Direct Delivery of piggyBac CD19 CAR T Cells Has Potent Anti-tumor Activity against ALL Cells in CNS in a Xenograft Mouse Model.
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ABSTRACT:
SUBMITTER: Tanaka K
PROVIDER: S-EPMC7321814 | biostudies-literature | 2020 Sep
REPOSITORIES: biostudies-literature
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Project description:Adoptive immunotherapy is a new potential method of tumour therapy, among which anti-CD19 chimeric antigen receptor T-cell therapy (CAR-T cell), is a typical treatment agent for haematological malignancies. Previous clinical trials showed that the quality and phenotype of CAR-T cells expanded ex vivo would seriously affect the tumour treatment efficacy. Although magnetic beads are currently widely used to expand CAR-T cells, the optimal expansion steps and methods have not been completely established. In this study, the differences between CAR-T cells expanded with anti-CD3/CD28 mAb-coated beads and those expanded with cell-based aAPCs expressing CD19/CD64/CD86/CD137L/mIL-15 counter-receptors were compared. The results showed that the number of CD19-specific CAR-T cells with a 4-1BB and CD28 co-stimulatory domain was much greater with stimulation by aAPCs than that with beads. In addition, the expression of memory marker CD45RO was higher, whereas expression of exhausted molecules was lower in CAR-T cells expanded with aAPCs comparing with the beads. Both CAR-T cells showed significant targeted tumoricidal effects. The CAR-T cells stimulated with aAPCs secreted apoptosis-related cytokines. Moreover, they also possessed marked anti-tumour effect on NAMALWA xenograft mouse model. The present findings provided evidence on the safety and advantage of two expansion methods for CAR-T cells genetically modified by piggyBac transposon system.
| S-EPMC7812273 | biostudies-literature
Project description:Anti-CD19 chimeric antigen receptor (CAR) T cell therapies can cause severe cytokine-release syndrome (CRS) and neurotoxicity, impeding their therapeutic application. Here we generated a new anti-CD19 CAR molecule (CD19-BBz(86)) derived from the CD19-BBz prototype bearing co-stimulatory 4-1BB and CD3ζ domains. We found that CD19-BBz(86) CAR T cells produced lower levels of cytokines, expressed higher levels of antiapoptotic molecules and proliferated more slowly than the prototype CD19-BBz CAR T cells, although they retained potent cytolytic activity. We performed a phase 1 trial of CD19-BBz(86) CAR T cell therapy in patients with B cell lymphoma (ClinicalTrials.gov identifier NCT02842138 ). Complete remission occurred in 6 of 11 patients (54.5%) who each received a dose of 2 × 108-4 × 108 CD19-BBz(86) CAR T cells. Notably, no neurological toxicity or CRS (greater than grade 1) occurred in any of the 25 patients treated. No significant elevation in serum cytokine levels after CAR T cell infusion was detected in the patients treated, including in those who achieved complete remission. CD19-BBz(86) CAR T cells persistently proliferated and differentiated into memory cells in vivo. Thus, therapy with the new CD19-BBz(86) CAR T cells produces a potent and durable antilymphoma response without causing neurotoxicity or severe CRS, representing a safe and potent anti-CD19 CAR T cell therapy.
