scholarly journals Foamy Virus Based Vectors for CAR-T Cell Development

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4646-4646
Author(s):  
Emmanouil Simantirakis ◽  
Vassilis Atsaves ◽  
Ioannis Tsironis ◽  
Margarita Gkyzi ◽  
Kostas Konstantopoulos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Peripheral Blood ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Ifn Γ

Introduction A novel approach that can cover the therapeutic gap in NHL treatment are the autologous T cells, expressing Chimeric Antigen Receptors (CAR-T cells) against tumor markers. Such clinical-grade products based on Lenti (LV) or Retro- vectors have hit the market. An alternative vector system for CAR gene transfer in T-cells are Foamy Viruses (FV). To evaluate the potential of FV vectors in CAR-T cell development, we synthesized an antiCD19 scFv cDNA and cloned it in both an FV and an LV backbone; both vectors were tested in paired experiments Material and Methods The anti-CD19 CAR was under the control of the EF1a promoter; EGFP expression was under the control of an IRES2 element. The anti-CD19 CAR sequence was deduced from published data. FV vectors were made with a 4-plasmid vector system in 293T cells. 2nd generation LV vectors were purchased from Addgene. Cord blood (CB), healthy donor peripheral blood (PB) and CLL patients' PB was used as a source for CD3+ cells using immunomagnetic enrichment. Informed consent has been obtained in all cases of human sample use. T cells were activated by antiCD3/CD28 beads and transduced with antiCD19 LV or FV vectors. Transduction efficiency was assayed by flow cytometry (FCM) using a PE-conjugated anti-mouse Fab antibody. FV and LV CAR-T cells were expanded with Rapid Expansion Protocol (REP) and their cytotoxicity assays was evaluated against the CD19+ cell lines Raji and Daudi. The CLL patient derived CAR-Ts were evaluated against autologous B cells. Cytotoxicity was evaluated with an FCM protocol using CFSE-stained target cells vs unstained effector CARTs in different ratios. At the end of the incubation cells were stained with 7AAD to discriminate against live/dead cells. CAR-T cell activation was also assayed by INF-γ ELISA, following cocultures with target cells at a ratio of 1:1 for 24h. Results Vector titers: LV vector titers were between 3-5x10^5 TU/ml for both LV vectors (with or without EGFP cassette). FV vector titers were between 2-4x10^5 TU/ml regardless of the presence of the EGFP cassette. Tx efficiency: FV can mediate efficient gene transfer on T cells in the presence of heparin at an effective dose of 20-40 U/ml using a spinoculation technique. Transduction efficiency ranged from 40-65% at MOI=3-5, and was comparable to the transduction efficiency of LV vectors at a much higher MOI (10 to 30). Cytotoxicity data on lines: Following REP, the cell population consisted mostly (close to 96% purity) of CAR-T cells regardless of the vector used or of the T cell source. Effector cells were cocultured with the CD19+ cell lines, Daudi and Raji at varying ratios. With cord blood derived FV-CAR-T cells, at 4h post coculture we observed a 39.4% cell lysis at a ratio of 10:1 effector to target (n=1). Similar results were obtained for LV vectors. Peripheral blood derived CAR-T cells at THE same ratio (10:1), demonstrated 83.9% and 93.1% cell lysis for FV-CART and LV-CART cells respectively (n=2). Cytotoxicity data on CLL cells: T-cells from peripheral blood of CLL patients were used to generate LV- and FV-CAR-T cells. At the ratio of 10:1, we observed 73.1% and 69,8% cytotoxicity for FV-CAR-Ts and 70.1% and 70.7% with LV-CAR-Ts, in 2 independent paired experiments. IFN as activation marker: In two paired activation experiments, CB-derived FV-CAR-T cells secrete 560 and 437pg/ml of IFN-γ; similarly, LV-CAR-Ts secrete 534 and 554pg/ml IFN-γ. Untransduced control cells, produced 68pg/ml and 12pg/ml for FV-CAR-T and LV-CAR-T experimental arm respectively. Conclusion In the current work, we developed and tested FV vectors for anti- CD19 CAR-T cell production. We proved that FV viral vectors are capable of mediating efficient gene transfer to human T cells. We developed a method to efficiently transfer FV vectors into T-cells, using a clinically relevant protocol with heparin. The FV-derived CAR T cells demonstrate the same cytotoxic properties in vitro as their LV-derived counterpart and the same activation levels in the presence of CD19 expressing target cells as measured by IFN-γ secretion. FV CARTs derived from PB of CLL patients were capable of mediating comparable cytotoxicity levels as their LV-derived counterparts. Overall, we provide a proof of concept that FVs could be a safe and efficient alternative to LV derived vectors for CAR-T cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals MDSC Suppression of CAR T Cells Can be Reduced By Targeted Signaling Disruption

