High efficiency closed-system gene transfer using automated spinoculation

Abstract Background Gene transfer is an important tool for cellular therapies. Lentiviral vectors are most effectively transferred into lymphocytes or hematopoietic progenitor cells using spinoculation. To enable cGMP (current Good Manufacturing Practice)-compliant cell therapy production, we developed and compared a closed-system spinoculation method that uses cell culture bags, and an automated closed system spinoculation method to decrease technician hands on time and reduce the likelihood for microbial contamination. Methods Sepax spinoculation, bag spinoculation, and static bag transduction without spinoculation were compared for lentiviral gene transfer in lymphocytes collected by apheresis. The lymphocytes were transduced once and cultured for 9 days. The lentiviral vectors tested encoded a CD19/CD22 Bispecific Chimeric Antigen Receptor (CAR), a FGFR4-CAR, or a CD22-CAR. Sepax spinoculation times were evaluated by testing against bag spinoculation and static transduction to optimize the Sepax spin time. The Sepax spinoculation was then used to test the transduction of different CAR vectors. The performance of the process using healthy donor and a patient sample was evaluated. Functional assessment was performed of the CD19/22 and CD22 CAR T-cells using killing assays against the NALM6 tumor cell line and cytokine secretion analysis. Finally, gene expression of the transduced T-cells was examined to determine if there were any major changes that may have occurred as a result of the spinoculation process. Results The process of spinoculation lead to significant enhancement in gene transfer. Sepax spinoculation using a 1-h spin time showed comparable transduction efficiency to the bag spinoculation, and much greater than the static bag transduction method (83.4%, 72.8%, 35.7% n = 3). The performance of three different methods were consistent for all lentiviral vectors tested and no significant difference was observed when using starting cells from healthy donor versus a patient sample. Sepax spinoculation does not affect the function of the CAR T-cells against tumor cells, as these cells appeared to kill target cells equally well. Spinoculation also does not appear to affect gene expression patterns that are necessary for imparting function on the cell. Conclusions Closed system-bag spinoculation resulted in more efficient lymphocyte gene transfer than standard bag transductions without spinoculation. This method is effective for both retroviral and lentiviral vector gene transfer in lymphocytes and may be a feasible approach for gene transfer into other cell types including hematopoietic and myeloid progenitors. Sepax spinoculation further improved upon the process by offering an automated, closed system approach that significantly decreased hands-on time while also decreasing the risk of culture bag tears and microbial contamination.

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Engineering of CD19-CAR T Cells from Non-Hodgkin Lymphoma Patients in a Closed System in Combination with Retronectin/OKT3 Stimulation

Blood ◽

10.1182/blood.v124.21.2446.2446 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2446-2446

Author(s):

Hideto Chono ◽

Kenichi Tahara ◽

Ikuei Nukaya ◽

Junichi Mineno ◽

Eisuke Uehara ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Closed System ◽

