scholarly journals Automated Generation and Phenotypic Characterization of Clinical Grade CD19 CAR-T Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1675-1675
Author(s):  
Ashish Sharma ◽  
Anne Roe ◽  
Filipa Blasco Lopes ◽  
Ruifu Liu ◽  
Jane Reese ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Central Memory ◽  
Car T Cells ◽  
Car T

Abstract BACKGROUND: Chimeric antigen receptor (CAR) T cells have shown enormous promise in the treatment of certain B cell malignancies. Access to treatment is still limited due to a variety of issues, including pricing and centralized manufacturing models. Generation of CAR-T cells using an automated platform, followed by rigorous functional phenotyping, may contribute to the development of a robust long-lasting therapy. METHODS: Here, we used the Miltenyi Prodigy (Miltenyi Biotech, Bergisch Gladbach, Germany) to automate the process of manufacturing genetically manipulated T cells in a closed system. The system obviates the need for clean room infrastructure. We tested the feasibility of utilizing the Miltenyi Prodigy to manufacture CAR-T cells using a CD19 scFV vector with the 4-1BB co-stimulatory domain. (Lentigen Technology, Inc, Gaithersburg, MD). The purity, differentiation capacity and effector function of the enriched CAR-T cells was studied using high-dimensional flow cytometry. Finally, the functional potential of these cells was tested in vitro and by treating NOD-SCID-gamma (NSG) mice infused with B cell lymphoma cells (Raji B cell), with the CAR-T cells. RESULTS: Starting with 1 x 108 CD4 and CD8 cells from donor apheresis products, CAR-T cells were expanded for 12 days in culture media containing with TransAct (Miltenyi Biotech), IL7 and IL15. The mean fold-expansion at day 12 was 44 ± 5.6, range 39-50 (n=3). The mean transduction efficiency of CAR-T vector was 20%, range 10-25% (n=3), which is similar to other reported methods. The CD19 CAR-T product was enriched in both the CD4 and CD8 T cells subsets, and showed high-level of cytotoxicity against CD19+ cell lines in vitro and in vivo (Figure 1: Mice treated with the CD19-CAR T demonstrated a marked reduction in disease burden as compared to T cell control as measured by bioluminescence imaging and flow cytometric analysis). The CAR-T product was enriched in cell subsets with both effector (CD27-CCR7-; ~20% of total cells) and central memory phenotypes (CD27+CCR7+; ~30% of total T cells). The effector CD4 and CD8 T cells showed increased expression of major functional T cell differentiation transcription factors (i.e. T-bet and GATA3) critical for the development of anti-tumor responses. Whereas, the central CD4 and CD8 T cells were enriched for the expression of TCF7 (a stemness related member of the WNT signaling known to increase longevity of these cells). The frequencies and phenotypes of these cells were maintained in peripheral blood of NSG mice infused with B cell lymphoma cells (Raji B cells), 1 week after treatment. A significant expansion of CD8+ effector T cells and a dramatic reduction in tumor burden was observed over the next 4 weeks in all major organs. Interestingly, we observed that smaller proportion of central-memory like cells (with higher TCF7 levels) continued to persist 6 weeks post-treatment, potentially contributing to a long-lived recallable response. Based on these data we have initiated a phase 1 clinical trial to test the therapeutic potential of the CAR-T product in patients with advanced/refractory B cell lymphoma. The first clinical grade manufacturing run resulted in a CD19 + cell yield of 1.4 x109. CONCLUSION: Our data highlight that the automated CAR-T generation platform (i.e. Miltenyi Prodigy) is effective at the generating purified functionally competent CAR-T cells. Further, findings from our phenotyping analyses show that the CAR-T product is enriched in both effector and central memory subsets and is effective at tumor clearance in vivo. Thus far, we have treated one patient with CD19 CAR-T manufactured on this platform and 2 more have been enrolled. Though this initial study is based on CD19 CAR-T cells, the approach described here could easily be utilized to genetically engineer T cells with gene constructs that are more relevant for specific cancers and infectious diseases. Disclosures No relevant conflicts of interest to declare.