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4438-4438 ◽  
Author(s):  
Estelle V Cervantes ◽  
Justin C. Boucher ◽  
Sae Bom Lee ◽  
Kristen Spitler ◽  
Kayla Reid ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Research Funding ◽  
Target Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Cell Counts ◽  
Car T

CAR T cells are genetically modified with an extracellular scFv, transmembrane domain, and intracellular costimulatory and CD3ζ domains. Two treatments received the approval from the FDA for the treatment of acute lymphoblastic leukemia and diffuse large B cell lymphoma. However, CAR T cell persistence remains a problem. A reason for this may be that myeloid cells such as myeloid derived suppressor cells (MDSCs) may be contributing to the reduced persistence of CAR T cells. MDSCs originate from myeloid cells and have been implicated in the suppression of the immune system in the tumor microenvironment. To determine what effect MDSCs might have during CAR T cell production we co-cultured MDSCs during CAR transduction. We found gene transfer was lower for m19z (38.7% vs 46.8%), m1928z (24.1% vs 39.1%), and m19hBBz (35.8% vs 46.2%) CAR T cells co-cultured with MDSCs compared to those that were not (Fig 1A). There was also a reduction in total T cell counts for m19z (58%), m1928z (88%), and m19hBBz (65%) after MDSC co-culture. This data suggests MDSCs present during CAR T cell production can alter gene transfer and total T cell counts. We also investigated the effect MDSCs can potentially have on CAR T cells when present during CAR T cell antigen stimulation. CAR T cells co-cultured with MDSCs in vitro had significant reductions in m19z, m1928z, and m19hBBz CAR T cell mediated killing against target cells (Fig 1B). We also found significantly lower production of IFNγ in m19z, m1928z, and m19hBBz CAR T cells cultured with MDSCs compared to cells cultured with total BM (Fig 1C). After 24hr stimulation with MDSCs and target cells, there was lower expression of activation markers PD1 and LAG3 by CAR T cells compared to culture without MDSCs. This suggests that MDSCs reduce CAR T cell activation, killing, and cytokine production. We also looked at how CAR proliferation after antigen stimulation is affected by MDSC co-culture and found MDSCs significantly reduced CAR proliferation in vitro. To evaluate if we could create a more resistant CAR T cell to MDSC suppression null mutations were incorporated into a CD28 CAR. We mutated the YMNM and PRRP subdomains of CD28 which signal through PI3K and ITK respectively leaving only PYAP active (mut06). When MDSCs were co-cultured with mut06 T cells during production mut06 had a smaller reduction in gene transfer (21% vs 38%) and T cell counts (80% vs 88%) compared to m1928z. Mut06 also had a significantly higher expression of PD1 and TIM3 compared to m1928z after production with MDSCs. In vitro when mut06 was co-cultured with MDSCs it had the same killing ability as m1928z without MDSCs and was significantly better at killing compared to m1928z co-cultured with MDSCs (Fig 1B). To examine the effect of MDSCs on CAR T cells in vivo we injected C57BL6 mice with CAR T cells followed by MDSCs a week later. In these ongoing experiments we found mut06 had significantly higher numbers of CAR T cells in the blood compared to m1928z (Fig1D). Overall our data shows that MDSCs can suppress CAR T cell function when present during production as well as CAR stimulation. It also suggests that by optimizing CD28 CAR signaling using mut06 that we were able to generate a CAR T cell that is more resistant to MDSCs. Furthermore, we may be able to recapitulate the effect of mut06 with targeted inhibitors. Figure 1 Disclosures Davila: GlaxoSmithKline: Consultancy; Novartis: Research Funding; Adaptive: Consultancy; Celgene: Research Funding; Precision Biosciences: Consultancy; Bellicum: Consultancy; Anixa: Consultancy; Atara: Research Funding.

