scholarly journals Very Rapid Production of CAR+ T-Cells upon Non-Viral Gene Transfer Using the Sleeping Beauty System

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2807-2807 ◽  
Author(s):  
Lenka V. Hurton ◽  
Harjeet Singh ◽  
Kirsten C. Switzer ◽  
Tiejuan Mi ◽  
Leo G. Flores ◽  
...  
Keyword(s):  
Tissue Culture ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Genetically Modified ◽  
Sleeping Beauty ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

Abstract T cells are genetically modified ex vivo to express chimeric antigen receptors (CARs) for in vivo clinical applications. CAR-modified T cells have demonstrated redirected specificity and, in several clinical trials, potent anti-tumor activity. Manufacture, to date, is based upon gene transfer in cycling T cells followed by a period of tissue culture to achieve stable expression of introduced CARs. In contrast, we have adapted the non-viral-based Sleeping Beauty (SB) system to avoid the need for (i) T-cell activation and (ii) extended ex vivo tissue culture; thereby developing an approach whereby T cells can be both manufactured and delivered at multiple points-of-care (POC). This shortened culture decreases the time frame for manufacturing CAR+ T cells compared with current protocols for viral- or non-viral-based methodologies and is a foundation of our POC technology. Furthermore, reducing the ex vivo culture time preserves the memory and sustained persistence of CAR+ T cells by avoiding the differentiation programming induced by activation events typically required before or after gene transfer. We have previously demonstrated that co-expressing a membrane-bound version of interleukin-15 (mbIL15) significantly enhances the in vivo persistence of CAR+ T cells that are generated following 28-day culture after electro-transfer of SB derived DNA plasmids. Herein, we incorporated mbIL15 to generate POC CD19-specific CAR+ T cells. Peripheral blood mononuclear cells were genetically modified with mbIL15 and 2nd generation CAR coded from individual SB DNA plasmids and placed in culture for less than 2 days prior to adoptive transfer. NSG mice burdened by established and disseminated CD19+ leukemia were intravenously injected with just 7.5 x 105 CAR+ T cells, or an equivalent total T-cell dose of CARneg (unmodified or mock-treated) T cells. The mbIL15-CAR T-cell infusion yielded excellent disease-free survival, anti-tumor activity (Figure), and T-cell persistence. This approach to expediting the generation of genetically modified T cells enables the administration of CAR-modified naïve T cells and demonstrates that POC T cells have potent anti-tumor effects, even at a reduced CAR+ T-cell dose. This improvement to non-viral gene transfer and T-cell production reduces the requirement for tissue culture and thus time to manufacture within a GMP facility which translates to improvements in scalability and reduced costs. In summary, these data provide a translational pathway to undertake clinical trials by rapidly infusing T cells after genetic modification using the SB system. Disclosures Hurton: Intrexon: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties. Singh:Immatics: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Switzer:Intrexon: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties. Mi:Intrexon: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties. Maiti:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Su:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Huls:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Employment, Equity Ownership, Patents & Royalties. Champlin:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Cooper:Immatics: Equity Ownership; City of Hope: Patents & Royalties; Targazyme, Inc.: Equity Ownership; Sangamo BioSciences: Patents & Royalties; Intrexon: Equity Ownership; Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties; MD Anderson Cancer Center: Employment; Miltenyi Biotec: Honoraria.

Manufacturing an Enhanced CAR T Cell Product By Inhibition of the PI3K/Akt Pathway During T Cell Expansion Results in Improved In Vivo Efficacy of Anti-BCMA CAR T Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1893-1893 ◽  
Author(s):  
Molly R. Perkins ◽  
Shannon Grande ◽  
Amanda Hamel ◽  
Holly M. Horton ◽  
Tracy E. Garrett ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership ◽  
Cells Cultured

