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.

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 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 121 ICAM-1-specific affinity tuned CAR T cells expressing SSTR2 for real-time imaging

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A130-A130
Author(s):  
Jingmei Hsu ◽  
Eric von Hofe ◽  
Michael Hsu ◽  
Koen Van Besien ◽  
Thomas Fahey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Phase I ◽  
Tumor Cells ◽  
Somatostatin Receptor ◽  
Laboratory Animals ◽  
Therapeutic Window ◽  
Car T Cells ◽  
Car T

BackgroundThe use of CAR T cells for solid tumors has a number of challenges, such as lack of tumor-specific targets, CAR T cell exhaustion, and the immunosuppressive tumor microenvironment. To address these challenges, AffyImmune has developed technologies to affinity tune and track CAR T cells in patients. The targeting moiety is affinity tuned to preferentially bind to tumor cells overexpressing the target while leaving normal cells with low basal levels untouched, thereby increasing the therapeutic window and allowing for more physiological T cell killing. The CAR T cells are designed to express SSTR2 (somatostatin receptor 2), which allows for the tracking of CAR T cells in vivo via PET/CT scan using FDA-approved DOTATATE.MethodsAIC100 was generated by affinity tuning the I-domain of LFA-1, the physiological ligand to ICAM-1. Various mutants with 106-fold difference in affinity were evaluated for affinity. This allowed structure activity relationships to be conducted using CAR T cells expressing the various affinity mutants against targets with varying antigen densities. The variant with micromolar affinity was clearly the most effective in non-clinical animal models. AIC100 is currently being evaluated to assess safety, CAR T expansion, tumor localization, and preliminary activity in patients with advanced thyroid cancer in a phase I study (NCT04420754). Our study uses a modified toxicity probability interval design with three dosage groups of 10 x 106, 100 x 106, and 500 x 106 cells.ResultsPreclinical studies demonstrated greater in vivo anti-tumor activity and safety with lower affinity CAR T cells. A single dose of AIC100 resulted in tumor elimination and significantly improved survival of animals. AIC100 activity was confirmed in other high ICAM-1 tumor models including breast, gastric, and multiple myeloma. In a Phase I patient given 10-million CAR T cells, near synchronous imaging of FDG and DOTATATE revealed preliminary evidence of transient CAR T expansion and tumor reduction at multiple tumor lesions, with the peak of CAR T density coinciding with the spike in CAR T numbers in blood.ConclusionsWe have developed affinity tuned CAR T cells designed to selectively target ICAM-1 overexpressing tumor cells and to spatiotemporally image CAR T cells. Near-synchronous FDG and DOTATATE scans will enhance patient safety by early detection of off-tumor CAR T activity and validation of tumor response. We anticipate that our ‘tune and track’ technology will be widely applicable to developing potent yet safe CAR T cells against hard-to-treat solid cancers.Trial RegistrationNCT04420754Ethics ApprovalIRB number19-12021154IACUC (animal welfare): All animal experiments were performed in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. Animal handling protocols were approved by the Institutional Laboratory Animal Use and Care Committee of Weill Cornell Medicine (Permit Number: 2012–0063).

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.

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 Piggybac Transposon Mediated CD19 CAR-T Cells Derived from CD45RA-Positive PBMCs Possess Potent and Sustained Antileukemic Function

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2807-2807
Author(s):  
Masaya Suematsu ◽  
Shigeki Yagyu ◽  
Nobuyoshi Nagao ◽  
Susumu Kubota ◽  
Yuto Shimizu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Research Funding ◽  
Stress Test ◽  
Transduction Efficiency ◽  
Antigen Stimulation ◽  
Car T Cells ◽  
Car T