scholarly journals Preclinical Evaluation of ALLO-819, an Allogeneic CAR T Cell Therapy Targeting FLT3 for the Treatment of Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3921-3921 ◽  
Author(s):  
Cesar Sommer ◽  
Hsin-Yuan Cheng ◽  
Yik Andy Yeung ◽  
Duy Nguyen ◽  
Janette Sutton ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Target Cells ◽  
Employment Equity ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Autologous chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B-cell leukemias, lymphomas and multiple myeloma, raising interest in using CAR T cell therapies in AML. These therapies are produced using a patient's own T cells, an approach that has inherent challenges, including requiring significant time for production, complex supply chain logistics, separate GMP manufacturing for each patient, and variability in performance of patient-derived cells. Given the rapid pace of disease progression combined with limitations associated with the autologous approach and treatment-induced lymphopenia, many patients with AML may not receive treatment. Allogeneic CAR T (AlloCAR T) cell therapies, which utilize cells from healthy donors, may provide greater convenience with readily available off-the-shelf CAR T cells on-demand, reliable product consistency, and accessibility at greater scale for more patients. To create an allogeneic product, the TRAC and CD52 genes are inactivated in CAR T cells using Transcription Activator-Like Effector Nuclease (TALEN®) technology. These genetic modifications are intended to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. We have previously described the functional screening of a library of anti-FLT3 single-chain variable fragments (scFvs) and the identification of a lead FLT3 CAR with optimal activity against AML cells and featuring an off-switch activated by rituximab. Here we characterize ALLO-819, an allogeneic FLT3 CAR T cell product, for its antitumor efficacy and expansion in orthotopic models of human AML, cytotoxicity in the presence of soluble FLT3 (sFLT3), performance compared with previously described anti-FLT3 CARs and potential for off-target binding of the scFv to normal human tissues. To produce ALLO-819, T cells derived from healthy donors were activated and transduced with a lentiviral construct for expression of the lead anti-FLT3 CAR followed by efficient knockout of TRAC and CD52. ALLO-819 manufactured from multiple donors was insensitive to ALLO-647 (100 µg/mL) in in vitro assays, suggesting that it would avoid elimination by the lymphodepletion regimen. In orthotopic models of AML (MV4-11 and EOL-1), ALLO-819 exhibited dose-dependent expansion and cytotoxic activity, with peak CAR T cell levels corresponding to maximal antitumor efficacy. Intriguingly, ALLO-819 showed earlier and more robust peak expansion in mice engrafted with MV4-11 target cells, which express lower levels of the antigen relative to EOL-1 cells (n=2 donors). To further assess the potency of ALLO-819, multiple anti-FLT3 scFvs that had been described in previous reports were cloned into lentiviral constructs that were used to generate CAR T cells following the standard protocol. In these comparative studies, the ALLO-819 CAR displayed high transduction efficiency and superior performance across different donors. Furthermore, the effector function of ALLO-819 was equivalent to that observed in FLT3 CAR T cells with normal expression of TCR and CD52, indicating no effects of TALEN® treatment on CAR T cell activity. Plasma levels of sFLT3 are frequently increased in patients with AML and correlate with tumor burden, raising the possibility that sFLT3 may act as a decoy for FLT3 CAR T cells. To rule out an inhibitory effect of sFLT3 on ALLO-819, effector and target cells were cultured overnight in the presence of increasing concentrations of recombinant sFLT3. We found that ALLO-819 retained its killing properties even in the presence of supraphysiological concentrations of sFLT3 (1 µg/mL). To investigate the potential for off-target binding of the ALLO-819 CAR to human tissues, tissue cross-reactivity studies were conducted using a recombinant protein consisting of the extracellular domain of the CAR fused to human IgG Fc. Consistent with the limited expression pattern of FLT3 and indicative of the high specificity of the lead scFv, no appreciable membrane staining was detected in any of the 36 normal tissues tested (n=3 donors). Taken together, our results support clinical development of ALLO-819 as a novel and effective CAR T cell therapy for the treatment of AML. Disclosures Sommer: Allogene Therapeutics, Inc.: Employment, Equity Ownership. Cheng:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Yeung:Pfizer Inc.: Employment, Equity Ownership. Nguyen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Sutton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Melton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Valton:Cellectis, Inc.: Employment, Equity Ownership. Poulsen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Djuretic:Pfizer, Inc.: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Chaparro-Riggers:Pfizer, Inc.: Employment, Equity Ownership. Sasu:Allogene Therapeutics, Inc.: Employment, Equity Ownership.