Immunomodulatory Agent Lenalidomide Enhances Antitumor Functions of Chimeric Receptor-Modified T Cells in Vitro and in Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 805-805 ◽  
Author(s):  
Otáhal Pavel ◽  
Dana Prukova ◽  
Vlastimil Král ◽  
Radek Jaksa ◽  
Lucie Lateckova ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Chimeric Receptor ◽  
Car T Cells ◽  
Car T

Abstract Tumor immunotherapy based on the use of Chimeric Receptor Modified T cells (CAR T cells) is a promising approach for the treatment of a refractory hematological cancer. However, a robust response mediated by CAR T cells is observed only in a minority of patients and the expansion and persistence of CAR T cells in vivo is mostly unpredictable. In order to enhance the effectiveness of CAR-based immunotherapy we tested the immunoadjuvant properities of lenalidomide in combination with CAR19 T cells in a mouse model of B cell lymphoma. CAR19 construct which was used is composed of anti-CD19scFv joined with signaling domain of 4-1BB and TCR zeta and was delivered into T cell via lentiviral transduction. Lenalidomide is an immunomodulatory drug used for the treatment of multiple myeloma and selected B-cell malignancies, e.g. mantle cell lymphoma (MCL) or activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL). However, the precise mechanism of action is not very well understood and it is believed that is mediated by a modulation of activity of E3 ubiquitin ligase cereblon which leads to increased ubiquitinylation of Ikaros and Aiolos transcription factors resulting in changes of expression of various receptors on the surface of tumor cells. To test our hypothesis, immunodeficient NSG mice (NOD-SCID-gamma chain null mice) were s.c. transplanted with various human B cells lymphoma cells (MCL or ABC-DLBCL) followed by i.v treatment with CAR19 T cells with or without daily i.p. lenalidomide. First, when we measured the growth of tumors following treatment with CAR19 T cells plus lenalidomide we found that this combination more effectively suppressed growth of s.c. B-NHL tumors than treatment with only CAR19 T cells or only lenalidomide (Figure 1, 1x10e7 Nemo tumor cell s.c., followed with 2 doses of 1x10(7) CAR19 T cells + Lenalidomide daily, tumor weight was measured 14 days after treatment). Additionally, in this experiment lenalidomide significantly enhanced infiltration of residual tumors by CD8+CAR19 T cells (not shown). Next, we tested the response of CAR19 T cells in vitro to B-NHL cells in the presence or, absence of lenalidomide to determine the costimulatory effect of lenalidomide on signaling via CAR, our data show that lenalidomide significantly enhanced functional response of CAR19 T cells following recognition of B cells in vitro which is demonstrated by enhanced production of IFN-gamma and by increased expression of CD69 by CAR19 T cells, interestingly, this effect was seen only if CAR19 T cells but not B-NHL cells were pre-treated with lenalidomide or, when we activated CAR19 T cell with antibody to CAR but not with antibody to CD3. Thus, our data indicate that lenalidomide might work through direct effects on T cells and specifically enhance signaling via CAR. The biochemical events underlying this costimulatory effect of lenalidomide on signaling by CAR are currently being investigated. In summary, our data support the use of lenalidomide for augmentation CAR-based immunotherapy in clinical settings. Figure 1 Figure 1. Disclosures Klener: Cellgene: Research Funding.

scholarly journals TGF-β/IL-7 Chimeric Switch Receptor-Expressing CAR-T Cells Inhibit Recurrence of CD19-Positive B Cell Lymphoma

2021 ◽  
Vol 22 (16) ◽  
pp. 8706
Author(s):  
Kyung-Eun Noh ◽  
Jun-Ho Lee ◽  
So-Yeon Choi ◽  
Nam-Chul Jung ◽  
Ji-Hee Nam ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Single Chain ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