Research Funding ◽

Healthy Donor ◽

Manufacturing Method ◽

Car T Cells ◽

Cell Manufacturing ◽

Healthy Donors ◽

Car T

Abstract Background; Adoptive immunotherapy with chimeric antigen receptor (CAR) gene transduced CD19-CAR T cells, which are engineered to express extracellular single-chain immunoglobulin variable fragments to CD19, linked to cytoplasmic T cell activation domains including CD3-ζ, showed remarkable therapeutic benefits toward CD19+ B cell malignancies including acute lymphoblastic leukemia, chronic lymphoblastic leukemia and non-Hodgkin lymphoma (B-NHL). For clinical setting, the phenotype of manufactured CAR T cells is an important factor; especially less differentiated T cells are anticipated to provide a long-lasting immune reconstitution. Furthermore, in order to avoid the risk of technical error and contamination during T cell manufacturing process, a closed system needs to be established. In this study, we addressed these issues and have established a novel CD19-CAR T cell manufacturing method from a small amount of blood in a closed system for clinical trial to treat patients with B-NHL. Methods; Peripheral blood was obtained from B-NHL patient volunteers and healthy donor volunteers who gave their written informed consents. Peripheral blood mononuclear cells (PBMCs) were isolated from 30 ml of blood using Ficoll-Paque PREMIUM density gradient centrifugation. PBMCs were stimulated in a plastic bag pre-coated with anti-CD3 monoclonal antibody (OKT3) and recombinant fibronectin fragment (RetroNectin®; RN). Following four days of stimulation, stimulated T cells were transferred into a 215 cm2 plastic bag pre-loaded with SFG-1928z retroviral vector (Brentjens et al., Clin Cancer Res. 2007) onto RN-coated substratum with low-temperature shaking (RBV-LTS method; Dodo et al., PLoS ONE, 2014). After one hour incubation, the bag was flipped over to facilitate more efficient utilization of the retroviral vector adsorbed on both top and bottom surfaces of the bag and further incubated. On Day 5, the transduction procedure was repeated, and the cells were transferred into 640 cm2 plastic bags until Day 10-14 for expansion. Closed system liquid handling was managed in all processes of manipulating T cells for stimulation, transduction, expansion and final product formulation. Results; We have previously reported that the fold expansion of T cells under stimulation with RN together with OKT3 enhanced cell proliferation while preserving the naïve phenotype of T cells in comparison to stimulation with OKT3 alone or OKT3 and anti-CD28 monoclonal antibody co-stimulation. Although B-NHL patients’ T cells showed much lower fold expansion compared to healthy donors’ T cells, 4/7 patients’ CAR T cells reached their target dose of 1 x 106 cells/kg from 30 ml of blood on Day 10. For the other 3 patients, 70-150 ml blood was estimated to be required to reach their target dose. The delay in proliferations was marked in B-NHL patients’ T cells compared to healthy donors’ T cells by Day 5, but B-NHL patients’ T cells represented significant catch-up growth, which was superior to healthy donor T cells during Day 7-14. Gene transfer efficiency of patients’ T cells (N = 7, 17.9 ± 4.7%) was equivalent to that of healthy donors’ T cells (N = 5, 22.2 ± 5.3%), and CAR T cells showed potent anti-tumor reactivity with cytokine productions against CD19 positive Raji cells in vitro. Comparing to CD3/CD28 beads stimulation method, RN/OKT3 stimulation method showed equivalent expansion. Furthermore, RN/OKT3 stimulated T cells preserved higher proportion of CD8+/CCR7+/CD45RA+/CD62L+ naïve phenotype T cells (43.6% in healthy donor and 30.9% in patient donor) compared to CD3/CD28 beads stimulated T cells (22.8% in healthy donor and 11.7% in patient donor). Conclusions; With our novel closed system manufacturing method utilizing RN/OKT3 stimulation combined with RBV-LTS transduction from a small amount of blood of B-NHL patients, we are able to manufacture a sufficient number of CAR T cells maintaining higher proportion of naïve phenotype, which is expected to improve the efficacy of adoptive immunotherapy. In our cell manufacturing, patients are not required to undergo leukapheresis, which is more invasive to patients. Based on our results, we employed our novel manufacturing method for the phase I/II clinical trial to treat patients with relapsed/refractory CD19+ B-NHL (clinicaltrials.gov, identifier; NCT02134262). Disclosures Chono: Takara Bio Inc.: Employment. Tahara:Takara Bio Inc.: Employment. Nukaya:Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Membership on an entity's Board of Directors or advisory committees. Tsukahara:Takara Bio Inc.: Research Funding. Ohmine:Takara Bio Inc.: Research Funding. Ozawa:Takara Bio Inc.: Research Funding. Takesako:Takara Bio Inc.: Membership on an entity's Board of Directors or advisory committees.

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Production of CAR T-cells by GMP-grade lentiviral vectors: latest advances and future prospects

Critical Reviews in Clinical Laboratory Sciences ◽

10.1080/10408363.2019.1633512 ◽

2019 ◽

Vol 56 (6) ◽

pp. 393-419 ◽

Cited By ~ 14

Author(s):

Mansour Poorebrahim ◽

Solmaz Sadeghi ◽

Elham Fakhr ◽

Mohammad Foad Abazari ◽

Vahdat Poortahmasebi ◽

...