Abstract Patients treated with chimeric antigen receptor (CAR) T cells targeting CD19 for B cell malignancies have experienced rapid and durable tumor regressions. Manufacture of CAR T cells is challenged by the necessity to produce a unique drug product for each patient. Each treatment requires ex vivo culture of patient T cells to facilitate CAR gene transfer and to achieve therapeutic amounts of T cells. Paradoxically, ex vivo culture with IL-2 also decreases CAR T cell activity. Some investigators have proposed isolating central memory T cells (thought to be enriched for therapeutic T cells), yet isolation techniques are cumbersome and costly to scale commercially. Culture of T cells in IL-7 and IL-15 has also been shown by several investigators to improve therapeutic activity. Here we explored the potential for culture modifications to improve the therapeutic potential of CAR T cells without adding complexity to manufacturing. We tested this hypothesis using CAR T cells specific to B cell maturation antigen (BCMA) manufactured using standard IL-2 culture with an inhibitor of PI3K added to the media, or with IL-7 and IL-15 in place of IL-2. The in vivo activity was studied in NSG mouse models of human Burkitt's lymphoma (Daudi), and multiple myeloma (RPMI-8226), both of which express BCMA. In the lymphoma model, NSG mice were injected intravenously (IV) with 2 x 106 Daudi cells and allowed to accumulate a large tumor burden before being treated with 4 x 106 CAR+ T cells on day 18 post-tumor injection. At this late time point post implantation, mice had highly disseminated Daudi tumor (our goal was to model late stage disease observed in relapsed and refractory lymphoma). In this model of advanced disease, IL-2 cultured anti-BCMA CAR T cells had no effect on tumor growth (p = 0.22) and all mice succumbed to the tumors within two weeks after treatment. Anti-BCMA CAR T cells grown in IL-7 and IL-15 also failed to control tumor growth (p = 0.23). In sharp contrast, all animals treated with anti-BCMA CAR T cells cultured with the PI3K inhibitor survived and experienced complete long-term tumor regression (p=0.003). The same anti-BCMA CAR T cells were used in a model of multiple myeloma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells, and at 22 days post-implantation mice received a single IV administration of anti-BCMA CAR T cells (4 x 105 CAR+ T cells/mouse) cultured under various conditions. In this model, all treatment groups demonstrated tumor regression, regardless of the in vitro culture conditions. To evaluate CAR T cell durability, two weeks after initial tumor clearance, surviving animals were then re-challenged with RPMI-8226 cells on the opposite flank to model tumor relapse. We found that only animals that had been treated with anti-BCMA CAR T cells cultured with PI3K inhibition were immune to subsequent tumor challenge (p=0.005). Given the superior in vivo efficacy of anti-BCMA CAR T cells cultured with PI3K inhibition, we sought to identify phenotypic characteristics associated with the improved therapeutic activity. Anti-BCMA CAR T cells cultured with PI3K inhibition contained an increased frequency of CD62L+ CD8 T cells in the final product (p < 0.001) suggesting improved expansion of a distinct CD8 T cell subset. These data suggest that inhibition of PI3K during ex vivo expansion with IL-2 may generate a superior anti-BCMA CAR T cell product for clinical use. Furthermore, this approach could potentially be used in the manufacture of other T cell therapies. Disclosures Perkins: bluebird bio: Employment, Equity Ownership. Grande:bluebird bio: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Miller:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

Minimally Ex Vivo Manipulated Gene-Modified T Cells Display Enhanced Tumor Control

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4549-4549 ◽  
Author(s):  
Saba Ghassemi ◽  
Patel Prachi ◽  
John Scholler ◽  
Selene Nunez-Cruz ◽  
David M. Barrett ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Low Dose ◽  
Ex Vivo ◽  
University Of Pennsylvania ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

Abstract Adoptive cell therapy employing T cells equipped with a chimeric antigen receptor (CAR) containing a single chain antibody fragment fused to T cell signaling domains 4-1BB and CD3zeta (CTL019) has shown great potency against various hematopoietic malignancies, e.g. B cell acute lymphoblastic leukemia (ALL). However, it has not shown the same response rate in other malignancies such as chronic lymphocytic leukemia (CLL). We recently demonstrated that the in vivo expansion and persistence of CAR T cells is an important predictor of response to CTL019 in CLL (PMID: 26333935) and ALL (Thudium et al., ASH 2016; Fraietta et al., ASH 2016). Furthermore, it is well known that prolonged culture of T cells negatively impacts the in vivo expansion of the adoptively transferred cells. We therefore hypothesized that minimizing the ex vivo manipulation of T cells would improve the efficacy of CAR T cells. We tested this hypothesis by generating CART19 cells using our standard 9-day manufacturing process plus two abbreviated versions. Cells from normal donors (n=9) and from patients with adult ALL (n=6) were stimulated on day 0 followed by transduction with the CAR19-encoding lentiviral vector on day 1. Cells were harvested on days 3, 5, and 9. Cryopreserved aliquots were evaluated for T cell differentiation using polychromatic flow cytometry, cytokine secretion profile using Luminex, cytolytic ability against a leukemia cell line (NALM6), proliferative ability upon restimulation with CD19-expressing target cells, and in vivo control of our well-established xenogeneic ALL model employing NALM6 as the target. Our data show that all cultures contain a substantial proportion (40%-80%) of na•ve-like CD45RO-CCR7+ T cells that progressively differentiate leading to the accumulation of predominantly (60%-90%) central memory T cells by the end of expansion. Comparative assessment of the CART19 cells at all three time points demonstrated that the cells from the shorter cultures displayed a superior in vitrocytolytic activity, and proliferative response compared to the standard process. In addition,the cells from our standard and shortened cultures all secreted comparable levels of type I cytokines (i.e. IFN-g, IL-2, and TNF-α). Importantly, we investigated the therapeutic potential of cells harvested at day 3 versus later time points. We treated NALM6 xenograftmice with a low dose (0.5 x106 CAR+ T cell I.V.) or standard dose (3 x106 CAR+ T cell I.V.).We demonstrate that day 3 CART19 cells show superior anti-leukemic activity compared to day 5 or day 9 cells. Additionally, we show that mice treated at a low dose with day 3 cells exhibit the greatest anti-leukemic efficacy compared with day 9 cells where the latter fail to control leukemia (Figure 1). Our preclinical findings provide evidence that extended ex vivo manipulation of T cells negatively affects their in vivo potency.In summary, we show that limiting T cell culture ex vivo to the minimum required for lentiviral transduction provides the most efficacious T cells for adoptive T cell immunotherapy. Figure 1 Figure 1. Disclosures Ghassemi: Novartis: Research Funding. Scholler:Novartis: Patents & Royalties; University of Pennsylvania: Patents & Royalties: FAP-CAR US Patent 9,365,641 for targeting tumor microenvironment. Nunez-Cruz:Novartis: Research Funding. Barrett:Novartis: Research Funding. Bedoya:Novartis: Patents & Royalties. Fraietta:Novartis: Patents & Royalties: Novartis, Research Funding. Lacey:Novartis: Research Funding. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Research Funding. June:Johnson & Johnson: Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder ; University of Pennsylvania: Patents & Royalties; Pfizer: Honoraria; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Immune Design: Consultancy, Equity Ownership; Celldex: Consultancy, Equity Ownership. Milone:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding.