scholarly journals Lenalidomide Enhances CAR-T Cell Activity Against Solid Tumor Cells

2020 ◽  
Vol 29 ◽  
pp. 096368972092082 ◽  
Author(s):  
Zhixiong Wang ◽  
Guomin Zhou ◽  
Na Risu ◽  
Jiayu Fu ◽  
Yan Zou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cytokine Secretion ◽  
Target Cells ◽  
Functional Assay ◽  
Cell Functions ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chimeric antigen receptor (CAR) T-cell immunotherapy still faces many challenges in the treatment of solid tumors, one of which is T-cell dysfunction or exhaustion. Immunomodulator lenalidomide may improve CAR T-cell function. In this study, the effects of lenalidomide on CAR T-cell functions (cytotoxicity, cytokine secretion, and cell proliferation) were investigated. Two different CAR T cells (CD133-specific CAR and HER2-specific CAR) were prepared, and the corresponding target cells including human glioma cell line U251 CD133-OE that overexpress CD133 and human breast cancer cell line MDA-MB-453 were used for functional assay. We found that lenalidomide promoted the killing of U251 CD133-OE by CD133-CAR T cells, the cytokine secretion, and the proliferation of CD133-CAR T cells. Lenalidomide also enhanced the cytotoxicity against MDA-MB-453 and the cytokine secretion of HER2-CAR T cells but did not affect their proliferation significantly. Furthermore, lenalidomide may regulate the function of CAR T cells by inducing the degradation of transcription factors Ikaros and Aiolos.

scholarly journals Enhanced CAR-T activity against established tumors by polarizing human T cells to secrete interleukin-9

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Lintao Liu ◽  
Enguang Bi ◽  
Xingzhe Ma ◽  
Wei Xiong ◽  
Jianfei Qian ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
Effector Memory ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Ifn Γ

AbstractCAR-T cell therapy is effective for hematologic malignancies. However, considerable numbers of patients relapse after the treatment, partially due to poor expansion and limited persistence of CAR-T cells in vivo. Here, we demonstrate that human CAR-T cells polarized and expanded under a Th9-culture condition (T9 CAR-T) have an enhanced antitumor activity against established tumors. Compared to IL2-polarized (T1) cells, T9 CAR-T cells secrete IL9 but little IFN-γ, express central memory phenotype and lower levels of exhaustion markers, and display robust proliferative capacity. Consequently, T9 CAR-T cells mediate a greater antitumor activity than T1 CAR-T cells against established hematologic and solid tumors in vivo. After transfer, T9 CAR-T cells migrate effectively to tumors, differentiate to IFN-γ and granzyme-B secreting effector memory T cells but remain as long-lived and hyperproliferative T cells. Our findings are important for the improvement of CAR-T cell-based immunotherapy for human cancers.

Update of CARTITUDE-1: A phase Ib/II study of JNJ-4528, a B-cell maturation antigen (BCMA)-directed CAR-T-cell therapy, in relapsed/refractory multiple myeloma.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 8505-8505 ◽  
Author(s):  
Jesus G. Berdeja ◽  
Deepu Madduri ◽  
Saad Zafar Usmani ◽  
Indrajeet Singh ◽  
Enrique Zudaire ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T ◽  
Phase 1B