Chimeric antigen receptor (CAR)-T cells are effective in the treatment of hematologic malignancies but have shown limited efficacy against solid tumors. Here, we demonstrated an approach to inhibit recurrence of B cell lymphoma by co-expressing both a human anti-CD19-specific single-chain variable fragment (scFv) CAR (CD19 CAR) and a TGF-β/IL-7 chimeric switch receptor (tTRII-I7R) in T cells (CD19 CAR-tTRII-I7R-T cells). The tTRII-I7R was designed to convert immunosuppressive TGF-β signaling into immune-activating IL-7 signaling. The effect of TGF-β on CD19 CAR-tTRII-I7R-T cells was assessed by western blotting. Target-specific killing by CD19 CAR-tTRII-I7R-T cells was evaluated by Eu-TDA assay. Daudi tumor-bearing NSG (NOD/SCID/IL2Rγ-/-) mice were treated with CD19 CAR-tTRII-I7R-T cells to analyze the in vivo anti-tumor effect. In vitro, CD19 CAR-tTRII-I7R-T cells had a lower level of phosphorylated SMAD2 and a higher level of target-specific cytotoxicity than controls in the presence of rhTGF-β1. In the animal model, the overall survival and recurrence-free survival of mice that received CD19 CAR-tTRII-I7R-T cells were significantly longer than in control mice. These findings strongly suggest that CD19 CAR-tTRII-I7R-T cell therapy provides a new strategy for long-lasting, TGF-β-resistant anti-tumor effects against B cell lymphoma, which may lead ultimately to increased clinical efficacy.

Direct Comparison of In Vivo Fate of Second and Third-Generation CD19-Specific Chimeric Antigen Receptor (CAR)-T Cells in Patients with B-Cell Lymphoma: Reversal of Toxicity from Tonic Signaling

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1851-1851 ◽  
Author(s):  
Diogo Gomes da Silva ◽  
Malini Mukherjee ◽  
Madhuwanti Srinivasan ◽  
Olga Dakhova ◽  
Hao Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
2Nd Generation ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

