F-Deletion Non-Integrating Measles Virus Vector: A Promising Tool for T-Cell Engineering and Naive iPSCs Generation

Although great successes of chimeric antigen receptor T-cell (CAR-T) therapy highlighted the importance of anti-cancer immunity for cancer treatment, there are still some problems remained, i.e., long preparation time, extremely high cost and potential risk by insertional mutagenesis. To developmore rapid and safer T-cell engineering systems than current procedures using retrovirus or lentiviral vectors, we have long been focusing on measles virus as a new vector because of its high infectivity to T cells including resting state and rapid gene expression without chromosome integration. Presently, Sendai virus vectors (SVs), which is also a Paramyxoviridaevirus-based vector, are widely used for gene delivery and induced pluripotent stem cells (iPSCs), but the transduction to undifferentiated T cells (UTs) is a big challenge for SVs. We, therefore, compared the gene transduction efficiency between our measles vector (MVs) and SVs. We also compared iPSCs generation efficiencies between MVs and SVs. We engineered our non-replicating and non-integrating measles virus vectors (MV)with F deletion to eliminate cell membrane fusion-associated cytotoxicity.Based on the original property of measles virus,our recombinant MVsallowed more efficient gene transduction to various hematopoietic cells including UTs and B cells than SVs. Importantly, MVs induced less apoptosis compared withSVs due to their slower amplification of viral RNA in transduced cells. Moreover, we could establish iPSCs from UTs with MVs harboring reprogramming genes 50 times more efficiently than SVs harboring the same reprogramming genes. MV-induced iPSCs derived from CD3+T cells (MV-TiPSCs) were similar to regular human pluripotent stem cells (hPSCs: embryonic stem cells and iPSCs), which are in primed state, in morphology, the expressions of pluripotent markers and the ability to differentiate into three germ layers. On the other hand, without using naive induction culture condition, MV-induced iPSCs derived from CD34+hematopoietic progenitor cells (MV-HPC-iPSCs) presented a dome shape and showed a transcriptome profile close to naive iPSCs. To further confirm naive-like properties of MV-HPC-iPSCs, we evaluated gene expression patterns of these cells for 22 common genes most differently expressed in naive and primed hPSCs reported in previous reports (Fig.1).As expected, MV-HPC-iPSCs were clustered in naive hPSCs group while MV-HPC-iPSCs after culturing in primed induction condition showed primed-like features (Fig.2). Moreover, whole genome bisulfite sequencing analysis showed that MV-HPC-iPSCs had lower methylation than primed MV-HPC-iPSCs. These results strongly suggested that MV could induce naive-like iPSCs directly, and primed induction culture changed the cells to primed state with increasing genomic methylation. Considering the very safe history of MV vaccine, the capabilities of simultaneous expressions of multiple genes and the high transduction efficiency for hematopoietic cells including UTs, our MVs will be useful to directly induce naive state iPSCs, and be a promising tool for developing new T-cell immunotherapies. Disclosures Liao: TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Soda:TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Miura:Neoprecision therapeutics: Research Funding. Tahara:TAKARA BIO, INC.: Research Funding. Miyamoto:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Takeda:TAKARA BIO, INC.: Research Funding. Tani:Oncolys BioPharma Inc.: Equity Ownership; SymBio Pharmaceuticals Limited: Equity Ownership; TAKARA BIO, INC.: Research Funding; Neoprecision therapeutics: Equity Ownership, Research Funding; neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Equity Ownership.

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Automated Lentiviral Transduction of T Cells with Cars Using the Clinimacs Prodigy

Blood ◽

10.1182/blood.v126.23.2043.2043 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2043-2043 ◽

Cited By ~ 1

Author(s):

Ulrike Mock* ◽

Lauren Nickolay* ◽

Gordon Weng-Kit Cheung ◽

Hong Zhan ◽

Karl Peggs ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Adoptive Immunotherapy ◽