scholarly journals Mitochondrial Biomass As a Measure of Fitness for T Cells Expressing Chimeric Antigen Receptors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3242-3242 ◽  
Author(s):  
Bipulendu Jena ◽  
David Rushworth ◽  
George T McNamara ◽  
Laurence JN Cooper
Keyword(s):  
T Cells ◽  
Research Funding ◽  
Genetically Modified ◽  
Sleeping Beauty ◽  
Mitochondrial Mass ◽  
Car T Cells ◽  
Mitochondrial Structure ◽  
Car T ◽  
Multiple Variables ◽  
Equity Ownership

Abstract Anti-tumor efficacy of genetically modified T cells depends on in vivo expansion and durable persistence of infused cells. Multiple variables including the structure of the CAR and characteristics of the recipient impact the anti-tumor effect of CAR+ T cells. However, a code for an optimal CAR design that would deliver clinically relevant result is yet to emerge. Here we propose a new measure of "fitness" for CAR+ T cells based on mitochondrial biomass that is quantifiable and could be translated to clinical settings. Spare respiratory capacity (SRC) is defined as the extra mitochondrial capacity available in a cell to produce energy under conditions of increased work or stress. Memory T cells capable of responding to infection has been shown to possess extra SRC (Windt et al., Immunity 2012). We therefore investigated whether subsets of CD19-specific CAR+ T cells after electro-transfer of Sleeping Beauty (SB) plasmids and propagation on activating and propagating cells (AaPC) could be identified based on SRC. Transmission electron microscopy revealed that genetically modified T cells revert to a condensed state of mitochondria after 2 weeks of activation through a second-generation CD19-specific CAR. However, mock-electroporated T cells activated by cross-linking CD3 (using AaPC loaded with OKT3) retain a classic mitochondrial structure. Moreover, antigen-driven numeric expansion in presence of membrane bound IL-15 led to an increase in mitochondrial biomass in CAR+ T cells. We extended these observations to various CAR+ T cells with unique specificity for tumor antigens and found similar changes in mitochondrial structure and distribution. Next, we examined if an increase in mitochondrial biomass influences functionality of genetically modified T cells. By SB mediated transposition CARs were co-expressed along with a fluorescence reporter protein (EYFP-GRX2) constituting yellow fluorescent protein fused to the mitochondrial localization sequence of GRX2 to track mitochondrial distribution in live cells. The genetically modified T cells were selectively propagated by stimulating the CARs using a proprietary monoclonal antibody that binds to a common extracellular stalk motif in CAR construct. CAR+ T cells that signaled through chimeric CD137z exhibited a high mitochondrial mass (EYFPhigh) and had superior rates of expansion ex vivo. In contrast, CAR+ T cells that signaled through chimeric CD28z had a low mitochondrial mass (EYFPdim), elevated levels of apoptosis, and inferior rates of numeric expansion. Confocal microscopy showed EYFP counts were higher for CAR+ T cells that signaled through CD137 signaling domain. We hypothesize that increased survival of CD137z-CAR T cells in a challenging cell culture environment could be due to reserve bio-energetic potential concomitant with the ability to meet metabolic demand of activated T cells. Further, SRC could be quantified using a fluorescent probe for mitochondrial mass pre-infusion which may be a defining criterion attesting to the fitness of CAR+ T cells for human applications. Disclosures Jena: Intrexon: Equity Ownership, Patents & Royalties: Potential royalties (Patent submitted); Ziopharm Oncology: Equity Ownership, Patents & Royalties: Potential roylaties (Patent submitted). Rushworth:Intrexon: Other: Potential Equity ownership; Ziopharam Oncology: Other: Potential Equity Ownership. McNamara:Ziopharm Oncology: Equity Ownership, Patents & Royalties: Potential royalties, Research Funding; Intrexon: Equity Ownership, Patents & Royalties: Potential royalties, Research Funding. Cooper:Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties, Research Funding; Intrexon: Equity Ownership, Patents & Royalties.

