P09.08 Clinical-grade manufacturing of ROR1 CAR T cells using a novel virus-free protocol

BackgroundImmunotherapy with T cells that were modified by gene-transfer to express a ROR1-specific chimeric antigen receptor (ROR1 CAR-T) has therapeutic potential in ROR1+ malignancies in hematology and oncology. The ROR1 tumor antigen has a favorable expression profile with absence in vital normal human tissues. In this study, we sought to establish and validate clinical-grade manufacturing of ROR1 CAR-T to enable a Phase I/IIa clinical trial. In particular, we sought to integrate virus-free gene-transfer based on Sleeping Beauty transposition into this manufacturing protocol to permit scale-up and export to point-of-care manufacturing, and to reduce turn-around time, complexity and regulatory burden associated with conventional viral gene-transfer (biosafety level 2 to biosafety level 1).Materials and MethodsBuffy coats or leukaphereses were obtained from healthy donors to perform protocol optimization (n=7) and scale-up runs (n=1). CD4+ and CD8+ T cells were isolated separately by magnetic selection and stimulated with CD3/CD28 TransACT® reagent. T cells were transfected with mRNA encoding hyperactive Sleeping Beauty transposase (SB100X) and minicircle DNA (MC) encoding a pT2 transposon comprising the ROR1 CAR and an EGFRt marker gene using the MaxCyte GTx ® electroporation platform. Following transfection, T cells were expanded for 10–13 days in G-REX® bioreactors and then harvested and formulated into the drug product at a 1:1 ratio of CAR-expressing CD4:CD8 T cells. The drug product underwent comprehensive phenotypic, functional and genomic analyses as part of product qualification.ResultsThe set of protocol optimization runs resulted in a highly robust process. On average, the stable gene-transfer rate at the end of the manufacturing process was 71% in CD4+ (n=5) and 54% in CD8+ T cells (n=7). The average yield of ROR1 CAR-T relative to the number of input T cells was 12.6-fold for CD4+ and 9.4-fold for CD8+ after 12–15 days of expansion, with an average viability of 84% for CD4+ and 82% of CD8+ T cells. The scale-up run was performed with a leukapheresis product from which 52.5 × 10^6 CD4+ and 109 × 10^6 CD8+ T cells were transfected. At the end of the manufacturing process (day 12), there were 844 × 10^6 CAR-expressing CD4+ (~16-fold expansion) and 857 × 10^6 CAR-expressing CD8+ T cells (8-fold expansion). In functional testing, ROR1 CAR-T showed specific recognition and potent elimination of ROR1+ target cells, as well as antigen-dependent cytokine production and productive proliferation in in vitro analyses. Experiments to determine the anti-tumor potency of the drug product in vivo and detailed genomic analyses are ongoing. Preliminary analyses suggest a favorable genomic insertion profile of the CAR transposon, and a transposon copy number that is well within the range acceptable for clinical use of the drug product.ConclusionsWith this novel protocol, we aim to obtain the first manufacturing license for CAR-T in Europe that integrates our optimized approach with SB100X mRNA and transposon MC for CAR gene-transfer on the MaxCyte transfection platform. The quality and yield of the drug product support the design and dose escalation of the proposed clinical trial with ROR1 CAR-T, and will serve as a blueprint for other CAR-T products from our pipeline.Disclosure InformationK. Mestermann: None. M. Eichler: None. M. Machwirth: None. K. Kebbel: None. U. Köhl: None. H. Einsele: None. C. Müller: None. J. Lehmann: None. T. Raskó: None. F. Lundberg: None. Z. Izsvák: None. G. Schmiedeknecht: None. M. Hudecek: None.

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CARAMBA: a first-in-human clinical trial with SLAMF7 CAR-T cells prepared by virus-free Sleeping Beauty gene transfer to treat multiple myeloma

Gene Therapy ◽

10.1038/s41434-021-00254-w ◽

2021 ◽

Author(s):

Sabrina Prommersberger ◽

Michael Reiser ◽

Julia Beckmann ◽

Sophia Danhof ◽

Maximilian Amberger ◽

...