Research Funding ◽

Transduction Efficiency ◽

Manufacturing Processes ◽

Antigen Receptors ◽

Chimeric Antigen Receptors ◽

Lentiviral Transduction ◽

Equity Ownership

Abstract BACKGROUND Genetically modified T cells have enormous potential for the treatment of relapsed and refractory haematopoietic malignancies. CD19-positive B-cell malignancies including acute lymphoblastic leukaemia (ALL), chronic lymphocytic leukaemia (CLL) or B cell non-Hodgkin lymphomas (NHL) have been shown to be an excellent target for adoptive immunotherapy with T cells expressing CD19-specific chimeric antigen receptors (CARs). The increasing need for genetically modified T cells is hampered by the limited number of centres with the required infrastructure and expertise to produce this complex therapeutic product. Ex vivo modification of T cells requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, Miltenyi Biotec has developed the CliniMACS Prodigy platform and is automating complex cell manufacturing processes. These have now been adapted for lentiviral transduction of T cells and we show the feasibility and effectiveness of the device for adoptive immunotherapy using chimeric antigen receptors. METHODS A self-inactivating third generation lentiviral vector encoding a CAR specific for CD19 (CAR19) was used for automated T-cell transductions (TCT). Using closed single-use tubing sets (TS520), fresh or cryopreserved peripheral blood mononuclear cells from non-mobilised leukapheresis collected from healthy donors were loaded onto the CliniMACS Prodigy, washed and activated in TexMACS media with TransAct, a polymeric nanomatrix activation reagent agonist for CD3 and CD28. Cells were transduced 24-48h after activation and expanded in the CentriCult-Unit of the tubing set, allowing for stable culture conditions as well as automated feeding and media exchange. Small and large scale comparison transductions were run in parallel to assess the efficiency of the automated T-cell modification. Finally, cells were harvested and cryopreserved to assess the functional capabilities of CAR19 T cells. RESULTS Three automated TCT runs were performed and continuously monitored to assess cell expansion, transduction efficiency and the phenotype of the final cell product. On average, expansion during automated cultivation was 11.7x (range: 5.4 - 22.8x) which was comparable to the expansion achieved in small scale controls (12.3x ± 1.2x). The average yield from the automated process was 11.8x108 total lymphocytes/run (ranging between 4 - 23.2x108 lymphocytes/run). Notably, this was comparable to existing CAR19 T cell manufacturing processes using a WAVE-Bioreactor. In all three runs in the Prodigy, successful transduction was observed with an average transduction efficiency of 32% CAR19-positive cells (range: 22- 45%). Again, this was similar to transduction efficiencies (32% CAR19-positive; range: 27-40%) in previous WAVE-production campaigns using X-Vivo15 media and magnetic beads conjugated with anti-CD3/CD28 antibodies for T-cell activation (Dynabeads). Flow cytometry analysis of the final cell product showed a high purity of CD45+/CD3+ cells (90%) as well as a relatively high frequency of CD8-positive cytotoxic T cells (56%). Immunophenotyping revealed high expression of CD45RA, CD62L, CD27 and CD95 with moderate expression of CCR7. Importantly, no significant difference in PD-1 expression was observed between automatically and manually processed cells. Finally, functional analysis showed cytotoxic activity as well as IFN-γ/TNF-α production upon co-cultivation with CD19-expressing target cells. CONCLUSION In summary, we have demonstrated the feasibility of the CliniMACS Prodigy for the generation of CAR+ T cells for adoptive immunotherapy. Automated activation, transduction and expansion resulted in clinically relevant doses of CAR19 T cells with very little 'hands-on' operator time. Given the closed-system nature of the device, and automated features, the CliniMACS Prodigy should widen applicability of T-cell engineering beyond centres with highly specialised infrastructures. Disclosures Mock*: Miltenyi Biotec GmbH: Research Funding. Nickolay*:Miltenyi Biotec GmbH: Research Funding. Peggs:Cellectis: Research Funding; Autolus: Consultancy, Equity Ownership. Johnston:Miltenyi Biotec GmbH: Employment. Kaiser:Miltenyi Biotec GmbH: Employment. Pule:CELLECTIS: Research Funding; AUTOLUS: Employment, Equity Ownership, Research Funding; AMGEN: Honoraria; UCLB: Patents & Royalties. Thrasher:Miltenyi Biotec GmbH: Research Funding; Autolus Ltd: Consultancy, Equity Ownership, Research Funding. Qasim:Cellectis: Research Funding; Miltenyi Biotec GmbH: Research Funding; Autolus Ltd: Consultancy, Equity Ownership, Research Funding; Cell Medica: Research Funding.