Abstract Background: The quality of chimeric antigen receptor (CAR)-T cell products, including the expression of memory and exhaustion markers, has been shown to influence their long-term functionality. We previously demonstrated that piggyBac (PB) transposon-mediated CD19 CAR-T cells exhibit memory-rich phenotype that is characterized by a high proportion of CD45RA+/CCR7+ T cell fraction. To further investigate the favorable phenotype of PB-CD19 CAR-T cells, we generated PB-CD19 CAR-T cells from CD45RA+ and CD45RA− peripheral blood mononuclear cells (PBMCs) (RA+ CAR and RA− CAR, respectively), and compared their phenotype and antitumor function. Methods: CD45RA+ and CD45RA− PBMCs were isolated by magnetic selection from whole PBMCs, then the CD19-CAR transgene was transduced into these cells using the PB transposon system, as described previously. Transduction efficiency of CD19 CAR transgene was determined 24 hours by flow cytometry after transduction. The phenotype of CD19 CAR-T was evaluated by flow cytometry on day 14. High throughput RNA sequencing was performed to see the T cell activation/exhaustion profile upon antigen stimulation. Sequential killing assays were performed by adding fresh tumor cells into CAR-T cells co-cultured with tumor cells every three days by restoring an effector target ratio of 1:1. To see the durable antitumor efficacy in vivo, we performed in vivo stress test, in which CAR T-cells dosage was lowered to the functional limits, so that these CAR-T cells should be maintained and expanded in vivo, to achieve the antitumor efficacy. We injected 5 x 10 5 of firefly luciferase-labeled CD19+ tumor cells (REH) into NSG mice via tail vein, then these mice were treated with 1 x 10 5 of CD19 RA+ CAR-T, RA− CAR-T, or control CAR-T cells, respectively, at day 6 after the tumor injection. Results: RA+ CAR T cells demonstrated better transient transduction efficiency 24 h after transduction (RA+ CAR-T: 77.5 ± 9.8% vs RA− CAR-T: 39.7 ± 3.8%), and superior expansion capacity after 14 days of culture than RA− CAR-T cells (RA+ CAR-T: 32.5 ± 9.3-fold vs RA− CAR-T: 11.1 ± 5.4-fold). RA+ CAR-T cells exhibited dominant CD8 expression (RA+ CAR-T: 84.0 ± 3.4% vs RA− CAR-T: 34.1 ± 10.6%), less expression of exhaustion marker PD-1 (RA+ CAR-T: 3.1 ± 2.5% vs RA− CAR-T: 19.2 ± 6.4%) and T cell senescence marker CD57 (RA+ CAR-T: 6.8 ± 3.6% vs RA− CAR-T: 20.2 ± 6.9%), and enrichment of naïve/stem cell memory fraction (CAR+/CD45RA+CCR7+ fraction; RA+ CAR-T: 71.9 ± 9.7% vs RA− CAR-T: 8.0 ± 5.3%), which were associated with longevity of CAR-T cells. Transcriptome analysis revealed that RA+ CAR-T cells exhibited the enrichment of naïve/memory phenotype and less expression of canonical exhaustion markers, and these exhaustion profiles even maintained after the antigen stimulation. RA+ CAR-T cells demonstrated sustained killing activity even after multiple tumor rechallenges in vitro, without inducing exhaustion marker expression of PD-1. Although antigen stimulation could increase CAR expression, leading to tonic CAR signaling and exhaustion, in our study, the expression of CAR molecule on the cell surface following antigen stimulation in RA+ CAR was controlled at a relatively lower level that in RA− CAR-T cells. RA+ CAR-T cells achieved prolonged tumor control with expansion of CAR-T cells than RA− CAR-T cells in in vivo stress test (Fig.1A-C). On day15, bone marrow studies in RA+ CAR group exhibited abundant human CD3 positive T cells with less expression of PD-1, and relatively smaller amount of REH cells than RA− CAR group (Fig.1D). Furthermore, in two of long-lived mice in RA+ CAR group, human CD3 positive T cells were expanded even day 50 after treatment as confirmed by sequential bone marrow studies (Fig.1E), which indicated the antigen-induced proliferation and long-term functionality of RA+ CAR-T cells in vivo. Conclusion: Our results suggest that PB-mediated RA+ CAR-T cells exhibit memory-rich phenotype and superior antitumor function, thereby indicating the usefulness of CD45RA+ PBMC as a starting material of PB-CAR-T cells. Figure 1 Figure 1. Disclosures Yagyu: AGC Inc.: Research Funding. Nagao: AGC Inc.: Current Employment. Kubota: AGC Inc.: Current Employment. Shimizu: AGC Inc.: Current Employment. Nakazawa: AGC Inc.: Research Funding; Toshiba Corporation: Research Funding.