Keyword(s):

T Cells ◽

Lentiviral Vectors ◽

Future Prospects ◽

Car T Cells ◽

Car T

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Identification of Small Molecule Modulators to Enhance the Therapeutic Properties of Chimeric Antigen Receptor T Cells

Blood ◽

10.1182/blood.v128.22.4712.4712 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4712-4712

Author(s):

Jonathan Rosen ◽

Betsy Rezner ◽

David Robbins ◽

Ian Hardy ◽

Eigen Peralta ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Small Molecules ◽

Small Molecule ◽

Employment Equity ◽

Car T Cells ◽

Car T ◽

Equity Ownership

Abstract Adoptive cellular therapies using engineered chimeric antigen receptor T cells (CAR-T cells) are rapidly emerging as a highly effective treatment option for a variety of life-threatening hematological malignancies. Small molecule-mediated modulation of T cell differentiation during the in vitro CAR-T manufacturing process has great potential as a method to optimize the therapeutic potential of cellular immunotherapies. In animal models, T cells with a central or stem memory (TCM/SCM) phenotype display enhanced in vivoefficacy and persistence relative to other T cell subpopulations. We sought to identify small molecules that promote skewing towards a TCM/SCM phenotype during the CAR-T manufacturing process, with associated enhanced viability, expansion and metabolic profiles of the engineered cells. To this end, we developed a high-throughput functional screening platform with primary human T cells using a combination of high-content immunophenotyping and gene expression-based readouts to analyze cells following a high-throughput T cell culture platform that represents a scaled-down model of clinical CAR-T cell production. Multicolor flow cytometry was used to measure expansion, cell viability and the expression levels of cell surface proteins that define TCM cells (e.g., CCR7, CD62L and CD27) and markers of T cell exhaustion (e.g., PD1, LAG3, and TIM3). In parallel, a portion of each sample was evaluated using high content RNA-Seq based gene expression analysis of ~100 genes representing key biological pathways of interest. A variety of known positive and negative control compounds were incorporated into the high-throughput screens to validate the functional assays and to assess the robustness of the 384-well-based screening. The ability to simultaneously correlate small molecule-induced changes in protein and gene expression levels with impacts on cell proliferation and viability of various T cell subsets, enabled us to identify multiple classes of small molecules that favorably enhance the therapeutic properties of CAR-T cells. Consistent with results previously presented by Perkins et al. (ASH, 2015), we identified multiple PI3K inhibitors that could modify expansion of T cells while retaining a TCM/SCM phenotype. In addition, we identified small molecules, and small molecule combinations, that have not been described previously in the literature that could improve CAR-T biology. Several of the top hits from the screens have been evaluated across multiple in vitro (e.g., expansion, viability, CAR expression, serial restimulation/killing, metabolic profiling, and evaluation of exhaustion markers) and in vivo (e.g., mouse tumor models for persistence and killing) assays. Results from the initial screening hits have enabled us to further refine the optimal target profile of a pharmacologically-enhanced CAR-T cell. In addition, we are extending this screening approach to identify small molecules that enhance the trafficking and persistence of CAR-T cells for treating solid tumors. In conclusion, the approach described here identifies unique small molecule modulators that can modify CAR-T cells during in vitro expansion, such that improved profiles can be tracked and selected from screening through in vitro and in vivo functional assays. Disclosures Rosen: Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment, Equity Ownership. Hardy:Fate Therapeutics: Employment, Equity Ownership. Peralta:Fate Therapeutics: Employment, Equity Ownership. Maine:Fate Therapeutics: Employment, Equity Ownership. Sabouri:Fate Therapeutics: Employment, Equity Ownership. Reynal:Fate Therapeutics: Employment. Truong:Fate Therapeutics: Employment, Equity Ownership. Moreno:Fate Therapeutics, Inc.: Employment, Equity Ownership. Foster:Fate Therapeutics: Employment, Equity Ownership. Borchelt:Fate Therapeutics: Employment, Equity Ownership. Meza:Fate Therapeutics: Employment, Equity Ownership. Thompson:Juno Therapeutics: Employment, Equity Ownership. Fontenot:Juno Therapeutics: Employment, Equity Ownership. Larson:Juno Therapeutics: Employment, Equity Ownership. Mujacic:Juno Therapeutics: Employment, Equity Ownership. Shoemaker:Fate Therapeutics: Employment, Equity Ownership.