8505 Background: JNJ-68284528 (JNJ-4528) is a chimeric antigen receptor T (CAR-T) cell therapy containing 2 BCMA-targeting single-domain antibodies. Here we present updated CARTITUDE-1 (NCT03548207) phase 1b results with longer follow-up. Methods: Pts had MM per IMWG criteria, measurable disease, received ≥3 prior regimens or were double refractory to a PI and IMiD, and received anti-CD38 antibody. Cyclophosphamide 300 mg/m2+ fludarabine 30 mg/m2 over 3 days were used for lymphodepletion. JNJ-4528 (median, 0.73x106 CAR+ viable T cells/kg) was given as a single infusion. Cytokine release syndrome (CRS) was graded by Lee et al2014 and neurotoxicity by CTCAE, v5.0 and ASTCT grading. Response was assessed per IMWG criteria. Results: As of 17 Jan 2020, median follow-up is 9 mo (3–17). Phase 1b enrollment is complete (N = 29 treated; median 5 (3–18) prior lines, 76% penta-exposed, 86% triple-refractory, 31% penta-refractory, 97% refractory to last line of therapy). Most frequent adverse events (AEs) were neutropenia (100%), CRS (93%), and thrombocytopenia (93%). Grade (Gr) ≥3 hematologic AEs were neutropenia (100%), thrombocytopenia (69%), and leukopenia (59%). 27 (93%) pts had CRS; 25 Gr 1–2, 1 Gr 3, and 1 Gr 5 (day 99 subsequent to dose-limiting toxicity of prolonged Gr 4 CRS). Median time to onset of CRS was 7 days (2–12). 4 pts had treatment-related neurotoxicity: 3 Gr 1–2 and 1 Gr 3. ORR was 100%, with 22 (76%) stringent complete responses (sCRs), 6 (21%) very good partial responses (VGPRs), and 1 (3%) PR. Median time to ≥CR was 2 mo (1–9). 26/29 pts are progression-free, with 6-mo progression-free survival rate of 93% and longest response ongoing at 15 mo. 1 death due to CRS and 1 to acute myeloid leukemia (not treatment-related) occurred during the study. All 16 pts (14 sCR, 2 VGPR) evaluable at 6 mo were minimal residual disease negative at 10−5 or 10−6. JNJ-4528 CAR+ T cell expansion peaked between day 10–14. At 6-mo individual follow-up, 22/28 pts had JNJ-4528 CAR+ T cells below the level of quantification (2 cells/µL) in peripheral blood, suggesting CAR-T persistence in peripheral blood did not seem to correlate with deepening of response. At peak expansion, preferential expansion of CD8+ CAR-T cells with a central memory phenotype was observed in peripheral blood. Conclusions: JNJ-4528 treatment led to responses in all pts. These responses were early, deep, and durable at a low dose of CAR-T cells with 26/29 (90%) pts progression free at median 9-mo follow-up. CRS was manageable in most pts, supporting outpatient dosing. Clinical trial information: NCT03548207 .

IL-18 Secreting CAR T Cells Enhance Cell Persistence, Induce Prolonged B Cell Aplasia and Eradicate CD19+ Tumor Cells without Need for Prior Conditioning

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 816-816 ◽  
Author(s):  
Mauro P. Avanzi ◽  
Dayenne G. van Leeuwen ◽  
Xinghuo Li ◽  
Kenneth Cheung ◽  
Hyebin Park ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Car T Cells ◽  
Car T Cell ◽  
Cytokine Analysis ◽  
Car T ◽  
Ifn Γ