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 EXTH-20. SYNGENEIC B7-H3-SPECIFIC CAR T-CELLS HAVE POTENT ANTI-BRAIN TUMOR ACTIVITY VIA LOCAL OR SYSTEMIC DELIVERY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii91-ii91
Author(s):  
Dalia Haydar ◽  
Zhongzhen Yi ◽  
Haley Houke ◽  
Martine F Roussel ◽  
Chris DeRenzo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Brain Tumors ◽  
Tumor Cells ◽  
Systemic Delivery ◽  
Car T Cells ◽  
Car T ◽  
Anti Tumor Activity ◽  
Gl261 Glioma

Abstract BACKGROUND We and others have identified B7-H3 (CD276) as a promising target for CAR T-cell-based immunotherapies for pediatric brain tumors. So far, B7-H3-CAR T cells have only been studied in xenograft models for brain tumors, which do not recapitulate the immunosuppressive tumor microenvironment (TME). To overcome this obstacle, we decided to adapt the immune competent GL261 murine glioma model which mimics human disease and host immune barriers. METHODS To evaluate their safety and efficacy, murine B7-H3-CAR T-cells were generated using retroviral particles encoding a 2nd generation B7-H3-CAR with a CD28.z signaling domain. Expansion, persistence, and anti-tumor activity were evaluated in vitro and in vivo. Components of the brain TME were then evaluated using flow cytometry and immunostaining. RESULTS B7-H3-CAR T cells only killed B7-H3+ tumor cells, secreted significant levels of IFNγ and IL-2 in an antigen-dependent manner and expanded an average of 85-fold in repeat stimulation assay with B7-H3+ tumor cells in contrast to control CAR T-cells. In vivo, intratumoral (2x106) or systemic (3x106) injection of syngeneic B7-H3-CAR T-cells into mice with orthotopic GL261 glioma induced complete regression in 60% of treated mice resulting in a significant survival advantage. Mice showed no evidence of acute or long-term toxicities related to CAR T-cell infusions. We confirmed this encouraging safety profile by systemic administration of a high dose (1x107) B7-H3-CAR T-cells and performing histological analyses of all major organs on day 14 post T-cell injection, which showed no notable signs of injury or on-target/off-tumor toxicities. CONCLUSIONS We successfully generated syngeneic B7-H3-CAR T-cells and have demonstrated that these cells have potent anti-tumor activity in the immune competent GL261 glioma model via local or systemic delivery without apparent toxicities. Our study paves the way for future testing of B7-H3-CAR T-cells in early phase clinical studies.

scholarly journals CD4-Targeted Fusosomes Are Capable of Transducing Resting T Helper Cells to Generate Highly Potent CAR-T Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2942-2942
Author(s):  
Christie Ciarlo ◽  
Zach Frye ◽  
Andre DeGroot ◽  
Walter Flores ◽  
Kutlu Elpek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cd4 T Cells ◽  
Transduction Efficiency ◽  
Publicly Traded ◽  
Car T Cells ◽  
Car T ◽  
Integrating Vector