Abstract Although adoptive transfer of T cells with second-generation CD19-specific CARs containing CD28 or 4-1BB costimulatory endodomains shows remarkable clinical efficacy against B cell malignancies, the optimal choice of costimulatory domains in these and other CARs remains controversial. Depending on the precise CAR structure and specificity, individual endodomains may be associated with deleterious ligand-independent tonic signaling in the transduced T cell. Long et al. (Nat Med 2015) established the CD28 co-stimulatory endodomain can have a toxic tonic signaling effect, but it is unclear if tonic 4-1BB signaling may have deleterious consequences as well, and if such effects can be reversed. We therefore modeled tonic CAR signaling in T cells by transducing them with gammaretroviral vectors expressing 2nd-generation CD19.CAR constructs containing either the CD28 or 4-1BB costimulatory endodomain (in addition to the CD3-ζ chain endodomain). Compared to CAR-T cells with the CD28 endodomain alone, those with 4-1BB alone expanded 70% more slowly following transduction. Impaired expansion of 4-1BB CD19.CAR-T cells was coupled with a 4-fold increase in apoptosis and a gradual downregulation of CAR expression, and was a consequence of 4-1BB-associated tonic TRAF2-dependent signaling, leading to activation of NF-κB, upregulation of Fas and augmented Fas-dependent activation-induced T cell death (AICD). Moreover, expression of 4-1BB CAR from a gammaretroviral vector increased tonic signaling through a self-amplifying/positive feedback effect on the retroviral LTR promoter. Because of the toxicity of 4-1BB in our gammaretroviral CAR.CD19 construct (manifest by delayed expansion and increased apoptosis) we could not directly compare the in vivo fate of T cells expressing CAR.CD19 4-1BB with that of co-administered CAR.CD19 CD28 T cells in patients with lymphoma. We found, however, that the adverse effects of tonic 4-1BB costimulation could be overcome in a 3rd-generation CAR.CD19 vector, containing both CD28 and 4-1BB costimulatory molecules in tandem. We thus compared the fate of a 3rd-generation vector containing both CD28 and 4-1BB costimulatory domains with that of a 2nd-generation vector containing CD28 alone. Six patients with refractory/relapsed diffuse large B-cell lymphoma received 2 cell populations, one expressing 2nd and one expressing 3rd generation vectors. To determine whether CD28 alone was optimal (which would suggest 4-1BB is antagonistic) or whether 4-1BB had an additive or synergistic effect contributing to superior persistence and expansion of the CD28-41BB combination, patients were simultaneously infused with 1-20×106 of both 2nd and 3rd generation CAR+ T cells/m2 48-72 hours after lymphodepletion with cyclophosphamide (500 mg/m2/d) and fludarabine (30 mg/m2/d) × 3. Persistence of infused T cells was assessed in blood by CD19.CAR qPCR assays specific for each CAR. Molecular signals peaked approximately 2 weeks post infusion, remaining detectable for up to 6 months. The 3rd-generation CAR-T cells had a mean 23-fold (range 1.1 to 109-fold) higher expansion than 2nd-generation CAR-T cells and correspondingly longer persistence. Two patients had grade 2 cytokine release syndrome, with elevation of proinflammatory cytokines, including IL-6, at the time of peak expansion of T cells. Of the 5 patients evaluable for response, 2 entered complete remission (the longest ongoing for 9 months), 1 has had continued complete remission after autologous stem cell transplantation, 1 had a partial response, and 1 progressed. In conclusion, our data indicate that infusion of T cells carrying a CD19.CAR containing CD28 and 4-1BB endodomains is safe and can have efficacy at every dose level tested. Additionally, in a side-by-side comparison, the 3rdgeneration vector produced greater in vivo expansion and persistence than an otherwise identical CAR-T cell population with CD28 alone. Disclosures Rooney: Cell Medica: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Viracyte: Equity Ownership. Heslop:Celgene: Patents & Royalties, Research Funding; Chimerix: Other: Endpoint adjudication committee; Viracyte: Equity Ownership; Cell Medica: Patents & Royalties: Licensing agreement EBV-specific T cells.

scholarly journals Analysis of CAR-T and Immune Cells within the Tumor Micro-Environment of Diffuse Large B-Cell Lymphoma Post CAR-T Treatment By Multiplex Immunofluorescence