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Single vector multiplexed shRNA provides a non-gene edited strategy to concurrently knockdown the expression of multiple genes in CAR T cells.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.3103 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. 3103-3103

Author(s):

David Edward Gilham ◽

Simon Bornschein ◽

Lorraine Springuel ◽

Alexandre Michaux ◽

Mikhail Steklov ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Retroviral Vector ◽

Vector System ◽

Car T Cells ◽

Shrna Vector ◽

Multiple Genes ◽

Car T

3103 Background: Engineered T cells expressing chimeric antigen receptors (CAR) are now delivering clinically relevant results in patients with advanced hematological malignancies. One critical area for future development is to modulate gene expression thereby endowing the engineered T cell with specific desired features that enhance anti-tumor activity. Methods: Short-hairpin RNA (shRNA) were cloned individually or multiplexed within micro-RNA scaffolds that enabled the co-expression of the individual shRNA with a CAR and a selectable marker all driven by a PolII promoter within a single retroviral vector. Primary human T cells transduced with the CAR-shRNA vectors were selected, expanded in vitro, subjected to negative selection to eliminate any remaining TCR+ cells and examined for target gene expression and functional activity. Results: A 500bp DNA fragment incorporating a shRNA-specific for CD3ζ cloned into a retroviral vectoreffectively knocked down expression of CD3ζ in transduced BCMA-specific CAR T cells. The consequent reduction of cell surface TCR expression resulted in minimal cytokine production upon TCR stimulation in vitro providing a potential allogeneic CAR T approach. These CAR T cells showed no demonstrable evidence of GvHD induction when infused in NSG mice yet maintained BCMA-specific CAR activity in KMS-11 and RPMI-8226 established myeloma models. Initial studies further confirmed that two shRNA could be expressed from a single retroviral vector to modulate the expression of multiple genes. Further engineering of the microRNA framework reduced the size of the transgene load to 394bp while enabling the expression of up to 4 shRNA within a single vector. shRNA specific for CD3ζ, beta-2-microglobulin, CD52 and diacylglycerol kinase alpha were engineered into the framework downstream of a CD19-CAR. Transduced Jurkat cells showed concurrent knockdown of the respective gene products at the mRNA and protein levels. Conclusions: A first-in-human clinical trial evaluating the first-generation single shRNA-vector in the context of a BCMA-targeting CAR as a non-gene edited approach to allogeneic CAR T cell therapy will be initiated in 2020. The proof of principle study here shows that multiple shRNAs are active within a single viral vector thereby avoiding the need for bespoke individual clinical reagents to target multiple genes. The multiplexed shRNA vector system is now in further development to explore whether this strategy can enhance the therapeutic potential of CAR T cells.

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Foamy Virus Based Vectors for CAR-T Cell Development

Blood ◽

10.1182/blood-2019-123022 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4646-4646

Author(s):

Emmanouil Simantirakis ◽

Vassilis Atsaves ◽

Ioannis Tsironis ◽

Margarita Gkyzi ◽

Kostas Konstantopoulos ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Gene Transfer ◽