Keyword(s):

Clinical Trial ◽

Multiple Myeloma ◽

T Cells ◽

Gene Transfer ◽

Drug Product ◽

Sleeping Beauty ◽

High Rate ◽

T Cell Therapy ◽

Car T Cells ◽

Car T

AbstractClinical development of chimeric antigen receptor (CAR)-T-cell therapy has been enabled by advances in synthetic biology, genetic engineering, clinical-grade manufacturing, and complex logistics to distribute the drug product to treatment sites. A key ambition of the CARAMBA project is to provide clinical proof-of-concept for virus-free CAR gene transfer using advanced Sleeping Beauty (SB) transposon technology. SB transposition in CAR-T engineering is attractive due to the high rate of stable CAR gene transfer enabled by optimized hyperactive SB100X transposase and transposon combinations, encoded by mRNA and minicircle DNA, respectively, as preferred vector embodiments. This approach bears the potential to facilitate and expedite vector procurement, CAR-T manufacturing and distribution, and the promise to provide a safe, effective, and economically sustainable treatment. As an exemplary and novel target for SB-based CAR-T cells, the CARAMBA consortium has selected the SLAMF7 antigen in multiple myeloma. SLAMF7 CAR-T cells confer potent and consistent anti-myeloma activity in preclinical assays in vitro and in vivo. The CARAMBA clinical trial (Phase-I/IIA; EudraCT: 2019-001264-30) investigates the feasibility, safety, and anti-myeloma efficacy of autologous SLAMF7 CAR-T cells. CARAMBA is the first clinical trial with virus-free CAR-T cells in Europe, and the first clinical trial that uses advanced SB technology worldwide.

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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 ◽

10.1182/blood.v122.21.166.166 ◽

2013 ◽

Vol 122 (21) ◽

pp. 166-166 ◽

Cited By ~ 7

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.

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CD19-Specific T Cells for Treatment of Pediatric Acute Lymphocytic Leukemia Using Sleeping Beauty Transposition.

Blood ◽

10.1182/blood.v110.11.2820.2820 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2820-2820

Author(s):

Harjeet Singh ◽

Pallavi R. Manuri ◽

Simon Olivares ◽

Navid Dara ◽

Margaret J. Dawson ◽

...

Keyword(s):

T Cells ◽

Sleeping Beauty ◽

Lymphocytic Leukemia ◽

Cell Phenotype ◽

Clinical Grade ◽

Drug Selection ◽

Next Generation ◽

Chromosomal Dna ◽

Car T

Abstract Genetic modification of clinical-grade T cells is undertaken to augment function, including redirecting specificity for desired antigen. We and others have introduced a chimeric antigen receptor (CAR) to enable T cells to recognize lineage-specific tumor antigen, such as CD19, and early-phase human trials are currently assessing safety and feasibility. However, a significant barrier to next-generation clinical studies is developing a suitable CAR-expression vector capable of genetically modifying a broad population of T cells. Transduction of T cells is relatively efficient, but it requires specialized manufacture of expensive clinical-grade recombinant virus. Electro-transfer of naked DNA plasmid offers a cost-effective alternative approach, but the inefficiency of transgene integration mandates ex vivo selection under cytocidal concentrations of drug to enforce expression of selection genes to achieve clinically-meaningful numbers of CARneg T cells. We now report an improved approach to efficiently generating T cells from peripheral blood with redirected specificity. This was accomplished by introducing DNA plasmids from the Sleeping Beauty transposon/transposase system to directly express a CD19-specific CAR in both memory and effector T cells without co-expression of immunogenic drug-selection genes. The success of this approach was based upon the rationale design ofa next-generation codon-optimized CD19-specific CAR capable of coordinated signaling through chimeric CD28,CD19+ artificial antigen-presenting cells (aAPC) derived from K562 and expressing desired co-stimulatory molecules, andelectro-transfer of two SB DNA plasmids expressing CAR transposon and an improved transposase. We report that introduction of a two-component SB system into primary human T cells results in efficient (∼60-fold improved expression compared with electro-transfer without transposase) and stable CAR gene transfer (60 fold as compared to single plasmid control) which can be numerically expanded to clinically-meaningful numbers within weeks on CD19+ aAPC, without the need for addition of drug-selection, and with the outgrowth of CD8+ and CD4+ CAR+ T-cell sub-populations. The improved CAR expression is due to SB transposon/transposase integration into chromosomal DNA compared with rare non-homologous end-joining process that mediates integration by electroporation alone. We demonstrate that the CAR+ T cells expressed memory cell markers (Figure 1A) as well as redirected-killing function of an effector-cell phenotype (Figure 1B). Our data have implications for improved in vivo therapeutic potential as memory T cells are associated with long-term persistence after adoptive transfer. Figure 1. (A) Phenotypic and (B) funtional characterization of CD 19-specific T cells. Figure 1. (A) Phenotypic and (B) funtional characterization of CD 19-specific T cells.