scholarly journals Rapid Personalized Manufacture (RPM) of Sleeping Beauty System-Generated NY-ESO-1-Specific TCR-T Cells Co-Expressing Membrane-Bound IL-15 Yields Anti-Tumor Responses

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3218-3218
Author(s):  
Lenka V Hurton ◽  
Tiejuan Mi ◽  
Kirsten C Switzer ◽  
Ling Zhang ◽  
Cuiping Dai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Genetic Modification ◽  
Sleeping Beauty ◽  
Cancer Center ◽  
Employment Equity ◽  
Car T ◽  
Membrane Bound ◽  
Equity Ownership

CAR-redirected T cells have demonstrated clinical effectiveness in early phase clinical trials, with persistence of adoptively transferred CD19-specific T cells correlated with positive outcomes. Notwithstanding the successes for some hematological malignancies, CAR-T targets a limited number of cell-surface antigens that curtails their appeal for solid tumors. This can be overcome by TCR gene transfer with specificity for intracellular tumor-associated antigens such as NY-ESO-1 expressed by hematologic malignancies and solid tumors. The predominant technologies for both CAR- and TCR-redirection of T cells utilize viral genetic modification as well as require a lengthy period of in vitro propagation with resultant deleterious differentiation to achieve clinically relevant cell numbers. A major impediment with current TCR-T is the high-cost and lengthy time associated with a viral-based manufacture of a library of TCRs that can address a multitude of desired targets and match HLA restriction to meet the need to infuse personalized TCR-T products with multiple specificities in each recipient. The Sleeping Beauty (SB) platform is the most clinically advanced non-viral gene transfer technology and overcomes the issues of scalability with viral based manufacture of TCR-T. We initially showed in pre-clinical models that co-expression of membrane-bound interleukin-15 (mbIL15) enhanced in vivo persistence of CAR-T (PMID: 27849617). This technology has been recently advanced to produce CD19-specific T cells in ≤ 2 days after electro-transfer of DNA plasmids using so-called "rapid personalized manufacture" (RPM). This was based on the SB system to stably co-express CAR and mbIL15 with a kill switch (HER1t). We have now adapted these technologies to address current limitations for T-cell therapy by using the RPM process to very rapidly generate TCR-modified T cells. The rationale for RPM of TCR-T is based on: (i) SB to genetically modify resting T cells thus eliminating the need to propagate cells prior to, or after, genetic modification, (ii) introduction of TCR to redirect T-cell specificity to tumor-associated antigens, (iii) mbIL15-HER1t to support T-cell persistence and enable selective elimination to increase safety, and (iv) manufacture within two days of gene transfer which limits T-cell differentiation and decreases time to manufacture. Mononuclear cells were electroporated with SB-derived DNA plasmids expressing (a) HLA A2-restricted NY-ESO-1-specific TCR or (b) the TCR and mbIL15-HER1t in separate plasmids. Following electroporation, cells were directly (unpropagated) injected into NSG (immunocompromised) mice bearing established HLA A2+ NY-ESO-1+ tumor. Administration of RPM TCR-mbIL15 T cells exhibited superior anti-tumor activity compared with RPM TCR T cells (Figure). Though engraftment of TCR+ T cells was not significantly different between the two groups, the RPM TCR-mbIL15 T cell-treated mice exhibited increased frequency of CD27+TCR+ T cells (p = 0.035, n = 6-7, Mann Whitney test), a phenotype that is correlated with improved therapeutic responses in human subjects. The RPM technology can thus be adapted to co-express TCR with mbIL15 (and HER1t), which can now be scaled to provide a cost-effective approach to manufacturing a multitude of TCR-T products from a library of TCRs with the necessary complexity to manage the range of specificities and HLA restrictions to treat multiple patients. Disclosures Hurton: • M.D. Anderson Cancer Center: Patents & Royalties; Intrexon: Patents & Royalties: US 9,629,877 B2 ; Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties: US 9,629,877 B2 . Zhang:Intrexon: Patents & Royalties: US 9,629,877 B2; Ziopharm Oncology: Patents & Royalties: US 9,629,877 B2. Deniger:Ziopharm Oncology: Employment, Equity Ownership. Olivares:Ziopharm Oncology: Patents & Royalties: US9629877B2, US20160158285A1, WO2009091826A2, US20190085079A1, US20170158749A1, US20170333480A1, US20190055299A1; Intrexon: Patents & Royalties: US9629877B2, US20160158285A1, WO2009091826A2, US20190085079A1, US20170158749A1, US20170333480A1, US20190055299A1. Cooper:CytoSen: Equity Ownership; Targazyme: Equity Ownership; MD Anderson Cancer Center: Patents & Royalties; Sangamo BioSciences: Patents & Royalties; Immatics: Equity Ownership, Patents & Royalties; City of Hope: Patents & Royalties; Ziopharm Oncology: Employment, Equity Ownership, Other: Contracted research, Patents & Royalties; Secure Transfusion Services: Equity Ownership; CellChorus: Equity Ownership. Singh:Ziopharm Oncology: Patents & Royalties: US9629877B2, US20160096902A1, US20170333480A1, US10125193B2; Intrexon: Patents & Royalties: US9629877B2, US20160096902A1, US20170333480A1, US10125193B2.