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Efficient Gene Transduction and Reprogramming of Hematopoietic Cells Including T-Cells By Using a Non-Integrating Measles Virus Vector

Blood ◽

10.1182/blood-2018-99-120350 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3494-3494

Author(s):

Jiyuan Liao ◽

Yasushi Soda ◽

Ai Sugawara ◽

Yoshie Miura ◽

Takafumi Hiramoto ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Gene Transfer ◽

Research Funding ◽

Measles Virus ◽

Gene Transduction ◽

Efficient Gene ◽

Car T ◽

Ipsc Generation ◽

Equity Ownership

Abstract By the ectopic expression of reprogramming genes OCT, KLF4, SOX2 and MYC (OKSM), somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs). Human iPSCs are considered a promising cell source to provide an import tool for the basic investigation and the advanced medicine including gene therapy and regenerative medicine. To establish iPSCs, integration-free Sendai virus (SV) vectors have been most widely used do far, but transduction and reprogramming of T cells without stimulation is still very challenging. On the other hand, a great success of chimeric antigen receptor T cell (CAR-T) therapies highlighted the importance of anti-cancer immunity for the cancer treatment. Particularly, many refractory patients with acute lymphoblastic leukemia and B-cell lymphoma were successfully treated with CD19-CAR-T therapies, however, some patients died before receiving the treatment due to long preparation time of CAR-Ts. Therefore, rapid production systems of CAR-Ts are desired, and for this purpose, efficient and safe gene transduction systems to T cells should be developed. In this study, we developed a new non-integrating measles virus (MV) vector-based delivery system with F deletion to eliminate cell membrane fusion-associated cytotoxicity. MV vectors transduced genes through MV receptors including CD46 and signaling lymphocyte activation molecule (CD150/SLAM). First, we examined transduction efficiencies of MV vectors and SV vectors in hematopoietic cells by using GFP expression vectors (MV-Gs and SV-Gs). Compared to SV-Gs, our MV-Gs allowed more efficient gene transfer into most hematopoietic cell type including T (3-fold) and B cells (7-fold) (Fig. 1). Furthermore, at the same multiplicity of infection (MOI) of viral transduction, MV-Gs induced less apoptosis in T cell subset compared to SV-Gs (Fig. 2) due to the slower kinetics of viral RNA amplification in the transduced cells 24 h ,48 h and 72 h post transduction. Those results encouraged us to examined if MV vectors are more potent than SV vectors in iPSC generation from unstimulated T cells. To address this question, we developed MV vectors harboring four reprogramming genes (MV-OKSMGs) and compared with SV vectors harboring these genes (SV-OKSMGs). As expected, with the MV-OKSMGs, we could generate high-quality iPSCs with the similar morphology, pluripotency markers, karyotype and differentiation capacity as human embryonic stem cells. Upon the less cytotoxicity, iPSC generation efficiency of MV-OKSMGs was much higher than that of SV-OKSMGs for unstimulated T cells (0.47 ± 0.25% vs 0.008 ± 0.009%). Considering the safe history of MV vaccine, carrying capabilities of multiple genes, more flexible receptors and higher transduction efficiency for resting T cells, our exclusive MV vector would be a potential gene transfer system for iPSC generation and lymphocyte-based-immunotherapies such as CAR-T therapies. Disclosures Liao: neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Soda:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Sugawara:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miura:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Tahara:TAKARA BIO, INC.: Research Funding. Takishima:neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Hirose:TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Hijikata:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miyamoto:Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; neopharma Japan Co. Ltd: Research Funding. Takeda:TAKARA BIO, INC.: Research Funding. Tani:neopharma Japan Co. Ltd: Research Funding; Oncolys BioPharma Inc.: Equity Ownership; SymBio Pharmaceuticals Limited: Equity Ownership; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding.