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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 ◽

10.1182/blood-2019-131419 ◽

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.

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

10.1101/675009 ◽

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).

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

Blood ◽

10.1182/blood.v128.22.2807.2807 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2807-2807 ◽

Cited By ~ 2

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.

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Stable Gene Transfer and Expression in Human Primary T-Cells by the Sleeping Beauty Transposon System.

Blood ◽

10.1182/blood.v106.11.5539.5539 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5539-5539

Author(s):

Xianzheng Zhou ◽

Xin Huang ◽

Andrew C. Wilber ◽

Lei Bao ◽

Dong Tuong ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Gene Transfer ◽

Transgene Expression ◽

Sleeping Beauty ◽

Stable Gene ◽

Cell Clones ◽

Stable Gene Expression

Abstract The Sleeping Beauty (SB) transposon system is a non-viral DNA delivery system in which a transposase directs integration of an SB transposon into TA-dinucleotide sites in the genome. To determine whether the SB transposon system can mediate integration and long-term transgene expression in human primary T-cells, freshly isolated peripheral blood lymphocytes (PBLs) without prior activation were nucleofected with SB vectors carrying a DsRed reporter gene. Plasmids containing the SB transposase on the same (cis) (n=10) or separate molecule (trans) (n=8) as the SB transposon mediated long-term and stable reporter gene expression in human primary T-cells. We observed that delivery of SB transposase-encoding plasmid in trans effectively mediated stable gene expression in primary T-cells, exhibiting about a 3-fold increase (11% vs. 3% with 10 microgram plasmid on day 21) in potency in comparison with the cis vector (p<0.0001). In addition, a transposase mutant construct was incapable of mediating stable gene expression in human PBLs (n=6, p<0.0001), confirming that catalytic DDE domain is necessary for transposition in human primary T-cells. Immunophenotyping analysis in transposed T-cells showed that both CD4 and CD8 T-cells were transgene positive. SB-mediated high level of transgene expression in human T-cells was maintained in culture for at least 4 months without losing observable expression. Southern hybridization analysis showed a variety of transposon integrants among the 6 DsRed positive T-cell clones and no transposon sequences identifiable in the 2 DsRed negative clones. Sequencing of transposon:chromosome junctions in 5 out of 6 transposed T-cell clones confirmed that stable gene expression was due to SB-mediated transposition. In other studies, PBLs were successfully transfected using the SB transposon system and shown to stably and functionally express a fusion protein consisting of a surface receptor useful for positive T-cell selection and a “suicide” gene useful for elimination of transfected T-cells after chemotherapy. This study is the first report demonstrating that the SB transposon system can mediate stable gene transfer in human primary PBLs, which may be more advantageous for T-cell based gene therapies over widely used virus-based or conventional mammalian DNA vectors in terms of simplicity, stability, efficiency and safety.

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Targeting An Ancient Retrovirus In Tumor Using Engineered T Cells

Blood ◽

10.1182/blood.v122.21.4206.4206 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4206-4206

Author(s):

Janani Krishnamurthy ◽

Brian Rabinovich ◽

Simon Olivares ◽

Mi Teijuan ◽

Kirsten Switzer ◽

...

Keyword(s):