First Clinical Trials Employing Sleeping Beauty Gene Transfer System and Artificial Antigen Presenting Cells To Generate and Infuse T Cells Expressing CD19-Specific Chimeric Antigen Receptor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 166-166 ◽  
Author(s):  
Partow Kebriaei ◽  
Helen Huls ◽  
Harjeet Singh ◽  
Simon Olivares ◽  
Matthew Figliola ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Transfer ◽  
Chimeric Antigen Receptor ◽  
Genetically Modified ◽  
Sleeping Beauty ◽  
Clinical Grade ◽  
Car T Cells ◽  
Artificial Antigen ◽  
Car T ◽  
Antigen Presenting

Abstract Background T cells can be genetically modified ex vivo to redirect specificity upon enforced expression of a chimeric antigen receptor (CAR) that recognizes tumor-associated antigen (TAA) independent of human leukocyte antigen. We report a new approach to non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific CAR- (designated CD19RCD28 that activates via CD3z/CD28) in autologous and allogeneic T cells manufactured in compliance with current good manufacturing practice (cGMP) for Phase I/II trials. Methods T cells were electroporated using a Nucleofector device to synchronously introduce DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of g-irradiated artificial antigen presenting cells (aAPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC (designated clone #4) were derived from K562 cells and genetically modified to co-express the TAA CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. The dual platforms of the SB system and aAPC are illustrated in figure below. Results To date we have enrolled and manufactured product for 25 patients with multiply-relapsed ALL (n=12) or B-cell lymphoma (n=13) on three investigator-initiated trials at MD Anderson Cancer Center to administer thawed patient- and donor-derived CD19-specific T cells as planned infusions in the adjuvant setting after autologous (n=7), allogeneic adult (n=14) or umbilical cord (n=4) hematopoietic stem-cell transplantation (HSCT). Each clinical-grade T-cell product was subjected to a battery of in-process testing to complement release testing under CLIA. Currently, five patients have been infused with the CAR+ T cells following allogeneic HSCT, including one patient with cord blood-derived T cells (ALL, n=4; NHL, n=1), beginning at a dose of 106 and escalating to 107 modified T cells/m2. Three patients treated at the first dose level of 106 T cells/m2 have progressed; the patient treated at the next dose level with 107 T cells/m2 remains in remission at 5 months following HSCT. Assessment for response too early for patient treated with UCB T cells. Four patients with non-Hodgkin’s lymphoma have been treated with patient-derived modified T cells following autologous HSCT at a dose of 5x107 T cells/m2, and all patients remain in remission at 3 months following HSCT. No acute or late toxicities have been noted to date. PCR testing for persistence of CAR-modified T cells is underway. Conclusion We report the first human application of the SB and aAPC systems to genetically modify clinical-grade cells. Importantly, infusing CD19-specific CAR+ T cells in the adjuvant HSCT setting and thus targeting minimal residual disease is feasible and safe, and may provide an effective approach for maintaining remission in patients with high risk, CD19+ lymphoid malignancies. Clinical data is accruing and will be updated at the meeting. This nimble manufacturing approach can be readily modified in a cost-effective manner to improve the availability, persistence and therapeutic potential of genetically modified T cells, as well as target tumor–associated antigens other than CD19. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Genomic Analyses of SLAMF7 CAR-T Cells Manufactured by Sleeping Beauty Transposon Gene Transfer for Immunotherapy of Multiple Myeloma

2019 ◽  
Author(s):  
Csaba Miskey ◽  
Maximilian Amberger ◽  
Michael Reiser ◽  
Sabrina Prommersberger ◽  
Julia Beckmann ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Copy Number ◽  
Sleeping Beauty ◽  
Vector System ◽  
Car T Cells ◽  
Car T