Abstract Chimeric antigen receptor (CAR) T cell therapy has consistently shown significant results against acute lymphoblastic leukemia (ALL) in clinical trials1. However, results with other hematological or solid malignancies have been far more modest2. These disparate outcomes could be partially due to an inhibitory tumor microenvironment that suppresses CAR T cell function3. Thus, in order to expand the anti-tumor CAR T cell applications, a novel strategy in which these cells are capable of overcoming the hostile tumor microenvironment is needed. The cytokine interleukin-18 (IL-18) induces IFN-γ secretion, enhances the Th1 immune response and activates natural killer and cytotoxic T cells4. Early phase clinical trials that utilized systemic administration of recombinant IL-18 for the treatment of both solid and hematological malignancies have demonstrated the safety of this therapy5. We hypothesize that CAR T cells that constitutively secrete IL-18 could enhance CAR T cell survival and anti-tumor activity, and also activate cells from the endogenous immune system. To generate CAR T cells that constitutively secrete IL-18, we modified SFG-1928z and SFG-19m28mz CAR T cell constructs and engineered bicistronic human and murine vectors with a P2A element to actively secrete the IL-18 protein (1928z-P2A-hIL18 and 19m28mz-P2A-mIL18, respectively). Human and mouse T cells were transduced with these constructs and in vitro CAR T cell function was validated by coculturing the CAR T cells with CD19+ tumor cells and collecting supernatant for cytokine analysis. Both human and mouse CAR T cells secreted increased levels of IL-18, IFN-γ and IL-2. Proliferation and anti-tumor cytotoxic experiments were conducted with human T cells by coculturing CAR T cells with hCD19+ expressing tumor cells. 1928z-P2A-hIL18 CAR T cells had enhanced proliferation over 7 days and enhanced anti-tumor cytotoxicity over 72 hours when compared to 1928z CAR T cells (p=0.03 and 0.01, respectively) Next, the in vivo anti-tumor efficacy of the IL-18 secreting CAR T cell was tested in xenograft and syngeneic mouse models. Experiments were conducted without any prior lympho-depleting regimen. In the human CAR T cell experiments, Scid-Beige mice were injected with 1x106 NALM-6 tumor cells on day 0 and 5x106 CAR T cells on day 1. Survival curves showed a significant improvement in mouse survival with the 1928z-P2A-hIL18 CAR T cell treatment when compared to 1928z CAR T cell (p=0.006). Subsequently, to determine if IL-18 secreting CAR T cells could also improve anti-tumor efficacy in immunocompetent mice, we tested the murine 19m28mz-P2A-mIL18 CAR T cells in a syngeneic mouse model. The C57BL/6 hCD19+/- mCD19+/- mouse model was utilized and injected with 1x106 EL4 hCD19+ tumor cells on day 0 and 2.5 x106 CAR T cells on day 1. Mice treated with 19m28mz-P2A-mIL18 CAR T cells had 100% long-term survival, when compared to 19m28mz (p<0.0001). 19m28mz-P2A-mIL18 CAR T cells were detected in peripheral blood for up to 30 days after injection, whereas the 19m28mz CAR T cells were not detectable at any time point. In addition, 19m28mz-P2A-mIL18 CAR T cells were capable of inducing B cell aplasia for greater than 70 days, whereas 19m28mz treatment was not capable of inducing B cell aplasia. In vivo serum cytokine analysis demonstrated that 19m28mz-P2A-mIL18 CAR T cells, as compared to 19m28mz, significantly increased the levels of IFN-γ and TNF-α in the peripheral blood for up to 14 days after injection (p<0.0001 and 0.01, respectively). Despite the increase in IFN-γ and TNF-α cytokines, there was no increase in IL-6 levels. Our findings demonstrate that anti-CD19 CAR T cells that constitutively secrete IL-18 significantly increase serum cytokine secretion, enhance CAR T cell persistence, induce long-term B cell aplasia and improve mouse survival, even without any prior preconditioning. To our knowledge, this is the first description of an anti-CD19 CAR T cell that constitutively secretes IL-18 and that induces such high levels of T cell proliferation, persistence and anti-tumor cytotoxicity. We are currently investigating other mechanisms by which this novel CAR T cell functions, its interactions with the endogenous immune system, as well as testing its applicability in other tumor types. We anticipate that the advances presented by this new technology will expand the applicability of CAR T cells to a wider array of malignancies. Disclosures Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Transposon-Based CAR T Cells in Acute Leukemias: Where Are We Going?

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1337 ◽  
Author(s):  
Chiara F. Magnani ◽  
Sarah Tettamanti ◽  
Gaia Alberti ◽  
Ilaria Pisani ◽  
Andrea Biondi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Cell Therapy ◽  
Viral Vector ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Chimeric Antigen Receptor (CAR) T-cell therapy has become a new therapeutic reality for refractory and relapsed leukemia patients and is also emerging as a potential therapeutic option in solid tumors. Viral vector-based CAR T-cells initially drove these successful efforts; however, high costs and cumbersome manufacturing processes have limited the widespread clinical implementation of CAR T-cell therapy. Here we will discuss the state of the art of the transposon-based gene transfer and its application in CAR T immunotherapy, specifically focusing on the Sleeping Beauty (SB) transposon system, as a valid cost-effective and safe option as compared to the viral vector-based systems. A general overview of SB transposon system applications will be provided, with an update of major developments, current clinical trials achievements and future perspectives exploiting SB for CAR T-cell engineering. After the first clinical successes achieved in the context of B-cell neoplasms, we are now facing a new era and it is paramount to advance gene transfer technology to fully exploit the potential of CAR T-cells towards next-generation immunotherapy.

scholarly journals Distribution of chimeric antigen receptor-modified T cells against CD19 in B-cell malignancies

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhitao Ying ◽  
Ting He ◽  
Xiaopei Wang ◽  
Wen Zheng ◽  
Ningjing Lin ◽  
...  
Keyword(s):  
Clinical Trial ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Target Cells ◽  
B Cell Malignancies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Over Time