Clinical Trials ◽

T Cells ◽

Tumor Cells ◽

Treatment Strategy ◽

Conflicts Of Interest ◽

Sleeping Beauty ◽

Normal Organ ◽

Car T Cells ◽

Car T ◽

Env Protein

Abstract Human endogenous retroviruses (HERVs) are ancient viruses forming 8% of human genome. One subset of HERVs, the HERV-K has recently been found to be expressed on tumor cells including melanoma, breast cancer and lymphoma but not on normal body cells. Thus, targeting HERV-K protein as a tumor associated antigen (TAA) may be a potential treatment strategy for tumors that are resistant to conventional therapies. One approach to improve therapeutic outcome is by infusing T cells rendered specific for such TAAs preferentially expressed on tumor cells. Recognition of cell-surface TAAs independent of major histocompatibility complex can be achieved by introducing a chimeric antigen receptor (CAR) on T cells using gene therapy. This approach is currently being used in our clinical trials adoptively transferring CD19-specific CAR+ T cells into patients with B-lineage malignancies. Preliminary analysis of HERV-K env protein expression in 268 melanoma samples and 139 normal organ donor tissues using immunohistochemistry demonstrated antigen expression in tumor cells and absence of expression in normal organ tissues. The scFv region from a mouse monoclonal antibody to target HERV-K env was used to generate a CAR and cloned into Sleeping Beauty (SB) plasmid for stable expression in T cells. HERV-K-specific CAR+T cells were selectively propagated ex vivo on artificial antigen presenting cells (aAPC) using an approach already in our clinical trials. Indeed, after genetic modification of T cells and selection on HERV-K+ aAPC, over 95% of propagated T cells stably expressed the introduced HERV-K-specific CAR and exhibited redirected specificity for HERV-K+ melanoma (Figure 1). Further, the adoptive transfer of HERV-K-specific CAR+T cells killed metastatic melanoma in a mouse xenograph model. While we have chosen melanoma as our tumor model, this study has the potential to be applied to other malignancies, including lymphoma and myeloma due to restricted expression of HERV-K envelope (env) protein on these tumor cells. These data demonstrate that it is feasible to generate T cells expressing a HERV-K-specific CAR using a clinically-appealing approach as a treatment strategy for HERV-K env+ tumors. Disclosures: No relevant conflicts of interest to declare.

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Adoptive Therapy Using Sleeping Beauty Gene Transfer System and Artificial Antigen Presenting Cells to Manufacture T Cells Expressing CD19-Specific Chimeric Antigen Receptor

Blood ◽

10.1182/blood.v124.21.311.311 ◽

2014 ◽

Vol 124 (21) ◽

pp. 311-311 ◽

Cited By ~ 13

Author(s):

Partow Kebriaei ◽

Helen Huls ◽

Harjeet Singh ◽

Simon Olivares ◽

Matthew Figliola ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cord Blood ◽

Umbilical Cord ◽

Mononuclear Cells ◽

Sleeping Beauty ◽

Car T Cells ◽

Car T ◽

Cord Blood Mononuclear Cells ◽

Active Disease

Abstract Objectives: T cells can be genetically modified ex vivo to redirect specificity upon expression of a chimeric antigen receptor (CAR) that recognizes tumor-associated antigen (TAA) independent of human leukocyte antigen. We employ 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 patient (pt)- or donor-derived T cells for patients with advanced B-cell malignancies. Methods: T cells were electroporated using a Nucleofector device to synchronously introduce two 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 designer g-irradiated activating and propagating cells (AaPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC 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 successfully manufactured product for 42 pts with multiply-relapsed ALL (n=19), NHL (n=17), or CLL (n=5) on 4 investigator-initiated trials at MD Anderson Cancer Center to administer thawed pt- and donor-derived CD19-specific T cells as planned infusions in the adjuvant setting after autologous (n=5), allogeneic (n=21) or umbilical cord (n=4) hematopoietic cell transplantation (HCT), or for the treatment of active disease (n=12). Each clinical-grade T-cell product was subjected to a battery of in-process and final release testing. Adjuvant trials: Twelve pts have been infused with donor-derived CAR+ T cells following allogeneic HCT, including 2 pts with cord blood-derived T cells (ALL, n=10; NHL, n=2), beginning at a dose of 106 and escalating to 5x107 modified T cells/m2. Three pts, all with ALL, remain alive and in remission at median 5 months following T cell infusion. Five pts with NHL have been treated with pt-derived modified T cells following autologous HCT at a dose of 5x108 T cells/m2, and 4 pts remain in remission at median 12 months following T-cell infusions. Relapse trials: Thirteen pts have been treated for active disease (ALL, n=8; NHL, n=3; CLL, n=2) with pt or donor-derived (if prior allo-HCT) modified T cells at doses 106-5x107/m2, and 3 remain alive and in remission at median 3 months following T-cell infusions. No acute or late toxicities, including excess GVHD, have been noted. Conclusion: We report the first human application of the SB and AaPC systems to genetically modify clinical-grade cells. Furthermore, infusing CD19-specific CAR+ T cells in the adjuvant HCT setting and thus targeting minimal residual disease may provide an effective and safe approach for maintaining remission in pts at high risk for relapse. Next steps: The SB system serves as a nimble and cost-effective platform for genetic engineering of T cells. We are implementing next-generation clinical T-cell trials targeting ROR1, releasing T cells for infusion within days after electro-transfer of SB DNA plasmid coding for CAR and mRNA coding for transposase, and infusing T cells modified with CAR designs with improved therapeutic potential. Figure: Manufacture of CD19-specific T cells from peripheral and umbilical cord blood mononuclear cells by electro-transfer of SB plasmids and selective propagation of CAR+ T cells on AaPC/IL-2/IL-21. Figure:. Manufacture of CD19-specific T cells from peripheral and umbilical cord blood mononuclear cells by electro-transfer of SB plasmids and selective propagation of CAR+ T cells on AaPC/IL-2/IL-21. Disclosures No relevant conflicts of interest to declare.