ABSTRACTWidespread treatment of human diseases with gene therapies necessitates the development of gene transfer vectors that integrate genetic information effectively, safely and economically. Accordingly, significant efforts have been devoted to engineer novel tools that i) achieve high-level stable gene transfer at low toxicity to the host cell; ii) induce low levels of genotoxicity and possess a ‘safe’ integration profile with a high proportion of integrations into safe genomic locations; and iii) are associated with acceptable cost per treatment and scalable/exportable vector production to serve large numbers of patients. The Sleeping Beauty (SB) transposon has been transformed into a vector system that is fulfilling these requirements.In the CARAMBA project, we use SB transposition to genetically modify T cells with a chimeric antigen receptor (CAR) specific for the SLAMF7 antigen, that is uniformly and highly expressed on malignant plasma cells in multiple myeloma. We have demonstrated that SLAMF7 CAR-T cells confer specific and very potent anti-myeloma reactivity in pre-clinical models, and are therefore preparing a Phase I/IIa clinical trial of adoptive immunotherapy with autologous, patient-derived SLAMF7-CAR T cells in multiple myeloma (EudraCT Nr. 2019-001264-30/CARAMBA-1).Here we report on the characterization of genomic safety attributes in SLAMF7 CAR-T cells that we prepared in three clinical-grade manufacturing campaigns under good manufacturing practice (GMP), using T cells that we obtained from three healthy donor volunteers. In the SLAMF7 CAR-T cell product, we determined the average transposon copy number, the genomic insertion profile, and presence of residual SB100X transposase. The data show that the SLAMF7 CAR transposon had been inserted into the T cell genome with the close-to-random distribution pattern that is typical for SB, and with an average transposon copy number ranging between 6 and 12 per T cell. No residual SB100X transposase could be detected by Western blotting in the infusion products. With these attributes, the SLAMF7 CAR-T products satisfy criteria set forth by competent regulatory authorities in order to justify administration of SLAMF7 CAR-T cells to humans in the context of a clinical trial. These data set the stage for the CARAMBA clinical trial, that will be the first in the European Union to use virus-free SB transposition for CAR-T engineering.DisclosuresThis project is receiving funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 754658 (CARAMBA).

scholarly journals Implications of Concurrent Ibrutinib Therapy on CAR T-Cell Manufacturing and Phenotype and on Clinical Outcomes Following CD19-Targeted CAR T-Cell Administration in Adults with Relapsed/Refractory CLL

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 58-58 ◽  
Author(s):  
Mark Blaine Geyer ◽  
Jae H. Park ◽  
Isabelle Riviere ◽  
Brigitte Senechal ◽  
Xiuyan Wang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Expansion ◽  
Research Funding ◽  
Ex Vivo ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership ◽  
T Cell Expansion

Abstract Introduction: CD19-targeted chimeric antigen receptor-modified (CAR) T cells have demonstrated considerable therapeutic efficacy in patients (pts) with relapsed and/or refractory (R/R) B cell ALL (B-ALL), resulting in rapid and often durable complete responses (CR). In contrast, a smaller subset of pts with R/R CLL have achieved CR following CD19-targeted CAR T cell therapy. Ibrutinib (IBR), which has considerable efficacy as a single agent in pts with R/R CLL, may modulate antitumor T cell immune responses. Others have observed enhanced ex vivo expansion of autologous T cells collected from pts with IBR exposure in response to CD3/CD28 bead stimulation, and improved CD19-targeted CAR T cell engraftment and antitumor efficacy in human xenograft models (Fraietta et al., Blood, 2016). Herein, we report on adults with CLL treated with IBR at the time of autologous T cell collection and/or around the time of CAR T cell infusion enrolled in our phase I clinical trial of CD19-targeted CAR T cells for adults with R/R CLL or B-cell NHL (NCT00466531). Methods: Eligible pts underwent leukapheresis and T cells were transduced with a retroviral vector encoding a CAR comprising a CD19-specific scFv and CD28 and CD3ζ signaling domains (19-28z). The present analysis is limited to pts with CLL. We identified pts with CLL treated with IBR at the time of leukapheresis and/or around the time of conditioning chemotherapy (CCT) and CAR T cell infusion. As a control group, we additionally identified all evaluable IBR-naïve pts with CLL treated on this study. Response was assessed by NCI-WG criteria. Cytokine levels were measured prospectively before and after CCT and CAR T cell infusion. Results: 5 pts (male, n=3), median age 58 at CAR T cell infusion (range, 43-66) with R/R CLL (TP53 loss, n=2) underwent therapy with IBR at leukapheresis (n=4) and/or immediately prior to or through CCT (cyclophosphamide [Cy], n=2; fludarabine [Flu]+Cy, n=3) and CAR T cell infusion (n=5). 6 additional evaluable pts with R/R CLL remained IBR-naïve through CCT (Cy, n=4; bendamustine, n=2) and CAR T cell infusion. A non-significant trend toward greater median cumulative fold T cell expansion ex vivo was noted in the 4 pts on IBR (vs the 7 not on IBR) at leukapheresis (374 [171-1518] vs 160 [49-468], p=0.13), with similar median manufacturing time (13.5 vs 15 days). End of process (EOP) T cells in pts undergoing collection while on IBR (vs those not on IBR) demonstrated a greater fraction of CD8+CAR+ T cells with a CD62L+CD127+ (central memory) phenotype (mean 29.0 vs 4.3%, p=0.10) and decreased fraction of CD62L- T cells (effector/effector memory phenotype) across CD8+CAR+ (mean 26.5 vs 54.4%, p=0.06) and CD4+CAR+ (mean 24.0 vs 57.8%, p=0.03) T cell subsets (Fig 1). IBR-treated pts received median 1x107 19-28z+ CAR T cells/kg (3x106-3x107/kg) and IBR-naïve pts received median 1x107 19-28z+ CAR T cells/kg (6x106-4x107/kg). Fevers developed in all 11 pts and began on the first day of infusion in 4/5 IBR-treated pts (vs 2/6 IBR-naïve pts); 2/5 IBR-treated pts (vs 0/6 IBR- naïve pts) developed severe CRS and required vasopressors for hypotension in addition to tocilizumab. IBR-treated pts additionally exhibited greater median peak levels of multiple immunoregulatory cytokines associated with CRS, including IL-6, IL-10, IL-2, IL-5, IFN-γ, FLT3L, fractalkine, and GM-CSF. In total, 5 of 11 enrolled pts with CLL (45%) treated with CCT and 19-28z CAR T cells achieved objective response (minimal residual disease [MRD]- CR, n=2; maintenance of MRD+ CR, n=1; PR, n=2); ORR was 4/5 among IBR-treated pts (1 MRD- CR, 1 MRD+ CR, 2 PR; p=0.08 for ORR between IBR-treated vs IBR-naïve pts). 2 pts remain in MRD- CR at 16 and 50 months. Maximal CAR T cell persistence observed to date is 159 days; peak vector copy levels by qPCR were highest in the 2 pts attaining MRD-negative CR. Conclusions: Prior therapy with IBR may influence EOP CAR T cell phenotypes. Prior ± concurrent IBR may improve antitumor responses following 19-28z CAR T cell administration, though small numbers of pts and differences in CCT regimens limit firm conclusions based on these data. Additionally, prior ± concurrent IBR may amplify CRS, though more intensive CCT (e.g. Flu/Cy vs Cy) may also enhance CAR T cell expansion in vivo and intensify CRS. Further strategies to overcome the inhibitory microenvironment and enhance CAR T cell expansion and efficacy in pts with R/R CLL are in preparation. Disclosures Park: Amgen: Consultancy; Genentech/Roche: Research Funding; Juno Therapeutics: Consultancy, Research Funding. Riviere:Juno Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Sadelain:Juno Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Brentjens:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Combination Immunotherapy with NY-ESO-1-Specific CAR+ T Cells with T-Cell Vaccine Improves Anti-Myeloma Effect