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 678-678 ◽  
Author(s):  
Pei-Hsuan Chen ◽  
Mikel Lipschitz ◽  
Kyle Wright ◽  
Philippe Armand ◽  
Caron A. Jacobson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract BACKGROUND: Axicabtagene ciloleucel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy that shows efficacy in patients with refractory diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma and transformed follicular lymphoma after failure of conventional therapy. However, the exact mechanism of anti-tumor immunity is poorly understood, in part due to the dearth of data on the events in the tumor micro-environment (TME) that occur upon exposure to CAR-T cells. We sought to quantify and characterize both CAR-T cells and non-CAR T cells within the TME of DLBCL using tissue biopsy samples collected in the ZUMA-1 multicenter trial of CAR-T cell therapy for patients with refractory DLBCL. METHODS: Tumor samples obtained from patients 5-30 days (median 10 days) after CAR-T infusion ("CAR-treated", n=14) and randomly-selected untreated ("untreated ", n=15) archival DLBCL tissue samples were analyzed by multiplex immunofluorescence using formalin-fixed, paraffin embedded tissue sections, with successive labeling by the primary antibodies KIP-1 and/or KIP-3 (recognizing separate CD19 CAR epitopes), PAX5, PD-1, CD4, and CD8, followed by secondary amplification and tyramide-conjugated fluorophores. For each case, at least 3 representative 20x fields of view were selected and imaged using a multispectral imaging platform. Two specific image analysis algorithms were designed to accurately identify CD4 and CD8 T cells and PAX5+ DLBCL cells simultaneously, then to threshold PD-1 and KIP-1/-3 by relative fluorescent units (RFU) in each phenotype. RESULTS: We identified CAR T-cells within the fixed biopsy samples of CAR-treated DLBCLs by immunostaining with CAR T-cell specific antibody KIP-1; at the timepoints analyzed, CAR T-cells comprised only a small minority of total T- cells (<2%) and included CD4+ and CD8+ T-cells. Immunostaining with a second antibody, KIP-3, validated the presence of CAR T-cells in these cases and confirmed the KIP-1 results. Expression of the T cell activation marker PD-1 was detected among majority of KIP-1+ cells. Further analysis that included KIP1-negative cells revealed that the percentage of CD8+ cells co-expressing PD-1 across all CD8+ cells was higher in the CAR-treated DLBCLs compared to the untreated DLBCLs (mean 50.1% vs 17.5%, p<0.0001 with unpaired t test ), indicating CD8 T cell activation within the tumor environment. In contrast, PD-1 positivity across CD4+ T cells were equivalent between the two groups (mean 21.8% vs 21.6%, ns with unpaired t test). The percentages of total, CD4+, and CD8+ T-cell populations in the TME were similar between the CAR-treated DLBCL and untreated biopsies. CONCLUSIONS: CD4+ and CD8+ CAR-T cells can be detected in CAR-treated DLBCL patient tissue biopsies by multiplex immunofluorescence. At the time points analyzed to date, CAR-T cells comprise only a small percentage of all T-cells (<2%) within the TME. However, the presence of gene marked T cells with downregulated CAR protein expression is also possible. The activation marker PD-1 is preferentially expressed by KIP-1-negative CD8+ T cells compared to CD4+ T cells in CAR-T treated DLBCLs relative to untreated DLBCLs. These data implicate preferential activation of CD8+ non-CAR "by-stander" T-cells in the post CAR-T TME, and the possible benefit of combining PD-1 blockade with CAR-T therapy in DLBCL. *PH.C and M.L share equal contribution. Disclosures Armand: Otsuka: Research Funding; Affimed: Consultancy, Research Funding; Pfizer: Consultancy; Infinity: Consultancy; Adaptive: Research Funding; Merck: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Roche: Research Funding; Tensha: Research Funding. Roberts:KITE: Employment. Rossi:KITE: Employment. Bot:KITE: Employment. Go:KITE: Employment. Rodig:Merck: Research Funding; Bristol Myers Squibb: Research Funding; Affimed: Research Funding; KITE: Research Funding.

scholarly journals Application of Novel T Cell Immunotherapeutics to Drive Antigen-Specific Activation, Expansion, and Differentiation of CD19 Chimeric Antigen Receptor T Cells (CAR T-cells)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-35
Author(s):  
Moriah Rabin ◽  
Mengyan Li ◽  
Scott Garforth ◽  
Jacqueline Marino ◽  
Jian Hua Zheng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Effector Memory ◽  
Mhc Molecules ◽  
Car T Cells ◽  
Car T