| S-EPMC7518381 | biostudies-literature
Project description:BackgroundAggressive B cell lymphoma with secondary central nervous system (CNS) involvement (SCNSL) carries a dismal prognosis. Chimeric antigen receptor (CAR) T cells (CAR-T) targeting CD19 have revolutionized the treatment for B cell lymphomas; however, only single cases with CNS manifestations successfully treated with CD19 CAR-T have been reported.MethodsWe prospectively enrolled 4 patients with SCNSL into our study to assess clinical responses and monitor T cell immunity.ResultsTwo of four SNCSL patients responded to the CD19-targeted CAR-T. Only one patient showed a substantial expansion of peripheral (PB) CAR-T cells with an almost 100-fold increase within the first week after CAR-T. The same patient also showed marked neurotoxicity and progression of the SNCSL despite continuous surface expression of CD19 on the lymphoma cells and an accumulation of CD4+ central memory-type CAR-T cells in the CNS. Our studies indicate that the local production of chemokine IP-10, possibly through its receptor CXCR3 expressed on our patient's CAR-T, could potentially have mediated the local accumulation of functionally suboptimal anti-tumor T cells.ConclusionsOur results demonstrate expansion and homing of CAR-T cells into the CNS in SNCSL patients. Local production of chemokines such as IP-10 may support CNS infiltration by CAR-T cells but also carry the potential of amplifying local toxicity. Future studies investigating numbers, phenotype, and function of CAR-T in the different body compartments of SNSCL patients receiving CAR-T will help to improve local delivery of "fit" and highly tumor-reactive CAR-T with low off-target reactivity into the CNS.
| S-EPMC10864416 | biostudies-literature
Project description:Adoptive T cell therapy using chimeric antigen receptor (CAR)-modified T cells is a promising cancer immunotherapy. We previously developed a non-viral method of gene transfer into T cells using a piggyBac transposon system to improve the cost-effectiveness of CAR-T cell therapy. Here, we have further improved our technology by a novel culture strategy to increase the transfection efficiency and to reduce the time of T cell manufacturing. Using a CH2CH3-free CD19-specific CAR transposon vector and combining irradiated activated T cells (ATCs) as feeder cells and virus-specific T cell receptor (TCR) stimulation, we achieved 51.4% ± 14% CAR+ T cells and 2.8-fold expansion after 14 culture days. Expanded CD19.CAR-T cells maintained a significant fraction of CD45RA+CCR7+ T cells and demonstrated potent antitumor activity against CD19+ leukemic cells both in vitro and in vivo. Therefore, piggyBac-based gene transfer may provide an alternative to viral gene transfer for CAR-T cell therapy.
| S-EPMC5907825 | biostudies-literature
Project description:Successful CAR T cell therapy for the treatment of solid tumors requires exemplary CAR T cell expansion, persistence and fitness, and the ability to target tumor antigens safely. Here we address this constellation of critical attributes for successful cellular therapy by using integrated technologies that simplify development and derisk clinical translation. We have developed a CAR-CD19 T cell that secretes a CD19-anti-Her2 bridging protein. This cell therapy strategy exploits the ability of CD19-targeting CAR T cells to interact with CD19 on normal B cells to drive expansion, persistence and fitness. The secreted bridging protein potently binds to Her2-positive tumor cells, mediating CAR-CD19 T cell cytotoxicity in vitro and in vivo. Because of its short half-life, the secreted bridging protein will selectively accumulate at the site of highest antigen expression, ie. at the tumor. Bridging proteins that bind to multiple different tumor antigens have been created. Therefore, antigen-bridging CAR-CD19 T cells incorporate critical attributes for successful solid tumor cell therapy. This platform can be exploited to attack tumor antigens on any cancer.
| S-EPMC7971483 | biostudies-literature
Project description:Background aimsAdoptive 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.MethodsT 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.ResultsThe 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.ConclusionsWe 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.
| S-EPMC4141724 | biostudies-literature
Project description:CD19-directed chimeric antigen receptor (CD19CAR) T-cell therapy has been successful in treating several B-cell lineage malignancies, including B-cell non-Hodgkin lymphoma (NHL). This modality has not yet been extended to NHL manifesting in the central nervous system (CNS), primarily as a result of concerns for potential toxicity. CD19CAR T cells administered IV are detectable in cerebrospinal fluid (CSF), suggesting that chimeric antigen receptor (CAR) T cells can migrate from the periphery into the CNS, where they can potentially mediate antilymphoma activity. Here, we report the outcome of a subset of patients with primary CNS lymphoma (PCNSL; n = 5) who were treated with CD19CAR T cells in our ongoing phase 1 clinical trial. All patients developed grade ≥ 1 cytokine release syndrome and neurotoxicity post-CAR T-cell infusion; toxicities were reversible and tolerable, and there were no treatment-related deaths. At initial disease response, 3 of 5 patients (60%; 90% confidence interval, 19-92%) seemed to achieve complete remission, as indicated by resolution of enhancing brain lesions; the remaining 2 patients had stable disease. Although the study cohort was small, we demonstrate that using CD19CAR T cells to treat PCNSL can be safe and feasible. This trial was registered at www.clinicaltrials.gov as #NCT02153580.
