti-CD19 chimeric antigen receptor (CAR) T cells, have produced significant clinical responses in otherwise refractory pre-B-cell acute lymphoblastic leukemia patients. Several groups have simultaneously reported robust response rates in children and adults alike. These early studies indicate an impending shift in paradigm for the treatment of acute lymphoblastic leukemia. Incorporating CD19 CAR T-cell therapy into upfront or salvage regimens has its challenges and opportunities. RECENT FINDINGS: Most CD19 CAR T-cell products in trial today are excellent at inducing minimal residual disease negative remissions, and most responding patients experience cytokine release syndrome and/or neurotoxicity. The challenges facing the CAR community involve how best to minimize the severity of cytokine release syndrome and neurotoxicity while maximizing antitumor efficacy, determining what role this therapy will play for the prophylaxis and treatment of central nervous system leukemia, and its implications on subsequent hematopoietic stem cell transplant given the emergence of CD19-negative relapses. SUMMARY: CD19 CAR T-cell therapy is a powerful new tool in the oncologist's arsenal. How it is incorporated into standard practice and how it will shift survival curves are the exciting questions that are waiting to be answered.

Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.

Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.

Targeting refractory/recurrent neuroblastoma and osteosarcoma with anti-CD3×anti-GD2 bispecific antibody armed T cells.

BACKGROUND: The survival benefit observed in children with neuroblastoma (NB) and minimal residual disease who received treatment with anti-GD2 monoclonal antibodies prompted our investigation into the safety and potential clinical benefits of anti-CD3×anti-GD2 bispecific antibody (GD2Bi) armed T cells (GD2BATs). Preclinical studies demonstrated the high cytotoxicity of GD2BATs against GD2+cell lines, leading to the initiation of a phase I/II study in recurrent/refractory patients. METHODS: The 3+3 dose escalation phase I study (NCT02173093) encompassed nine evaluable patients with NB (n=5), osteosarcoma (n=3), and desmoplastic small round cell tumors (n=1). Patients received twice-weekly infusions of GD2BATs at 40, 80, or 160×106 GD2BATs/kg/infusion complemented by daily interleukin-2 (300,000 IU/m2) and twice-weekly granulocyte macrophage colony-stimulating factor (250 µg/m2). The phase II segment focused on patients with NB at the dose 3 level of 160×106 GD2BATs/kg/infusion. RESULTS: Of the 12 patients enrolled, 9 completed therapy in phase I with no dose-limiting toxicities. Mild and manageable cytokine release syndrome occurred in all patients, presenting as grade 2-3 fevers/chills, headaches, and occasional hypotension up to 72 hours after GD2BAT infusions. GD2-antibody-associated pain was minimal. Median overall survival (OS) for phase I and the limited phase II was 18.0 and 31.2 months, respectively, with a combined OS of 21.1 months. A phase I NB patient had a complete bone marrow response with overall stable disease. In phase II, 10 of 12 patients were evaluable: 1 achieved partial response, and 3 showed clinical benefit with prolonged stable disease. Over 50% of evaluable patients exhibited augmented immune responses to GD2+targets post-GD2BATs, as indicated by interferon-gamma (IFN-γ) EliSpots, Th1 cytokines, and/or chemokines. CONCLUSIONS: This study demonstrated the safety of GD2BATs up to 160×106 cells/kg/infusion. Coupled with evidence of post-treatment endogenous immune responses, our findings support further investigation of GD2BATs in larger phase II clinical trials.

Simplified process for the production of anti-CD19-CAR-engineered T cells.

BACKGROUND AIMS: Adoptive immunotherapy with the use of chimeric antigen receptor (CAR)-engineered T cells specific for CD19 has shown promising results for the treatment of B-cell lymphomas and leukemia. This therapy involves the transduction of autologous T cells with a viral vector and the subsequent cell expansion. We describe a new, simplified method to produce anti-CD19-CAR T cells. METHODS: T cells were isolated from peripheral blood mononuclear cell (PBMC) with anti-CD3/anti-CD28 paramagnetic beads. After 2 days, the T cells were added to culture bags pre-treated with RetroNectin and loaded with the retroviral anti-CD19 CAR vector. The cells, beads and vector were incubated for 24 h, and a second transduction was then performed. No spinoculation was used. Cells were then expanded for an additional 9 days. RESULTS: The method was validated through the use of two PBMC products from a patient with B-cell chronic lymphoblastic leukemia and one PBMC product from a healthy subject. The two PBMC products from the patient with B-cell chronic lymphoblastic leukemia contained 11.4% and 12.9% T cells. The manufacturing process led to final products highly enriched in T cells with a mean CD3+ cell content of 98%, a mean expansion of 10.6-fold and a mean transduction efficiency of 68%. Similar results were obtained from the PBMCs of the first four patients with acute lymphoblastic leukemia treated at our institution. CONCLUSIONS: We developed a simplified, semi-closed system for the initial selection, activation, transduction and expansion of T cells with the use of anti-CD3/anti-CD28 beads and bags to produce autologous anti-CD19 CAR-transduced T cells to support an ongoing clinical trial.

Clustering of phosphatase RPTPα promotes Src signaling and the arthritogenic action of synovial fibroblasts.

Receptor-type protein phosphatase α (RPTPα) promotes fibroblast-dependent arthritis and fibrosis, in part, by enhancing the activation of the kinase SRC. Synovial fibroblasts lining joint tissue mediate inflammation and tissue damage, and their infiltration into adjacent tissues promotes disease progression. RPTPα includes an ectodomain and two intracellular catalytic domains (D1 and D2) and, in cancer cells, undergoes inhibitory homodimerization, which is dependent on a D1 wedge motif. Through single-molecule localization and labeled molecule interaction microscopy of migrating synovial fibroblasts, we investigated the role of RPTPα dimerization in the activation of SRC, the migration of synovial fibroblasts, and joint damage in a mouse model of arthritis. RPTPα clustered with other RPTPα and with SRC molecules in the context of actin-rich structures. A known dimerization-impairing mutation in the wedge motif (P210L/P211L) and the deletion of the D2 domain reduced RPTPα-RPTPα clustering; however, it also unexpectedly reduced RPTPα-SRC association. The same mutations also reduced recruitment of RPTPα to actin-rich structures and inhibited SRC activation and cellular migration. An antibody against the RPTPα ectodomain that prevented the clustering of RPTPα also inhibited RPTPα-SRC association and SRC activation and attenuated fibroblast migration and joint damage in arthritic mice. A catalytically inactivating RPTPα-C469S mutation protected mice from arthritis and reduced SRC activation in synovial fibroblasts. We conclude that RPTPα clustering retains it to actin-rich structures to promote SRC-mediated fibroblast migration and can be modulated through the extracellular domain.