Peripheral Blood ◽

Transduction Efficiency ◽

Target Cells ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Ifn Γ

Introduction A novel approach that can cover the therapeutic gap in NHL treatment are the autologous T cells, expressing Chimeric Antigen Receptors (CAR-T cells) against tumor markers. Such clinical-grade products based on Lenti (LV) or Retro- vectors have hit the market. An alternative vector system for CAR gene transfer in T-cells are Foamy Viruses (FV). To evaluate the potential of FV vectors in CAR-T cell development, we synthesized an antiCD19 scFv cDNA and cloned it in both an FV and an LV backbone; both vectors were tested in paired experiments Material and Methods The anti-CD19 CAR was under the control of the EF1a promoter; EGFP expression was under the control of an IRES2 element. The anti-CD19 CAR sequence was deduced from published data. FV vectors were made with a 4-plasmid vector system in 293T cells. 2nd generation LV vectors were purchased from Addgene. Cord blood (CB), healthy donor peripheral blood (PB) and CLL patients' PB was used as a source for CD3+ cells using immunomagnetic enrichment. Informed consent has been obtained in all cases of human sample use. T cells were activated by antiCD3/CD28 beads and transduced with antiCD19 LV or FV vectors. Transduction efficiency was assayed by flow cytometry (FCM) using a PE-conjugated anti-mouse Fab antibody. FV and LV CAR-T cells were expanded with Rapid Expansion Protocol (REP) and their cytotoxicity assays was evaluated against the CD19+ cell lines Raji and Daudi. The CLL patient derived CAR-Ts were evaluated against autologous B cells. Cytotoxicity was evaluated with an FCM protocol using CFSE-stained target cells vs unstained effector CARTs in different ratios. At the end of the incubation cells were stained with 7AAD to discriminate against live/dead cells. CAR-T cell activation was also assayed by INF-γ ELISA, following cocultures with target cells at a ratio of 1:1 for 24h. Results Vector titers: LV vector titers were between 3-5x10^5 TU/ml for both LV vectors (with or without EGFP cassette). FV vector titers were between 2-4x10^5 TU/ml regardless of the presence of the EGFP cassette. Tx efficiency: FV can mediate efficient gene transfer on T cells in the presence of heparin at an effective dose of 20-40 U/ml using a spinoculation technique. Transduction efficiency ranged from 40-65% at MOI=3-5, and was comparable to the transduction efficiency of LV vectors at a much higher MOI (10 to 30). Cytotoxicity data on lines: Following REP, the cell population consisted mostly (close to 96% purity) of CAR-T cells regardless of the vector used or of the T cell source. Effector cells were cocultured with the CD19+ cell lines, Daudi and Raji at varying ratios. With cord blood derived FV-CAR-T cells, at 4h post coculture we observed a 39.4% cell lysis at a ratio of 10:1 effector to target (n=1). Similar results were obtained for LV vectors. Peripheral blood derived CAR-T cells at THE same ratio (10:1), demonstrated 83.9% and 93.1% cell lysis for FV-CART and LV-CART cells respectively (n=2). Cytotoxicity data on CLL cells: T-cells from peripheral blood of CLL patients were used to generate LV- and FV-CAR-T cells. At the ratio of 10:1, we observed 73.1% and 69,8% cytotoxicity for FV-CAR-Ts and 70.1% and 70.7% with LV-CAR-Ts, in 2 independent paired experiments. IFN as activation marker: In two paired activation experiments, CB-derived FV-CAR-T cells secrete 560 and 437pg/ml of IFN-γ; similarly, LV-CAR-Ts secrete 534 and 554pg/ml IFN-γ. Untransduced control cells, produced 68pg/ml and 12pg/ml for FV-CAR-T and LV-CAR-T experimental arm respectively. Conclusion In the current work, we developed and tested FV vectors for anti- CD19 CAR-T cell production. We proved that FV viral vectors are capable of mediating efficient gene transfer to human T cells. We developed a method to efficiently transfer FV vectors into T-cells, using a clinically relevant protocol with heparin. The FV-derived CAR T cells demonstrate the same cytotoxic properties in vitro as their LV-derived counterpart and the same activation levels in the presence of CD19 expressing target cells as measured by IFN-γ secretion. FV CARTs derived from PB of CLL patients were capable of mediating comparable cytotoxicity levels as their LV-derived counterparts. Overall, we provide a proof of concept that FVs could be a safe and efficient alternative to LV derived vectors for CAR-T cells. Disclosures No relevant conflicts of interest to declare.

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Closed-system transposon-mediated manufacture of GMP grade CAR T-cells via the Lonza Nucleofector LV XL

Cytotherapy ◽

10.1016/j.jcyt.2020.03.385 ◽

2020 ◽

Vol 22 (5) ◽

pp. S183 ◽

Cited By ~ 1

Author(s):

L.M. Brownrigg ◽

S. Nichols ◽

E. Bosio ◽

B. Carnley ◽

M. Sturm

Keyword(s):

T Cells ◽

Closed System ◽

Car T Cells ◽

Car T

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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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Transposon-Based CAR T Cells in Acute Leukemias: Where Are We Going?