| S-EPMC8945630 | biostudies-literature
Project description:Cryopreservation of chimeric antigen receptor (CAR) T cells facilitates shipment, timing of infusions, and storage of subsequent doses. However, reports on the impact of cryopreservation on CAR T cell efficacy have been mixed. We retrospectively compared clinical outcomes between patients who received cryopreserved versus fresh CAR T cells for treatment of B cell leukemia across two cohorts of pediatric and young adult patients: those who received anti-CD22 CAR T cells and those who received bispecific anti-CD19/22 CAR T cells. Manufacturing methods were consistent within each trial but differed between the two trials, allowing for exploration of cryopreservation within different manufacturing platforms. Among 40 patients who received anti-CD22 CAR T cells (21 cryopreserved cells and 19 fresh), there were no differences in in vivo expansion, persistence, incidence of toxicities, or disease response between groups with cryopreserved and fresh CAR T cells. Among 19 patients who received anti-CD19/22 CAR T cells (11 cryopreserved and 8 fresh), patients with cryopreserved cells had similar expansion, toxicity incidence, and disease response, with decreased CAR T cell persistence. Overall, our data demonstrate efficacy of cryopreserved CAR T cells as comparable to fresh infusions, supporting cryopreservation, which will be crucial for advancing the field of cell therapy.
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Project description:Our study provides compelling support to an effective CAR-T cell anti-aging intervention using NMN, a key NAD+ intermediate. We found the clinical potential of NMN for CAR-T cell immunotherapy for human cancers, attributed to their central memory phenotype. The cells are more viable, proliferative, and long-lived in vivo. Furthermore, these CAR-T cells exerted great antitumor efficacy against tumors in human xenograft mouse models. Detailed molecular mechanisms responsible for the pleiotropic effects of NMN need to be investigated further. So RNA-seq was used to analyze the different gene expression of these CAR-T cells.
2023-04-01 | GSE200367 | GEO
Project description:Clinical trials of CD19-specific chimeric antigen receptor (CAR19) T cells have demonstrated remarkable efficacy against relapsed and refractory B cell malignancies. The piggyBac transposon system offers a less complex and more economical means for generating CAR19 T cells compared to viral vectors. We have previously optimized a protocol for the generation of CAR19 T cells using the piggyBac system, but we found that CAR19 T cells had poor in vivo efficacy and persistence, probably due to deleterious FcγR interactions with the CAR's IgG1 Fc-containing spacer domain. We therefore designed three CD19-specifc CARs that lacked the IgG1 Fc region, and we incorporated combinations of CD28 or 4-1BB transmembrane and co-stimulatory domains. PiggyBac-generated CAR19 T cells expressing these re-designed constructs all demonstrated reactivity in vitro specifically against CD19+ cell lines. However, those combining CD28 transmembrane and co-stimulatory domains showed CD4 predominance and inferior cytotoxicity. At high doses, CAR19 T cells were effective against B-ALL in a xenograft mouse model, regardless of co-stimulatory domain. At diminishing doses, 4-1BB co-stimulation led to greater potency and persistence of CAR19 T cells, and it provided protection against B-ALL re-challenge. Production of potent CAR T cells using piggyBac is simple and cost-effective, and it may enable wider access to CAR T cell therapy.
| S-EPMC6094355 | biostudies-literature