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3366-3366 ◽  
Author(s):  
Krina Patel ◽  
Simon Olivares ◽  
Harjeet Singh ◽  
Lenka V. Hurton ◽  
Mary Helen Huls ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Sleeping Beauty ◽  
Employment Equity ◽  
Effector Cells ◽  
T Effector Cells ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

Abstract Adoptive transfer of T cells expressing chimeric antigen receptor (CAR) has demonstrated clinical effectiveness in early phase clinical trials, with persistence of effector cells typically leading to improved outcomes. Most CARs directly dock with cell-surface antigens, but this limits the number of tumor-derived targets. Thus, we have adapted two technologies to target intracellular antigens and improve survival of infused T cells. This was accomplished by expressing a CAR on T effector cells that functions as a mimetic of T-cell receptor (TCR) to recognize NY-ESO-1 in the context of HLA A2 and adapting HLA-A2+ T cells to serve as antigen presenting cells (T-APC) by expressing NY-ESO-1 antigen. NY-ESO-1 is a desirable target for T-cell therapy of high risk multiple myeloma (MM) with efficacy in trials infusing T cells expressing TCR recognizing this antigen. We hypothesized combined immunotherapy with NY-ESO-1-specific CAR+ T cells and an NY-ESO-1+ T-APC vaccine will lead to enhanced anti-myeloma efficacy due to improved persistence of the CAR+ T effector cells. An NY-ESO-1-specific CAR and control TCR were expressed on primary T cells using the Sleeping Beauty (SB) transposon/transposase system. T-APC was generated by electro-transfer of DNA plasmids from SB system coding for NY-ESO-1 and membrane-bound IL-15 (mbIL15). The tethered cytokine functions as co-stimulatory molecule to improve the potency of the vaccine. In vitro studies confirmed the NY-ESO-1-specific CAR+ (and TCR+) T cells could be numerically expanded upon co-culture with T-APC. A mouse model of NY-ESO-1+HLA-A2+(CD19neg) multiple myeloma was used to compare tumor growth for CAR+ T effector cells with and without T-APC. The NY-ESO-1-specific CAR+ T effector cells displayed anti-tumor effect that was superior to control mice without T cells and mice receiving CD19-specific control CAR+ T cells. Mice receiving both NY-ESO-1-specific CAR+T effector cells and T-APC exhibited further improvement in anti-myeloma activity. This group demonstrated superior persistence of T effector cells with recovered cells exhibiting a memory phenotype. In summary, T cells can target intracellular NY-ESO-1 using a TCR mimetic CAR. Improved anti-tumor effect attributed to better persistence can be achieved by co-infusion of T-APC vaccine. These data provide the foundation to assess T cells targeting NY-ESO-1 in a clinical trial. Disclosures Patel: Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Olivares:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Singh:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Immatics: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Hurton:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Huls:Ziopharm Oncology: Equity Ownership, Patents & Royalties; Intrexon: Employment, Equity Ownership, Patents & Royalties. Cooper:City of Hope: Patents & Royalties; Intrexon: Equity Ownership; Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties; Targazyme, Inc.,: Equity Ownership; Immatics: Equity Ownership; Sangamo BioSciences: Patents & Royalties; MD Anderson Cancer Center: Employment; Miltenyi Biotec: Honoraria.

