In Vivo Proof of Concept of Activity and Safety of UCART19, an Allogeneic “Off-the-Shelf” Adoptive T-Cell Immunotherapy Against CD19+ B-Cell Leukemias

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4689-4689 ◽  
Author(s):  
Agnes Gouble ◽  
Brian Philip ◽  
Laurent Poirot ◽  
Cecile Schiffer-Mannioui ◽  
Roman Galetto ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
B Cell ◽  
Graft Versus Host Reaction ◽  
Proof Of Concept ◽  
Graft Versus Host ◽  
Car T Cells ◽  
Car T

Abstract Chimeric antigen receptor (CAR)-redirected T-cells have given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. A limitation of the current autologous approach is that CAR T-cells must be manufactured on a "per patient basis". We have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic "off-the-shelf" engineered CD19-CAR+ T-cell–based frozen products. Our platform involves the use of transcription activator-like effector nucleases (TALEN™), which mediate the simultaneous inactivation of two genes through genome editing. The knockout of the TCR alpha gene eliminates TCR expression and is intended to abrogate the donor T-cell’s potential for graft-versus-host disease (GvHD), while knocking out the CD52 gene makes donor T-cells resistant to the lymphodepleting agent alemtuzumab. In addition, our T-cells are engineered to coexpress the RQR8 gene as a safety feature, with the aim of rendering them sensitive to the monoclonal antibody rituximab. We previously provided proof-of-concept for the application of this approach by manufacturing TCR/CD52-deficient RQR8+ and CD19-CAR+ T-cells (UCART19) using a good manufacturing practice–compatible process, and we also demonstrated that the resulting UCART19 cells were functional using in vitro assays. Here we report the ability of UCART19 cells to engraft into an orthotopic human CD19+ lymphoma xenograft immunodeficient mouse model. UCART19 cells exhibited antitumor activity equivalent to that of standard CD19 CAR T-cells. We also demonstrated that UCART19 cells did not mediate alloreactivity in a xeno-GvHD mouse model. Furthermore, the effectiveness of the rituximab-induced depletion mechanism of RQR8+ cells was shown in an immunocompetent mouse model. In conclusion, our work significantly enlarges upon previous results by showing in vivo that (1) concomitant inactivation of a second gene has no deleterious effects on T-cells, (2) the antitumor potency of manufactured TCR/CD52-deficient CD19–CAR+ T-cells is similar to that of standard CD19-CAR+ T-cells, (3) TCR gene inactivation is efficient at preventing potential graft-versus-host reaction, and (4) allogeneic T-cells can be depleted by the use of rituximab. This valuable dataset supports the development of allogeneic CAR T-cells, and UCART19 will be investigated in an exploratory, first-in-human, clinical trial where refractory/relapsed CD19+ B-cell leukemia patients are to be enrolled. Disclosures Gouble: Cellectis SA: Employment. Poirot:Cellectis SA: Employment. Schiffer-Mannioui:Cellectis SA: Employment. Galetto:Cellectis SA: Employment. Derniame:Cellectis SA: Employment. Arnould:Cellectis SA: Employment. Desseaux:Cellectis SA: Employment. Smith:Cellectis SA: Employment.

scholarly journals Switchable control over in vivo CAR T expansion, B cell depletion, and induction of memory

2018 ◽  
Vol 115 (46) ◽  
pp. E10898-E10906 ◽  
Author(s):  
Sophie Viaud ◽  
Jennifer S. Y. Ma ◽  
Ian R. Hardy ◽  
Eric N. Hampton ◽  
Brent Benish ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Rest Period ◽  
Cell System ◽  
Cellular Activity ◽  
Dosing Regimen ◽  
Car T Cells ◽  
Car T