Background: While chimeric antigen receptor T cells (CAR T-cells) induce dramatic remissions of refractory or recurrent B cell malignancies, the durability of these remissions is frequently limited by subsequent reduction in circulating CAR T-cells and/or by diminution of their effector function. We hypothesized that we could overcome this therapeutic limitation and increase the functional activity and longevity of CAR T-cells by selectively deriving them from virus-specific effector memory T cells. We have developed biologics we termed synTacs (artificial immunological synapse for T-cell activation), which selectively activate and expand antigen-specific CD8+ T cells in vitro and in vivo by recapitulating signals delivered at the immunological synapse. The synTacs consist of dimeric Fc domain scaffolds linking CD28- or 4-1BB-specific ligands to HLA-A2 MHC molecules covalently tethered to virus-derived peptides. Treatment of PBMCs from CMV-exposed donors with synTacs presenting a CMV-derived peptide (pp65-NLVPMVATV) induce vigorous and selective ex vivo and in vivo expansion of highly functional CMV-specific CD8+ T cells, with potent antiviral activity. We used these synTacs to selectively generate CAR T-cells from CMV-specific effector memory CD8+ T cells, which could be further expanded by restimulation with the CMV-specific synTacs. Methods: We treated PBMCs from CMV-exposed donors in media supplemented with either IL-2 or IL-7/12/15 with a synTac containing the CMV-derived pp65 peptide presented by HLA-A2 MHC molecules linked to ligands capable of stimulating CD28- or 4-1BB-dependent costimulatory pathways. PBMCs activated either with anti-CD3/CD28 or the CMV-specific synTacs were transduced with lentivirus expressing an anti-CD19 CAR and a GFP reporter gene. CMV-specific CD8+ T cells were quantified by tetramer staining and CAR T-cells were detected by GFP expression determined by flow cytometric analysis. The functional activity of the CD19 CAR T-cells was determined by a B cell-specific cytotoxic assay. Results: After 7 days, treatment of PBMCs with CMV-specific synTacs rapidly induced robust activation and &gt;50-fold expansion of CMV-specific CD8+ T cells expressing effector memory markers. Treatment of the PBMCs with CMV-specific synTacs selectively activated CMV-specific T cells and enabled them to be specifically transduced with a CD19-specific CAR lentivirus and converted into CD19 CAR T-cells. These CMV-specific CD19 CAR T-cells displayed potent dose-responsive cytotoxic activity targeting purified primary B cells. Furthermore, these CMV-specific CD19 CAR T-cells could be selectively expanded by in vitro treatment with CMV-specific synTacs. Conclusions: SynTacs are versatile immunotherapeutics capable of selective in vitro and in vivo activation and expansion of virus-specific CD8+ T cells with potent antiviral cytotoxic activity. After selective lentiviral transduction and conversion into CD19 CAR T-cells, their co-expression of the CMV-specific T cell receptor enabled them to be potently stimulated and activated by in vitro treatment with CMV synTacs. The modular design of synTacs facilitates efficient coupling of other costimulatory ligands - such as OX40 or GITRL - or cytokines, such as IL-2, IL-7, or IL-15, to enable the selective in vivo delivery of defined costimulatory signals or cytokines to the CAR T-cells expressing CMV-specific TCR. This strategy has the potential to boost the in vivo activity of tumor-specific CAR T-cells after infusion and enable more durable and potent treatment of refractory/recurrent B cell malignancies. Disclosures Almo: Cue Biopharma: Current equity holder in publicly-traded company, Patents & Royalties: Patent number: 62/013,715, Research Funding. Goldstein:Cue Biopharma: Research Funding.

scholarly journals Third-generation anti-CD19 chimeric antigen receptor T-cells incorporating a TLR2 domain for relapsed or refractory B-cell lymphoma: a phase I clinical trial protocol (ENABLE)

BMJ Open ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. e034629 ◽  
Author(s):  
Philip George ◽  
Nathaniel Dasyam ◽  
Giulia Giunti ◽  
Brigitta Mester ◽  
Evelyn Bauer ◽  
...  
Keyword(s):  
T Cells ◽  
Phase I ◽  
B Cell ◽  
Chimeric Antigen Receptor ◽  
Dose Escalation ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Third Generation ◽  
Car T Cells ◽  
Car T