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A Novel Second Generation CD19 CAR for Therapy of High Risk/Relapsed Paediatric CD19+ Acute Lymphoblastic Leukaemia and Other Haematological Malignancies: Preliminary Results from the Carpall Study

Blood ◽

10.1182/blood.v128.22.4026.4026 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4026-4026

Author(s):

Sara Ghorashian ◽

Anne Marijn Kramer ◽

Sarah Jayne Albon ◽

Catherine Irving ◽

Lucas Chan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Memory T Cells ◽

Transduction Efficiency ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Post Infusion ◽

Equity Ownership

Abstract Introduction: Recent clinical trials with T cells engineered to express 2nd generation CD19 chimeric antigen receptors (CARs) unprecedented anti-leukemic responses. We have developed a novel CD19CAR with a new scFv in the 41BBz format (CAT-41BBz CAR) which confers enhanced cytotoxicity and cytokine secretion in response to stimulation with CD19+ targets in vitro as well as equivalent in vivo anti-tumour efficacy to the FMC63 41BBZ CAR in use in clinical studies. We have designed, optimized and validated GMP-grade CAR T cell production using this novel CAR. Based on these data, we have recently initiated a Phase I clinical study (CARPALL) of this novel CAR in pediatric patients with relapsed ALL and other CD19+ hematological malignancies to determine the safety profile and durability of responses to CD19CART therapy. This will be critical in determining whether CD19CAR T cells are best used as a stand-alone therapy or as a bridge to stem cell transplant (SCT). Methods: We initially optimized our GMP production methodology in terms of activation method, cytokine milieu and expansion conditions on healthy donor peripheral blood mononuclear cells (PBMCs) to give optimal transduction efficiency and preserve early memory subsets within the CAR T cell product. We have subsequently validated this methodology using unstimulated leucaphereses from 5 lymphopenic patients with ALL. PBMCs were activated with anti-CD3/CD28 microbeads (Dynabeads CTS) and then lentivirally transduced with the CAT CAR vector. T cells were then expanded in the WAVE bioreactor before bead removal on a magnetic system and cryopreservation. Patients on study receive lymphodepletion with fludarabine and cyclophosphamide followed by a single dose of 106 CAR+ T cells/kg and are then monitored as an in-patient for 14 days post infusion for toxicities such as cytokine release syndrome or neurotoxicity. The primary end-points of the study are toxicity and the proportion of patients achieving molecular CR at 1 month post CD19CAR T cell infusion. Following this, patients undergo intensive monitoring of disease status for a total of 2 years post infusion. To determine the durability of responses, patients achieving a molecular CR will be monitored closely for the re-emergence of molecular level disease without additional consolidative therapy or SCT Results: We were able to generate the target dose of 1x106 CAR+ T cells/kg in 6 of 7 production runs (involving 2 healthy donors and 5 patients) to date, all of which met sterility release criteria. Transduction efficiency was on average 37% (range 7-84%, see table 1). Mean viral copy was 4.2 (range 1.2-5.8). Memory T cells of stem cell-like phenotype (CAR+ CCR7+ CD45RA+ CD95+ CD127+) formed a mean of 9% (range 0-31%), central memory T cells (CAR+ CCR7+ CD45RA-) formed a mean of 43% (range 16-70%) and effector memory T cells formed a mean of 31% (range 0-77%) of the final CAR T cell product. The percentage of CAR T cells expressing dual exhaustion markers (TIM3+ PD-1+) was on average 5% (range 2-8%). So far 2 patients have been treated. Conclusions We have optimized and successfully validated a robust GMP production method for CD19CAR T cells lentivirally transduced with a novel CD19CAR. Preliminary results of therapy with CAT-41BBz CAR T cells in initial patients on the clinical study will be presented. Disclosures Qasim: Autolus: Consultancy, Equity Ownership, Research Funding; Cellectis: Research Funding; Calimmune: Research Funding; Catapult: Research Funding. Pule:Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL Business: Patents & Royalties; Amgen: Honoraria; Roche: Honoraria.