Chimeric antigen receptor (CAR) T cells with a long-lived memory phenotype are correlated with durable, complete remissions in patients with leukemia. However, not all CAR T cell products form robust memory populations, and those that do can induce chronic B cell aplasia in patients. To address these challenges, we previously developed a switchable CAR (sCAR) T cell system that allows fully tunable, on/off control over engineered cellular activity. To further evaluate the platform, we generated and assessed different murine sCAR constructs to determine the factors that afford efficacy, persistence, and expansion of sCAR T cells in a competent immune system. We find that sCAR T cells undergo significant in vivo expansion, which is correlated with potent antitumor efficacy. Most importantly, we show that the switch dosing regimen not only allows control over B cell populations through iterative depletion and repopulation, but that the “rest” period between dosing cycles is the key for induction of memory and expansion of sCAR T cells. These findings introduce rest as a paradigm in enhancing memory and improving the efficacy and persistence of engineered T cell products.

scholarly journals GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

2019 ◽  
Vol 11 (485) ◽  
pp. eaau7746 ◽  
Author(s):  
Eric L. Smith ◽  
Kim Harrington ◽  
Mette Staehr ◽  
Reed Masakayan ◽  
Jon Jones ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
B Cell ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell Therapy ◽  
Car T

Early clinical results of chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) for multiple myeloma (MM) appear promising, but relapses associated with residual low-to-negative BCMA-expressing MM cells have been reported, necessitating identification of additional targets. The orphan G protein–coupled receptor, class C group 5 member D (GPRC5D), normally expressed only in the hair follicle, was previously identified as expressed by mRNA in marrow aspirates from patients with MM, but confirmation of protein expression remained elusive. Using quantitative immunofluorescence, we determined that GPRC5D protein is expressed on CD138+ MM cells from primary marrow samples with a distribution that was similar to, but independent of, BCMA. Panning a human B cell–derived phage display library identified seven GPRC5D-specific single-chain variable fragments (scFvs). Incorporation of these into multiple CAR formats yielded 42 different constructs, which were screened for antigen-specific and antigen-independent (tonic) signaling using a Nur77-based reporter system. Nur77 reporter screen results were confirmed in vivo using a marrow-tropic MM xenograft in mice. CAR T cells incorporating GPRC5D-targeted scFv clone 109 eradicated MM and enabled long-term survival, including in a BCMA antigen escape model. GPRC5D(109) is specific for GPRC5D and resulted in MM cell line and primary MM cytotoxicity, cytokine release, and in vivo activity comparable to anti-BCMA CAR T cells. Murine and cynomolgus cross-reactive CAR T cells did not cause alopecia or other signs of GPRC5D-mediated toxicity in these species. Thus, GPRC5D(109) CAR T cell therapy shows potential for the treatment of advanced MM irrespective of previous BCMA-targeted therapy.

CD19-Targeted Donor T Cells Exert Potent Graft Versus Lymphoma Activity and Attenuated Gvhd

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 451-451 ◽  
Author(s):  
Arnab Ghosh ◽  
Marco L. Davila ◽  
Lauren F. Young ◽  
Christopher Kloss ◽  
Gertrude Gunset ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Acute Gvhd ◽  
Tcr Signaling ◽  
Car T Cells ◽  
Donor T Cells ◽  
Car T