Cells ◽

10.3390/cells9061337 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1337 ◽

Cited By ~ 1

Author(s):

Chiara F. Magnani ◽

Sarah Tettamanti ◽

Gaia Alberti ◽

Ilaria Pisani ◽

Andrea Biondi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Gene Transfer ◽

Cell Therapy ◽

Viral Vector ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T

Chimeric Antigen Receptor (CAR) T-cell therapy has become a new therapeutic reality for refractory and relapsed leukemia patients and is also emerging as a potential therapeutic option in solid tumors. Viral vector-based CAR T-cells initially drove these successful efforts; however, high costs and cumbersome manufacturing processes have limited the widespread clinical implementation of CAR T-cell therapy. Here we will discuss the state of the art of the transposon-based gene transfer and its application in CAR T immunotherapy, specifically focusing on the Sleeping Beauty (SB) transposon system, as a valid cost-effective and safe option as compared to the viral vector-based systems. A general overview of SB transposon system applications will be provided, with an update of major developments, current clinical trials achievements and future perspectives exploiting SB for CAR T-cell engineering. After the first clinical successes achieved in the context of B-cell neoplasms, we are now facing a new era and it is paramount to advance gene transfer technology to fully exploit the potential of CAR T-cells towards next-generation immunotherapy.

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A Phase 1/1b Safety Study of Prgn-3006 Ultracar-T™ in Patients with Relapsed or Refractory CD33-Positive Acute Myeloid Leukemia and Higher Risk Myelodysplastic Syndrome

Blood ◽

10.1182/blood-2020-139417 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 17-17

Author(s):

David A. Sallman ◽

Hany Elmariah ◽

Kendra L. Sweet ◽

Chetasi Talati ◽

Asmita Mishra ◽

...

Keyword(s):