IntroductionAutologous T-cells transduced to express a chimeric antigen receptor (CAR) directed against CD19 elicit high response rates in relapsed or refractory (r/r) B-cell non-Hodgkin lymphoma (B-NHL). However, r/r B-NHL remissions are durable in fewer than half of recipients of second-generation CAR T-cells. Third-generation (3G) CARs employ two costimulatory domains, resulting in improved CAR T-cell efficacy in vitro and in animal models in vivo. This investigator-initiated, phase I dose escalation trial, termed ENABLE, will investigate the safety and preliminary efficacy of WZTL-002, comprising autologous T-cells expressing a 3G anti-CD19 CAR incorporating the intracellular signalling domains of CD28 and Toll-like receptor 2 (TLR2) for the treatment of r/r B-NHL.Methods and analysisEligible participants will be adults with r/r B-NHL including diffuse large B-cell lymphoma and its variants, follicular lymphoma, transformed follicular lymphoma and mantle cell lymphoma. Participants must have satisfactory organ function, and lack other curative options. Autologous T-cells will be obtained by leukapheresis. Following WZTL-002 manufacture and product release, participants will receive lymphodepleting chemotherapy comprising intravenous fludarabine and cyclophosphamide. A single dose of WZTL-002 will be administered intravenously 2 days later. Targeted assessments for cytokine release syndrome and immune cell effector-associated neurotoxicity syndrome, graded by the American Society Transplantation and Cellular Therapy criteria, will be made. A modified 3+3 dose escalation scheme is planned starting at 5×104 CAR T-cells/kg with a maximum dose of 1×106 CAR T-cells/kg. The primary outcome of this trial is safety of WZTL-002. Secondary outcomes include feasibility of WZTL-002 manufacture and preliminary measures of efficacy.Ethics and disseminationEthical approval for the study was granted by the New Zealand Health and Disability Ethics Committee (reference 19/STH/69) on 23 June 2019 for Protocol V.1.2. Trial results will be reported in a peer-reviewed journal, and results presented at scientific conferences or meetings.Trial registration numberNCT04049513

G-CSF does not worsen toxicities and efficacy of CAR-T cells in refractory/relapsed B-cell lymphoma

2020 ◽  
Vol 55 (12) ◽  
pp. 2347-2349
Author(s):  
Eugenio Galli ◽  
Vincent Allain ◽  
Roberta Di Blasi ◽  
Sophie Bernard ◽  
Laetitia Vercellino ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Car T Cells ◽  
Car T

scholarly journals Long-term in vivo microscopy of CAR T cell dynamics during eradication of CNS lymphoma in mice

2019 ◽  
Vol 116 (48) ◽  
pp. 24275-24284 ◽  
Author(s):  
Matthias Mulazzani ◽  
Simon P. Fräßle ◽  
Iven von Mücke-Heim ◽  
Sigrid Langer ◽  
Xiaolan Zhou ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Growth ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Therapeutic Effects ◽  
Term Survival ◽  
Car T Cells ◽  
Car T

T cells expressing anti-CD19 chimeric antigen receptors (CARs) demonstrate impressive efficacy in the treatment of systemic B cell malignancies, including B cell lymphoma. However, their effect on primary central nervous system lymphoma (PCNSL) is unknown. Additionally, the detailed cellular dynamics of CAR T cells during their antitumor reaction remain unclear, including their intratumoral infiltration depth, mobility, and persistence. Studying these processes in detail requires repeated intravital imaging of precisely defined tumor regions during weeks of tumor growth and regression. Here, we have combined a model of PCNSL with in vivo intracerebral 2-photon microscopy. Thereby, we were able to visualize intracranial PCNSL growth and therapeutic effects of CAR T cells longitudinally in the same animal over several weeks. Intravenous (i.v.) injection resulted in poor tumor infiltration of anti-CD19 CAR T cells and could not sufficiently control tumor growth. After intracerebral injection, however, anti-CD19 CAR T cells invaded deeply into the solid tumor, reduced tumor growth, and induced regression of PCNSL, which was associated with long-term survival. Intracerebral anti-CD19 CAR T cells entered the circulation and infiltrated distant, nondraining lymph nodes more efficiently than mock CAR T cells. After complete regression of tumors, anti-CD19 CAR T cells remained detectable intracranially and intravascularly for up to 159 d. Collectively, these results demonstrate the great potential of anti-CD19 CAR T cells for the treatment of PCNSL.

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