Abstract Abstract 451 Chimeric antigen receptors (CAR) represent a potent strategy to target T cells against selected tumor antigens. Ongoing clinical trials indicate that autologous T cells expressing CARs targeting CD19, a B cell-associated antigen, can induce complete remission and B cell aplasia in patients with B cell malignancies. Donor CD19-CAR+ T cells could potentially be used to treat recipients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the risk of alloreactivity mediated by endogenous T cell receptors (TCR) triggering an acute GVHD is not known. This is partly due to the absence of in vivo models to study the relative effects of CAR and endogenous TCR signaling. For the first time, we have evaluated the relative effects of CD19-targeted donor T cells on the elimination of CD19+ B cells and endogenous TCR-mediated alloreactivity in mouse models of allo-HSCT. We generated a panel of retroviral vectors encoding mouse CD19-specific CARs: as a control, CD19-delta, a tail-less CAR lacking the CD3ζ signaling domain; CD19z1, which signals through its CD3ζ endodomain; and CD19-28z, which signals through CD28 and CD3ζ (Figure 1A). CD19z1+ and CD19-28z+ T cells mediated specific lysis of CD19-expressing tumors in vitro, while CD19-delta+ T cells did not. In order to assess the anti-tumor capacity of CD19-CAR+ T cells in vivo, we transferred the transduced B6 donor T cells into lethally irradiated BALB/c recipients that were administered T cell-depleted allografts and CD19+ lymphoma A20-TGL (B6–> BALB/c+A20-TGL). CD19-CAR+ T cells (CD19z1 and CD19-28z) mediated clearance of A20 tumor cells visualized by in vivo imaging of luciferase-expressing tumor cells (Figure 1B and data not shown) and significantly improved tumor free survival. CD19-CAR+ B6 T cells could sustain prolonged B cell hypoplasia when adoptively transferred into lethally irradiated haploidentical CBF1 recipients of T cell-depleted allografts (B6–> CBF1, Figure 1C). These data indicate that under alloreactive conditions, donor CD19-CAR+ T cell signaled through the CAR leading to specific elimination of CD19+ tumors and B lineage cells. In order to determine the risk of GVHD, we transferred the donor CD19-CAR+ T cells into haploidentical HSCT recipients. Interestingly, CD19-CAR+ T cells mediated significantly less acute GVHD, resulting in improved survival and lower GVHD scores (Figure 1D). Donor CD19-delta+ T cells however mediated lethal GVHD, indicating that the endogenous TCR mediated strong alloreactivity in the absence of CAR signaling. Similar results were obtained from experiments using MHC-mismatched (B6–> BALB/c) models. It is known that signaling through endogenous TCR is accompanied by down-regulation of surface TCR expression. We found significant decreases in surface CD3ϵ, TCRβ and CD90 expressions in donor CD19-delta+ T cells under alloreactive conditions. In contrast, donor CD1928z+ T cells failed to down-regulate surface TCR expression under similar conditions, suggesting that endogenous TCR function was altered in CAR-activated T cells. In the context of allo-HSCT, preferential CAR signaling at the expense of alloreactive endogenous TCR signaling may thus lead to reduced alloreactivity and attenuation of GVHD. These results provide the first pre-clinical evidence suggesting that CAR-modified, unselected donor T cells may be safely applied in an allogeneic context. Disclosures: No relevant conflicts of interest to declare.

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.

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 Development of CAR-T Cell Therapy for B-ALL Using a Point-of-Care Approach

2019 ◽  
Author(s):  
Luiza de Macedo Abdo ◽  
Luciana Rodrigues Carvalho Barros ◽  
Mariana Saldanha Viegas ◽  
Luisa Vieira Codeço Marques ◽  
Priscila de Sousa Ferreira ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
B Cell ◽  
Point Of Care ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell Therapy ◽  
Car T

AbstractRecently approved by the FDA and European Medicines Agency, CAR-T cell therapy is a new treatment option for B-cell malignancies. Currently, CAR-T cells are manufactured in centralized facilities and face bottlenecks like complex scaling up, high costs and logistic operations. These difficulties are mainly related to the use of viral vectors and the requirement to expand CAR-T cells to reach the therapeutic dose. In this paper, by using Sleeping Beauty-mediated genetic modification delivered by electroporation, we show that CAR-T cells can be generated and used without the need for ex vivo activation and expansion, consistent with a point-of-care (POC) approach. Our results show that minimally manipulated CAR-T cells are effective in vivo against RS4;11 leukemia cells engrafted in NSG mice even when inoculated after only 4 hours of gene transfer. In an effort to better characterize the infused CAR-T cells, we show that 19BBz T lymphocytes infused after 24h of electroporation (where CAR expression is already detectable) can improve the overall survival and reduce tumor burden in organs of mice engrafted with RS4;11 or Nalm-6 B cell leukemia. A side-by-side comparison of POC approach with a conventional 8-day expansion protocol using Transact beads demonstrated that both approaches have equivalent antitumor activity in vivo. Our data suggests that POC approach is a viable alternative for the generation and use of CAR-T cells, overcoming the limitations of current manufacturing protocols. Its use has the potential to expand CAR immunotherapy to a higher number of patients, especially in the context of low-income countries.