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

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

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

scholarly journals Combination of NKTR-255, a Polymer Conjugated Human IL-15, with CD19 CAR T Cell Immunotherapy in a Preclinical Lymphoma Model

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2866-2866 ◽  
Author(s):  
Cassie Chou ◽  
Simon Fraessle ◽  
Rachel Steinmetz ◽  
Reed M. Hawkins ◽  
Tinh-Doan Phi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Board Member ◽  
Ad Hoc ◽  
Advisory Board ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Background CD19 CAR T immunotherapy has been successful in achieving durable remissions in some patients with relapsed/refractory B cell lymphomas, but disease progression and loss of CAR T cell persistence remains problematic. Interleukin 15 (IL-15) is known to support T cell proliferation and survival, and therefore may enhance CAR T cell efficacy, however, utilizing native IL-15 is challenging due to its short half-life and poor tolerability in the clinical setting. NKTR-255 is a polymer-conjugated IL-15 that retains binding affinity to IL15Rα and exhibits reduced clearance, providing sustained pharmacodynamic responses. We investigated the effects of NKTR-255 on human CD19 CAR T cells both in vitro and in an in vivo xenogeneic B cell lymphoma model and found improved survival of lymphoma bearing mice receiving NKTR-255 and CAR T cells compared to CAR T cells alone. Here, we extend upon these findings to further characterize CAR T cells in vivo and examine potential mechanisms underlying improved anti-tumor efficacy. Methods CD19 CAR T cells incorporating 4-1BB co-stimulation were generated from CD8 and CD4 T cells isolated from healthy donors. For in vitro studies, CAR T cells were incubated with NKTR-255 or native IL-15 with and without CD19 antigen. STAT5 phosphorylation, CAR T cell phenotype and CFSE dilution were assessed by flow cytometry and cytokine production by Luminex. For in vivo studies, NSG mice received 5x105 Raji lymphoma cells IV on day (D)-7 and a subtherapeutic dose (0.8x106) of CAR T cells (1:1 CD4:CD8) on D0. To determine optimal start date of NKTR-255, mice were treated weekly starting on D-1, 7, or 14 post CAR T cell infusion. Tumors were assessed by bioluminescence imaging. Tumor-free mice were re-challenged with Raji cells. For necropsy studies mice received NKTR-255 every 7 days following CAR T cell infusion and were euthanized at various timepoints post CAR T cell infusion. Results Treatment of CD8 and CD4 CAR T cells in vitro with NKTR-255 resulted in dose dependent STAT5 phosphorylation and antigen independent proliferation. Co-culture of CD8 CAR T cells with CD19 positive targets and NKTR-255 led to enhanced proliferation, expansion and TNFα and IFNγ production, particularly at lower effector to target ratios. Further studies showed that treatment of CD8 CAR T cells with NKTR-255 led to decreased expression of activated caspase 3 and increased expression of bcl-2. In Raji lymphoma bearing NSG mice, administration of NKTR-255 in combination with CAR T cells increased peak CAR T cell numbers, Ki-67 expression and persistence in the bone marrow compared to mice receiving CAR T cells alone. There was a higher percentage of EMRA like (CD45RA+CCR7-) CD4 and CD8 CAR T cells in NKTR-255 treated mice compared to mice treated with CAR T cells alone and persistent CAR T cells in mice treated with NKTR-255 were able to reject re-challenge of Raji tumor cells. Additionally, starting NKTR-255 on D7 post T cell infusion resulted in superior tumor control and survival compared to starting NKTR-255 on D-1 or D14. Conclusion Administration of NKTR-255 in combination with CD19 CAR T cells leads to improved anti-tumor efficacy making NKTR-255 an attractive candidate for enhancing CAR T cell therapy in the clinic. Disclosures Chou: Nektar Therapeutics: Other: Travel grant. Fraessle:Technical University of Munich: Patents & Royalties. Busch:Juno Therapeutics/Celgene: Consultancy, Equity Ownership, Research Funding; Kite Pharma: Equity Ownership; Technical University of Munich: Patents & Royalties. Miyazaki:Nektar Therapeutics: Employment, Equity Ownership. Marcondes:Nektar Therapeutics: Employment, Equity Ownership. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; Lyell Immunopharma: Equity Ownership, Patents & Royalties, Research Funding. Turtle:Allogene: Other: Ad hoc advisory board member; Novartis: Other: Ad hoc advisory board member; Humanigen: Other: Ad hoc advisory board member; Nektar Therapeutics: Other: Ad hoc advisory board member, Research Funding; Caribou Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; T-CURX: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Patents & Royalties: Co-inventor with staff from Juno Therapeutics; pending, Research Funding; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Other: Ad hoc advisory board member.

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