T Cells ◽

Gene Transfer ◽

Board Of Directors ◽

Dose Escalation ◽

Myeloid Leukemia ◽

Research Funding ◽

Publicly Traded ◽

Advisory Committees ◽

Car T Cells ◽

Car T

Background: Therapeutic options for relapse or refractory (r/r) acute myeloid leukemia (AML) and hypomethylating agent (HMA) failure higher risk myelodysplastic syndrome (MDS) pts are limited with median overall survival of < 6 months. Consequently, novel therapies are urgently needed. CD33 is highly expressed on most myeloid leukemia stem cells with lesser expression on normal hematopoietic stem cell populations and minimal non-hematopoietic expression making CD33 a leading target in chimeric antigen receptor therapy (CAR-T) development for myeloid malignancies. However, additional barriers of CAR-T development in myeloid malignancies include long manufacturing period, expansion of CAR-T cells and potential toxicity related to on-target, off-tumor toxicity. Scientific Rationale: Current CAR-T cells utilize viral vectors for gene transfer and subsequent lengthy ex vivo expansion at centralized manufacturing facilities, which is costly and leads to cell product that is exhausted and short lived in vivo. Time is of the essence for pts with rapidly progressing disease such as r/r AML and the prolonged interval between apheresis to product infusion with current CAR-T cell therapies can be a disadvantage. Although allogeneic "off-the-shelf" products allow for rapid administration, challenges remain with rapid rejection. Precigen has developed UltraCAR-T platform to overcome these limitations by utilizing an advanced non-viral gene delivery system and a rapid, decentralized manufacturing process. UltraCAR-T cells are manufactured overnight at medical center's cGMP facility using patient's autologous T cells and administered back to patient only one day after gene transfer with no need for ex vivo expansion. PRGN-3006 UltraCAR-T cells co-express CD33 CAR, membrane bound IL-15 (mbIL15) and a kill switch. Preclinical studies have demonstrated that the expression of the mbIL15 on UltraCAR-T cells leads to maintenance of preferred stem-like memory phenotype (TSCM). Superior efficacy of UltraCAR-T cells was demonstrated in an aggressive murine xenograft model of AML where a single administration of PRGN-3006, only one day after gene transfer, showed significantly higher expansion and persistence; effectively eliminated tumor burden; and significantly improved overall survival compared to traditional CD33 CAR-T cells lacking mbIL15 expression (Blood (2019) 134(S1): 2660). Study Design: The PRGN-3006 UltraCAR-T cells are currently being evaluated in a Phase 1/1b first-in-human dose escalation/dose expansion clinical trial (NCT03927261). The study population includes adult pts (≥ 18 years) with relapsed or refractory AML and HMA failure higher risk MDS or chronic myelomonocytic leukemia (CMML) with ≥ 5% blasts. Pts who have relapsed post allogeneic stem cell transplant are allowed if > 3 months out from transplant without evidence of active graft versus host disease and off immunosuppression for 6 weeks. Key inclusion criteria include an absolute lymphocyte count ≥ 0.2k/µL, KPS > 60%, absence of other active malignancy within 1 year of study entry, daily corticosteroid dose < 10mg of prednisone daily, adequate organ function and a backup allogeneic donor should bone marrow aplasia occur. Hydroxyurea is allowed for cytoreduction with cessation 3 days prior to apheresis/infusion but can be reinitiated post-infusion. To test the hypothesis that expression of mbIL15 on PRGN-3006 cells is sufficient to promote CAR-T cell expansion and persistence, study subjects will receive PRGN-3006 infusion either without prior lymphodepletion (Cohort 1) or following lymphodepleting chemotherapy (Cohort 2 with fludarabine 30mg/m2 and cyclophosphamide 500mg/m2 days -5 to -3). Up to 5 dose levels are planned in dose escalation. All subjects will be followed for adverse events, CAR-T-related toxicities, disease response and PRGN-3006 cell expansion and persistence in blood and bone marrow compartments. In addition, the mechanisms of safety and effectiveness of PRGN-3006 cells will be evaluated with correlative assays of specific immune response pathways. Currently, the study is in the dose escalation phase and has cleared the lower dose level while demonstrating successful manufacturing of UltraCAR-T cells. Additionally, multi-center expansion of the trial is in progress. Disclosures Sallman: Celgene, Jazz Pharma: Research Funding; Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy. Sweet:Agios: Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Stemline: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding. Talati:Astellas: Speakers Bureau; Jazz: Speakers Bureau; AbbVie: Honoraria; Pfizer: Honoraria; BMS: Honoraria. Lankford:Precigen, Inc.: Current Employment. Chan:Precigen, Inc.: Current Employment, Current equity holder in publicly-traded company. Shah:Precigen, Inc.: Current Employment; Intrexon Corporation: Current equity holder in publicly-traded company. Padron:BMS: Research Funding; Novartis: Honoraria; Kura: Research Funding; Incyte: Research Funding. Komrokji:Novartis: Honoraria; Agios: Honoraria, Speakers Bureau; Acceleron: Honoraria; AbbVie: Honoraria; JAZZ: Honoraria, Speakers Bureau; Incyte: Honoraria; Geron: Honoraria; BMS: Honoraria, Speakers Bureau. Lancet:Abbvie: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Astellas Pharma: Consultancy; Celgene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy. Sabzevari:Precigen, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Compass Therapeutics: Current equity holder in publicly-traded company. Bejanyan:Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees.

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Allogeneic CAR-T Cells: More than Ease of Access?

Cells ◽

10.3390/cells7100155 ◽

2018 ◽

Vol 7 (10) ◽

pp. 155 ◽

Cited By ~ 41

Author(s):

Charlotte Graham ◽

Agnieszka Jozwik ◽

Andrea Pepper ◽

Reuben Benjamin

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Healthy Donor ◽

B Cell Lymphoma ◽

Cell Dysfunction ◽

Car T Cells ◽

Car T ◽

T Cell Dysfunction ◽

Ease Of Access

Patient derived anti-CD19 chimeric antigen receptor-T (CAR-T) cells are a powerful tool in achieving a complete remission in a range of B-cell malignancies, most notably B-acute lymphoblastic leukaemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL). However, there are limitations, including inability to manufacture CAR-T cells from the patient’s own T cells, disease progression and death prior to return of engineered cells. T cell dysfunction is known to occur in cancer patients, and several groups have recently described differences in CAR-T cells generated from chronic lymphocytic leukaemia (CLL) patients compared with those from a healthy donor. This is thought to contribute to the low response rate in this disease group. Healthy donor, gene-edited CAR-T cells which do not require human leucocyte antigen (HLA) matching have the potential to provide an ‘off the shelf’ product, overcoming the manufacturing difficulties of producing CAR-T cells for each individual patient. They may also provide a more functional, potent product for malignancies such as CLL, where T cell dysfunction is common and frequently cannot be fully reversed during the manufacturing process. Here we review the potential benefits and obstacles for healthy donor, allogeneic CAR-T cells.

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