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 Ibrutinib before Apheresis May Improve Tisagenlecleucel Manufacturing in Relapsed/Refractory Adult Diffuse Large B-Cell Lymphoma: Initial Results from a Phase 1b Study

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-4
Author(s):  
Julio C. Chavez ◽  
Frederick L. Locke ◽  
Ellen Napier ◽  
Carl Simon ◽  
Andrew Lewandowski ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Biomedical Research ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Background: Tisagenlecleucel (tisa-cel), an autologous anti-CD19 chimeric antigen receptor (CAR)-T cell therapy, has demonstrated durable responses and a manageable safety profile in adult patients (pts) with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). It has previously been suggested that prior therapy with ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, may improve tisa-cel manufacturing, in vivo cellular kinetics, and antitumor efficacy (Fraietta et al. Blood. 2016). Moreover, since BTK signaling is involved in direct pro-inflammatory polarization of macrophages, as well as indirectly by T cells, it is hypothesized that ibrutinib may mitigate CAR-T cell-related toxicities such as cytokine release syndrome (CRS) and neurological events (NE). We report the initial results from a Phase Ib, multicenter, open-label trial evaluating the safety and tolerability of tisa-cel in combination with ibrutinib in adult pts with r/r DLBCL. Methods: Adult pts with r/r DLBCL who received &gt;2 prior lines of systemic therapy, including pts who progressed after or were ineligible for autologous stem cell transplant, were enrolled. The study design has 2 nonrandomized arms. In Arm 1, pts received ibrutinib 560 mg/d for ~4 weeks prior to leukapheresis; in Arm 2, pts were exposed to ibrutinib after leukapheresis. In both arms, ibrutinib was continued throughout lymphodepleting chemotherapy, tisa-cel infusion, and post infusion for up to 24 months. Lymphodepleting chemotherapy, ending at least 2 days before tisa-cel infusion, was either fludarabine (25 mg/m2) and cyclophosphamide (250 mg/m2) daily for 3 days or bendamustine (90 mg/m2) daily for 2 days. Pts received a single infusion of tisa-cel (target dose: 0.6-6.0×108 viable CAR+ T cells). Primary endpoints are incidence and severity of adverse events and ibrutinib dose interruptions/modifications. Secondary endpoints include best overall response (BOR) by Lugano criteria and cellular kinetics of tisa-cel. Results: As of June 9, 2020, 10 pts have been treated and observed through at least the Day 28 assessment: 4 in Arm 1 and 6 in Arm 2. Median age was 59 (range, 32-67) in Arm 1 and 64 (range, 58-76) in Arm 2. Median number of prior therapies was 3.5 (range, 2-5) in Arm 1 and 2 (range, 2-3) in Arm 2. Three of 10 pts (Arm 1, n=1; Arm 2, n=2) had an activated B-cell-like subtype of DLBCL. Six of 10 pts (Arm 1, n=1; Arm 2, n=5) had grade 1 CRS (by Lee scale) and 1 pt had NE (Arm 2, grade 1 by ASTCT criteria; Table). One pt in Arm 2 had grade 3 neutropenia lasting &gt;28 days post tisa-cel infusion. No other pts had grade 3 or 4 