Abstract Background The unprecedented efficacy of chimeric antigen receptor T (CAR-T) cell immunotherapy of CD19+ B-cell malignancies has opened a new and useful way for the treatment of malignant tumors. Nonetheless, there are still formidable challenges in the field of CAR-T cell therapy, such as the biodistribution of CAR-T cells in vivo. Methods NALM-6, a human B-cell acute lymphoblastic leukemia (B-ALL) cell line, was used as target cells. CAR-T cells were injected into a mice model with or without target cells. Then we measured the distribution of CAR-T cells in mice. In addition, an exploratory clinical trial was conducted in 13 r/r B-cell non-Hodgkin lymphoma (B-NHL) patients, who received CAR-T cell infusion. The dynamic changes in patient blood parameters over time after infusion were detected by qPCR and flow cytometry. Results CAR-T cells still proliferated over time after being infused into the mice without target cells within 2 weeks. However, CAR-T cells did not increase significantly in the presence of target cells within 2 weeks after infusion, but expanded at week 6. In the clinical trial, we found that CAR-T cells peaked at 7–21 days after infusion and lasted for 420 days in peripheral blood of patients. Simultaneously, mild side effects were observed, which could be effectively controlled within 2 months in these patients. Conclusions CAR-T cells can expand themselves with or without target cells in mice, and persist for a long time in NHL patients without serious side effects. Trial registration The registration date of the clinical trial is May 17, 2018 and the trial registration numbers is NCT03528421.

scholarly journals Addition of Fludarabine to Cyclophosphamide Lymphodepletion Improves In Vivo Expansion of CD19 Chimeric Antigen Receptor-Modified T Cells and Clinical Outcome in Adults with B Cell Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3773-3773 ◽  
Author(s):  
Cameron J Turtle ◽  
Laila-Aicha Hanafi ◽  
Carolina Berger ◽  
Daniel Sommermeyer ◽  
Barbara Pender ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Expansion ◽  
Research Funding ◽  
Car T Cells ◽  
Cell Dose ◽  
Car T Cell ◽  
Car T ◽  
Ifn Γ ◽  
T Cell Expansion