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The MEK Inhibitor Trametinib Enhances Diverse T Cell Reconstitution with Suppressing Xenogeneic Graft-Versus-Host-Disease

Blood ◽

10.1182/blood-2019-128913 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1929-1929

Author(s):

Hidekazu Itamura ◽

Hiroyuki Muranushi ◽

Takero Shindo ◽

Kazutaka Kitaura ◽

Seiji Okada ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Mek Inhibitor ◽

Effector Memory ◽

Graft Versus Host ◽

T Cell Reconstitution ◽

Tcr Repertoire ◽

Repertoire Diversity ◽

Equity Ownership

Introduction: Early immune reconstitution without severe graft-versus-host disease (GVHD) is required for the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We showed that MEK inhibitors suppress GVHD but retain antiviral immunity and graft-versus-tumor (GVT) effects (Shindo, Blood2013; Itamura, Shindo, JCI Insight2016). Furthermore, we have shown that they attenuate graft rejection but spare thymic function following rat lung transplantation (Takahagi, Shindo, Am J Respir Cell Mol Biol2019). Here we analyzed their effects on human polyclonal T cell reconstitution in xenogeneic transplant by evaluating T-cell receptor (TCR) repertoire diversity. Methods: As a xenogeneic GVHD model, human PBMCs were infused to NOD/Scid/JAK3null mice, immunodeficient mice lacking T/B/NK cells, after total body irradiation. Vehicle, tacrolimus, or the MEK inhibitor trametinib was administered from day 0 through 28 or day 15 through 28. Human TCR repertoire diversity was evaluated by an adapter ligation PCR method with next generation sequencing (Shindo, Oncoimmunol2018) in the liver, lung, and spleen. The assignment and frequencies of TCRαV/J clones were determined at the single-cell level. Their diversity and clonality were evaluated by Inv. Simpson's index 1/λ. Results: Trametinib prolonged their survival compared with vehicle (median survival: 88 vs 46 days, p<0.05). It enhanced engraftment of human leukocytes in peripheral blood (human CD45+cells: 11.0 vs 2.5%), but prevented their infiltration into the lung (human CD45+cells on day 60: 1.5 vs 6.5%). Treatment with vehicle resulted in skewed TCR repertoire with limited clones in the spleen, liver and lung. Interestingly, expansion of one specific clone (TRAV20/J10) was commonly observed, which might reflect the GVHD-inducing pathological clone (Fig. 1: 3D graphs show the frequencies of TCRαV/J clones). However, trametinib enabled diverse and polyclonal T cell engraftment without the TRAV20/J10 clone. While CD4+and CD8+T cells within injected human PBMCs mainly consisted of naïve (CD45RA+CD27+) and central memory (CD45RA-CD27+) T cells, infiltrating T cells in each organ showed effector memory (CD45RA-CD27-) T cell phenotype. Of note, CD8+T cells in the bone marrow, spleen, and lung of trametinib-treated recipients showed reduced effector memory T cells (CD45RA-CD27-) compared with vehicle-treated mice at day 28 (bone marrow 21.7 vs 74.7%, p<0.01; spleen 66.3 vs 88.7%, p<0.05; lung 33.0 vs 72.5%, p<0.05), which indicating that MEK inhibition suppresses functional differentiation of human T cells in vivo. Furthermore, trametinib treatment from day 14 to 28 still ameliorated clinical GVHD score, and maintained polyclonal T cell repertoire. Conclusions:GVHD can be characterized with skewed TCR repertoire diversity and expansion of pathological T cell clones in the target tissues. Trametinib suppresses GVHD but maintains polyclonal T cell reconstitution, even in established GVHD. These results explain the facts that MEK inhibitors separate GVHD from GVT effects/antimicrobial immunity. Furthermore, MEK inhibition enhances immune reconstitution after allo-HSCT, which would avoid post-transplant complications. Disclosures Shindo: Novartis: Research Funding. Kitaura:Repertoire Genesis Inc.: Employment. Okada:Bristol-Myers Squibb: Research Funding; Japan Agency for Medical Research and Development: Research Funding. Shin-I:BITS Co., Ltd: Equity Ownership. Suzuki:Repertoire Genesis Inc.: Equity Ownership. Takaori-Kondo:Celgene: Honoraria, Research Funding; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Ono: Research Funding; Takeda: Research Funding; Kyowa Kirin: Research Funding; Chugai: Research Funding; Janssen: Honoraria; Pfizer: Honoraria. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.