neutropenia or thrombocytopenia lasting &gt;28 days. No major bleeding events were observed. Ibrutinib-related bradycardia and atrial fibrillation (both grade 2) were each observed in 1 pt in Arm 1; supraventricular tachycardia (grade 1) related to tisa-cel was observed in 1 pt in Arm 2. No pt required tocilizumab or ICU admission. As of data cutoff, BOR in Arm 1 was complete response (CR) in 2 pts and partial response (PR) in 2 pts, with no relapses. BOR in Arm 2 was CR in 2 pts, PR in 1 pt, and progressive disease in 3 pts (Table). CAR-T cell expansion in vivo by qPCR was in line with data from the pivotal JULIET trial, except for 1 pt in Arm 2 whose transgene levels were below the limit of quantification at all points in time and who progressed at Day 28. Median viability of the leukapheresis material was 96.80% (range, 88.8-97.3) in Arm 1 and 90.95% (range, 88.1-94.7) in Arm 2. A naïve/stem cell-like central memory phenotype (CD45RA+/CCR7+) was observed in 24.05% (median; range, 15.9-37.0) of CD8+ T cells in the leukapheresis material for Arm 1 and in 8.12% (median; range, 1.3-20.4) for Arm 2 (Fig.1A). Fig.1B shows total CAR+ manufactured cells in each arm. The median dose of the final product was 3.9×108 CAR+ T cells in Arm 1 (range, 3.4-4.6×108 CAR+ T cells; median viability 92.25%) and 1.7×108 CAR+ T cells in Arm 2 (range, 1.2-3.0×108 CAR+ T cells; median viability 85.8%; Fig.1C). IFNγ secretion of tisa-cel in vitro in response to CD19+ target cells was similar between the 2 arms, whereas median normalized IL-2 responses were 23.1 fg/CAR+ cell in Arm 1 (range, 16.7-43.8) and 1.1 fg/CAR+ cell in Arm 2 (range, 0-17.3). Conclusions: These results support the feasibility of administering ibrutinib to pts with DLBCL throughout tisa-cel therapy. When given before apheresis, ibrutinib may improve CAR-T cell manufacturing, although further studies are needed to confirm this finding. Disclosures Chavez: AstraZeneca: Speakers Bureau; Morphosys: Consultancy, Speakers Bureau; Merck: Research Funding; Bayer: Consultancy; BeiGene: Speakers Bureau; Karyopharm: Consultancy; Genentech: Speakers Bureau; AbbVie: Consultancy; Epizyme: Speakers Bureau; Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Kite, a Gilead Company: Consultancy, Speakers Bureau; Verastem: Consultancy; Pfizer: Consultancy. Locke:Kite, a Gilead Company: Consultancy, Research Funding; Calibr: Consultancy; Celgene/Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; GammaDelta Therapeutics: Consultancy; Cellular Biomedicine Group: Other: Consultancy with grant options; Allogene: Consultancy; Wugen: Consultancy. Simon:Novartis: Current Employment. Lewandowski:Novartis Institutes for BioMedical Research: Current Employment. Awasthi:Novartis Institutes for BioMedical Research: Current Employment. Engels:Novartis Institutes for BioMedical Research: Current Employment. Georgala:Novartis Pharmaceuticals Corporation: Current Employment. Bondanza:Novartis Institutes for BioMedical Research: Current Employment. Schuster:AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria; Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding.