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A Novel and Highly Potent CAR T Cell Drug Product for Treatment of BCMA-Expressing Hematological Malignances

Blood ◽

10.1182/blood.v126.23.3094.3094 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3094-3094 ◽

Cited By ~ 5

Author(s):

Alena A. Chekmasova ◽

Holly M. Horton ◽

Tracy E. Garrett ◽

John W. Evans ◽

Johanna Griecci ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Cell Lines ◽

Drug Product ◽

Single Chain ◽

Employment Equity ◽

Car T Cells ◽

Car T ◽

Equity Ownership

Abstract Recently, B cell maturation antigen (BCMA) expression has been proposed as a marker for identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM and some lymphoma tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Targeting BCMA maybe a therapeutic option for treatment of patients with MM and some lymphomas. We are developing a chimeric antigen receptor (CAR)-based therapy for the treatment of BCMA-expressing MM. Our anti-BCMA CAR consists of an extracellular single chain variable fragment (scFv) antigen recognition domain derived from an antibody specific to BCMA, fused to CD137 (4-1BB) co-stimulatory and CD3zeta chain signaling domains. Selection of our development candidate was based on the screening of four distinct anti-BCMA CARs (BCMA01-04) each comprised of unique single chain variable fragments. One candidate, BCMA02 (drug product name bb2121) was selected for further studies based on the robust frequency of CAR-positive cells, increased surface expression of the CAR molecule, and superior in vitro cytokine release and cytolytic activity against the MM cell lines. In addition to displaying specific activity against MM (U226-B1, RPMI-8226 and H929) and plasmacytoma (H929) cell lines, bb2121 was demonstrated to react to lymphoma cell lines, including Burkitt's (Raji, Daudi, Ramos), chronic lymphocytic leukemia (Mec-1), diffuse large B cell (Toledo), and a Mantle cell lymphoma (JeKo-1). Based on receptor density quantification, bb2121 can recognize tumor cells expressing less than 1000 BCMA molecules per cell. The in vivo pharmacology of bb2121 was studied in NSG mouse models of human MM and Burkitt's lymphoma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells. After 18 days, mice received a single intravenous (IV) administration of vehicle or anti-CD19Δ (negative control, anti-CD19 CAR lacking signaling domain) or anti-BCMA CAR T cells, or repeated IV administration of bortezomib (Velcade®; 1 mg/kg twice weekly for 4 weeks). Bortezomib, which is a standard of care for MM, induced only transient reductions in tumor size and was associated with toxicity, as indicated by substantial weight loss during dosing. The vehicle and anti-CD19Δ CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors, increased body weights, and 100% survival. Flow cytometry and immunohistochemical analysis of bb2121 T cells demonstrated trafficking of CAR+ T cells to the tumors (by Day 5) followed by significant expansion of anti-BCMA CAR+ T cells within the tumor and peripheral blood (Days 8-10), accompanied by tumor clearance and subsequent reductions in circulating CAR+ T cell numbers (Days 22-29). To further test the potency of bb2121, we used the CD19+ Daudi cell line, which has a low level of BCMA expression detectable by flow cytometry and receptor quantification analysis, but is negative by immunohistochemistry. NSG mice were injected IV with Daudi cells and allowed to accumulate a large systemic tumor burden before being treated with CAR+ T cells. Treatment with vehicle or anti-CD19Δ CAR T cells failed to prevent tumor growth. In contrast, anti-CD19 CAR T cells and anti-BCMA bb2121 demonstrated tumor clearance. Adoptive T cell immunotherapy approaches designed to modify a patient's own lymphocytes to target the BCMA antigen have clear indications as a possible therapy for MM and could be an alternative method for treatment of other chemotherapy-refractory B-cell malignancies. Based on these results, we will be initiating a phase I clinical trial of bb2121 for the treatment of patients with MM. Disclosures Chekmasova: bluebird bio, Inc: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Evans:bluebird bio, Inc: Employment, Equity Ownership. Griecci:bluebird bio, Inc: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Seidel:bluebird bio, Inc: Employment, Equity Ownership. Ryu:bluebird bio, Inc: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

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