Abstract BACKGROUND: Chemotherapy followed by autologous T cells that are genetically modified to express a CD19-specific chimeric antigen receptor (CAR) has shown promise as a novel therapy for patients with relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL); however, the risk of severe cytokine release syndrome (sCRS) and neurotoxicity has tempered enthusiasm for widespread application of this approach. The functional heterogeneity that is inherent in CAR-T cell products that are manufactured from undefined T cell subsets has hindered definition of dose-response relationships and identification of factors that may impact efficacy and toxicity. METHODS: We are conducting the first clinical trial that administers CD19 CAR-T cells manufactured from a defined composition of T cell subsets to adults with relapsed or refractory B-ALL. CD8+ and CD4+ T cells were enriched from each patient, transduced with a CD19 CAR lentivirus and separately expanded in vitro before formulation for infusion in a 1:1 ratio of CD8+:CD4+ CAR+ T cells at 2x105, 2x106 or 2x107 CAR-T cells/kg. Prior to CAR-T cell infusion, patients underwent lymphodepletion with a high-dose cyclophosphamide (Cy)-based regimen with or without fludarabine (Flu). RESULTS: Twenty-nine adults with B-ALL (median age 40, range 22 - 73 years; median 17% marrow blasts, range 0 - 97%), including 10 patients who had relapsed after allogeneic transplantation, received at least one CAR-T cell infusion. Twenty-four of 26 restaged patients (92%) achieved bone marrow (BM) complete remission (CR) by flow cytometry. CD4+ and CD8+ CAR-T cells expanded in vivo after infusion and their number in blood correlated with the infused CAR-T cell dose. Thirteen patients received lymphodepletion with Cy-based regimens without Flu. Ten of 12 restaged patients (83%) achieved BM CR by flow cytometry; however, 7 of these (70%) relapsed a median of 66 days after CAR-T cell infusion. Disease relapse correlated with a loss of CAR-T cell persistence in blood. We observed a CD8 cytotoxic T cell response to the murine scFv component of the CAR transgene that contributed to CAR-T cell rejection, and resulted in lack of CAR-T cell expansion after a second CAR-T cell infusion in 5 patients treated for persistent or relapsed disease. To minimize immune-mediated CAR-T cell rejection 14 patients were treated with Cy followed by Flu lymphodepletion (Cy/Flu, Cy 60 mg/kg x 1 and Flu 25 mg/m2 x 3-5) before CAR-T cell infusion. All patients (100%) who received Cy/Flu lymphodepletion achieved BM CR after CAR-T cell infusion. CAR-T cell expansion and persistence in blood was higher in Cy/Flu-lymphodepleted patients compared to their counterparts who received Cy alone (Day 28 after 2x106 CAR-T cells/kg: CD8+ CAR-T cells, mean 55.8/μL vs 0.10/μL, p<0.01; CD4+ CAR-T cells, 2.1/μL vs 0.02/μL, p<0.01), enabling reduction in CAR-T cell dose for Cy/Flu-treated patients. Patients who received Cy/Flu lymphodepletion appear to have longer disease-free survival (DFS) than those who received Cy alone (Cy/Flu, median, not reached; Cy alone, 150 days, p=0.09). CAR-T cell infusion was associated with sCRS, characterized by fever and hypotension requiring intensive care in 7 of 27 patients (26%) and neurotoxicity (≥ grade 3 CTCAE v4.03) in 13 of 27 patients (48%). Two patients died following complications of sCRS. Patients with sCRS or neurotoxicity had higher peak serum levels of IL-6, IFN-γ, ferritin and C-reactive protein compared to those without serious toxicity. Importantly IL-6, IFN-γ and TNF-α levels in serum collected on day 1 after CAR-T cell infusion from those who subsequently developed neurotoxicity were higher than those collected from their counterparts who did not develop neurotoxicity (IL-6, p<0.01; IFN-γ, p=0.05; TNF-α, p=0.04), providing potential biomarkers to test early intervention strategies to prevent neurotoxicity. The risks of sCRS and neurotoxicity correlated with higher leukemic marrow infiltration and increasing CAR-T cell dose. We have now adopted a risk-stratified approach to CAR-T cell dosing in which the CAR-T cell dose inversely correlates to the patient's bone marrow tumor burden. CONCLUSION: Risk-stratified dosing of CD19 CAR-T cells of defined subset composition is feasible and safe in a majority of patients with refractory B-ALL, and results in a CR rate of 92%. Addition of Flu to Cy-based lymphodepletion improves CAR-T cell expansion, persistence and DFS. Disclosures Turtle: Juno Therapeutics: Patents & Royalties, Research Funding. Berger:Juno Therapeutics: Patents & Royalties. Jensen:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Riddell:Adaptive Biotechnologies: Consultancy; Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Membership on an entity's Board of Directors or advisory committees. Maloney:Seattle Genetics: Honoraria; Janssen Scientific Affairs: Honoraria; Roche/Genentech: Honoraria; Juno Therapeutics: Research Funding.

scholarly journals CAR T cell therapy in B-cell acute lymphoblastic leukaemia: Insights from mathematical models

2020 ◽  
Author(s):  
Odelaisy León-Triana ◽  
Soukaina Sabir ◽  
Gabriel F. Calvo ◽  
Juan Belmonte-Beitia ◽  
Salvador Chulián ◽  
...  
Keyword(s):  
Mathematical Model ◽  
T Cells ◽  
T Cell ◽  
Side Effects ◽  
B Cell ◽  
Target Cells ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

AbstractImmunotherapies use components of the patient immune system to selectively target cancer cells. The use of CAR T cells to treat B-cell malignancies – leukaemias and lymphomas– is one of the most successful examples, with many patients experiencing long-lasting complete responses to this therapy. This treatment works by extracting the patient’s T cells and adding them the CAR group, which enables them to recognize and target cells carrying the antigen CD19+, that is expressed in these haematological tumors.Here we put forward a mathematical model describing the time response of leukaemias to the injection of CAR T-cells. The model accounts for mature and progenitor B-cells, tumor cells, CAR T cells and side effects by incorporating the main biological processes involved. The model explains the early post-injection dynamics of the different compartments and the fact that the number of CAR T cells injected does not critically affect the treatment outcome. An explicit formula is found that provides the maximum CAR T cell expansion in-vivo and the severity of side effects. Our mathematical model captures other known features of the response to this immunotherapy. It also predicts that CD19+ tumor relapses could be the result of the competition between tumor and CAR T cells analogous to predator-prey dynamics. We discuss this fact on the light of available evidences and the possibility of controlling relapses by early re-challenging of the tumor with stored CAR T cells.

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