Abstract Introduction: Chimeric antigen receptor T cell therapy (CAR T) is a successful treatment for B cell malignancies; however, the time, complexity and cost of manufacturing autologous CAR T products limits the availability of these therapies to patients. Furthermore, ex vivo manipulation of T cells is likely to have a negative impact on quality. In vivo gene delivery of CAR T transgenes by systemic infusion of standard lentiviral vectors may increase therapeutic accessibility but is limited by off-target transduction and the requirement for T cell activation. Here, we demonstrate that a paramyxovirus-based integrating vector (fusosome) engineered with a CD4 re-targeted envelop (CD4 fusogen) can efficiently and specifically transduce resting and activated CD4+ T cells to generate functional CD4+ CD19-specific CAR T cells capable of eliminating CD19+ lymphoma cells. Methods: Anti-CD4 single chain variable fragments () and single variable domain (VHHs) were screened for CD4 binding, specificity, and NHP cross-reactivity and inserted into receptor binding paramyxovirus fusogen. CD4-targeted fusosomes expressing GFP were screened for high on-target titer against the CD4+ SupT1 cell line and low off-target transduction on non-CD4 expressing cells. Subsequently, a CD19-specific CAR encoding 4-1BB and the CD3z endo-domains (CD19 CAR) was generated to examine CD4+ CAR T transduction efficiency and functionality. PBMCs were thawed and activated with anti-CD3/anti-CD28 beads and exposed to GFP, CD4-targeted fusosomes and specificity of targeting CD4+ T cells was measured by flow cytometry. Subsequently, CD19 CAR fusosomes targeting CD4 were used to test transduction efficiency against activated (CD3/CD28 or IL-7 treated) or resting T cells, and to measure T cell function against CD19+ and CD19 knockout (CRISPR/Cas9-edited) Nalm-6 lymphoma cells (e.g., tumor co-culture and rechallenge assays and cytokine production) in vitro. Vector copy number (VCN) was determined by a multiplex ddPCR assay and reported as copies per diploid genome (c/dg). Results: To target CD4+ T cells, we generated fusogens encoding scFvs and VHHs specific to the CD4 T cell co-receptor (n = 399). Using fusosomes carrying the GFP transgene, NHP cross-reactive CD4-targeted fusogens that efficiently transduced CD4+ SupT1 cells were selected (n = 12 with crude SupT1 titers &gt;1E6). Activated PBMCs transduced with a CD4-targeted fusosomes exhibited specific CD4 T cell transduction whereas VSV-G pseudotyped vectors showed broad transduction including CD4+ and CD8+ T cells. CD4-targeted CD19 CAR fusosomes could efficiently transduce both activated (34% ± 1.5% CD4+CAR+; 0.54 ± 0.18 c/dg) and resting T cells, albeit at a lower expression and integration rate (20% ± 0.5% CD4+CAR+; 0.28 ± 0.14 c/dg). Resting CD4-transduced CAR T cells demonstrated specific cytotoxicity and cytokine production (GM-CSF, IFN-g, TNF-a, IL-2, IL-6, and IL-10) against CD19+ Nalm-6 but did not recognize CD19 knockout tumor cells. In long-term co-culture assays with repetitive stimulation with fresh tumor cells, resting CD4+ CD19 CAR T cells continued to show potent tumor cell killing. Future experiments will evaluate the efficacy of CD4 fusosomes against CD19+ tumors in vivo. Summary: CD4-specific fusosomes can efficiently deliver an integrating CAR payload to resting and activated CD4+ T cells. Modified CD4+ CAR T cells demonstrate potent anti-tumor activity against CD19+ tumor cells. These data suggest that targeting the CD4 co-receptor through in vivo delivery using a novel pseudotyped integrating vector can produce functional CAR T cells to target cancer. Disclosures Ciarlo: Sana Biotechnology: Current Employment. Frye: Sana Biotechnology: Current Employment. DeGroot: Sana Biotechnology: Current Employment. Flores: Sana Biotechnology: Current Employment. Elpek: Sana Biotechnology: Current Employment. Pepper: Sana Biotechnology: Current Employment. Johnson: Sana Biotechnology: Current Employment. Shah: Sana Biotechnology: Current Employment. Foster: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

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.

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