scholarly journals CD58 Aberrations Limit Durable Responses to CD19 CAR in Large B Cell Lymphoma Patients Treated with Axicabtagene Ciloleucel but Can be Overcome through Novel CAR Engineering

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 53-54
Author(s):  
Robbie G. Majzner ◽  
Matthew J. Frank ◽  
Christopher Mount ◽  
Aidan Tousley ◽  
David M. Kurtz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Research Funding ◽  
Private Company ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T ◽  
Support Research

CD19 CAR T cells have revolutionized the treatment of relapsed and refractory (R/R) large B cell lymphomas (LBCL), mediating durable complete responses in approximately 40-50% of patients. Besides a loss or decrease in CD19 expression, no studies have identified tumor specific factors driving inherent or acquired resistance to CAR T cells in LBCL. Mutations in and loss of expression of LFA-3 (CD58) have been described in approximately 20% of cases of LBCL. As the ligand for CD2 on T cells, CD58 provides costimulation to T cells and CD58 loss or mutation has been linked to immune resistance in LBCL. We evaluated CD58 status in fifty-one R/R LBCL patients treated at Stanford with commercial axicabtagene ciloleucel (axi-cel) through immunohistochemistry (IHC) on tumor biopsy samples and/or deep sequencing of circulating tumor DNA by CAPP-Seq. We identified 12/51 (24%) patients with a CD58 aberration (lack of expression by IHC or mutation by CAPP-Seq). Progression-free survival (PFS) was significantly decreased in patients with a CD58 aberration (median PFS for CD58 aberration 3 months vs. not reached for CD58 intact, p&lt;0.0001). In fact, only 1/12 patients with a CD58 alteration achieved a durable, complete response to axi-cel, while the remaining 11 patients progressed, most commonly after a period of initial response. Partial responses were more common among patients with CD58 aberrations (58% for CD58 aberration vs 10% for CD58 intact, p&lt;0.001), and complete responses were less common (25% for CD58 aberration vs 82% for CD58 intact, p&lt;0.0001). To probe the biology of CAR T cell responses towards tumors lacking functional CD58, we generated a CD58 knockout Nalm6 model. CD19.CD28.ζ, CD19.4-1BB.ζ, and CD22.4-1BB.ζ CAR T cells demonstrated significantly reduced cytokine production and cytolytic activity in response to CD58 KO vs wildtype (WT) tumor cells. Additionally, while mice inoculated with WT Nalm6 and treated with any of the three CARs demonstrate complete responses and prolonged leukemia-free survival, mice inoculated with CD58KO Nalm6 demonstrated only partial responses, eventual tumor progression, and death from leukemia. CD2, the T cell ligand for CD58, plays both an adhesive role and a costimulatory role in T cells. CD2 knockout resulted in significantly reduced cytokine production after CAR stimulation. Re-expression of only the CD2 extracellular domain did not rescue CAR function, indicating that CD2 signaling is essential for full CAR activation. Additionally, when we stimulated CD19 CAR T cells with anti-idiotype antibody (CAR stimulation), soluble CD58 (CD2 stimulation), or both, we observed significantly enhanced phosphorylation of both CD3ζ and ERK by western blot in CAR T cells stimulated through both the CAR and CD2. Phosphorylation analysis by mass spectrometry revealed that CD2 stimulation enhances phosphorylation of proximal signaling molecules in the TCR pathway (LCK, LAT, CD3ε among others) and also mediators of actin-cytoskeletal rearrangement in CAR T cells, consistent with effects in natural T cell responses. To overcome CD58 loss in LBCL, we generated second- and third-generation CAR T cell constructs integrating CD2 costimulatory domains within the CAR molecule. While these cis constructs demonstrated increased potency against CD58KO cells in vitro, they were unable to ultimately overcome CD58 loss in vivo. However, when CARs were co-expressed with an additional CD2 receptor in trans, they mediated significant anti-tumor activity in vivo, overcoming CD58 knockout in tumor cells. In conclusion, we have identified that CD58 status is an important biomarker for durable response to CAR T cells in LBCL. We modeled the biologic basis for this finding and generated CAR T cells capable of overcoming CD58 loss in B cell malignancies. CD58 mutations have been reported in many cancers, including multiple myeloma and colon cancer, and are likely to play a role in immune evasion for CAR T cells as they are developed for additional histologies. These data provide rationale for investigating CD58 status for patients receiving CAR based therapeutics and devising next generation CARs capable of overcoming this newly discovered mechanism of resistance. Disclosures Majzner: Xyphos Biopharma: Consultancy; Zai Lab: Consultancy; Lyell Immunopharma: Consultancy; GammaDelta Therapeutics: Membership on an entity's Board of Directors or advisory committees; Aprotum Group: Consultancy; Illumina Radiopharmaceuticals: Consultancy. Kurtz:Roche: Consultancy; Genentech: Consultancy; Foresight Diagnostics: Other: Ownership. Sotillo:Lyell Immunopharma: Consultancy, Other: Consultancy. Alizadeh:Janssen: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; Pfizer: Research Funding. Miklos:Miltenyi Biotec: Research Funding; Janssen: Consultancy, Other: Travel support; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Novartis: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding. Mackall:BMS: Consultancy; Allogene: Current equity holder in publicly-traded company; Apricity Health: Consultancy, Current equity holder in private company; Nektar Therapeutics: Consultancy; NeoImmune Tech: Consultancy; Lyell Immunopharma: Consultancy, Current equity holder in private company.

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.

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