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Dissecting the Immune Cell Progeny of CAR-Expressing Hematopoietic Stem Cells in a Humanized Mouse Model

Blood ◽

10.1182/blood-2019-129746 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4640-4640

Author(s):

Heng-Yi Liu ◽

Nezia Rahman ◽

Tzu-Ting Chiou ◽

Satiro N. De Oliveira

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

Hematopoietic Stem Cells ◽

Cell Lines ◽

Immune Cells ◽

Research Funding ◽

Humanized Mice ◽

Tumor Challenge ◽

Hematopoietic Stem

Background: Chemotherapy-refractory or recurrent B-lineage leukemias and lymphomas yield less than 50% of chance of cure. Therapy with autologous T-cells expressing chimeric antigen receptors (CAR) have led to complete remissions, but the effector cells may not persist, limiting clinical efficacy. Our hypothesis is the modification of hematopoietic stem cells (HSC) with anti-CD19 CAR will lead to persistent generation of multilineage target-specific immune cells, enhancing graft-versus-cancer activity and leading to development of immunological memory. Design/Methods: We generated second-generation CD28- and 4-1BB-costimulated CD19-specific CAR constructs using third-generation lentiviral vectors for modification of human HSC for assessment in vivo in NSG mice engrafted neonatally with human CD34-positive cells. Cells were harvested from bone marrows, spleens, thymus and peripheral blood at different time points for evaluation by flow cytometry and ddPCR for vector copy numbers. Cohorts of mice received tumor challenge with subcutaneous injection of lymphoma cell lines. Results: Gene modification of HSC with CD19-specific CAR did not impair differentiation or proliferation in humanized mice, leading to CAR-expressing cell progeny in myeloid, NK and T-cells. Humanized NSG engrafted with CAR-modified HSC presented similar humanization rates to non-modified HSC, with multilineage CAR-expressing cells present in all tissues with stable levels up to 44 weeks post-transplant. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. T-cell populations were identified at higher rates in humanized mice with CAR-modified HSC in comparison to mice engrafted with non-modified HSC. CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice engrafted with CAR-modified HSC successfully presented tumor growth inhibition and survival advantage at tumor challenge with lymphoma cell lines, with no difference between both constructs (62.5% survival for CD28-costimulated CAR and 66.6% for 41BB-costimulated CAR). In mice sacrificed due to tumor development, survival post-tumor injection was directly correlated with tumor infiltration by CAR T-cells. Conclusions: CAR modification of human HSC for cancer immunotherapy is feasible and continuously generates CAR-bearing cells in multiple lineages of immune cells. Targeting of different malignancies can be achieved by adjusting target specificity, and this approach can augment the anti-lymphoma activity in autologous HSC recipients. It bears decreased morbidity and mortality and offers alternative therapeutic approach for patients with no available sources for allogeneic transplantation, benefiting ethnic minorities. Disclosures De Oliveira: National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding.

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

Blood ◽

10.1182/blood-2019-125960 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2866-2866 ◽

Cited By ~ 2

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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Itolizumab As a Potential Therapeutic for the Prevention and Treatment of Graft Vs Host Disease

Blood ◽

10.1182/blood-2019-122787 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5603-5603 ◽

Cited By ~ 1

Author(s):

Cherie Tracy Ng ◽

Jeanette Ampudia ◽

Robert J. Soiffer ◽

Jerome Ritz ◽

Stephen Connelly

Keyword(s):

Weight Loss ◽

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Research Funding ◽

Chronic Gvhd ◽

Vehicle Control ◽

Control Group ◽

Employment Equity ◽

Equity Ownership

Background: CD6 is a co-stimulatory receptor, predominantly expressed on T cells, that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation, differentiation and trafficking and is central to inflammation. While effector T cell (Teff) are CD6hi and upregulate expression upon activation, regulatory T cells (Treg) remain CD6lo/-, making this an attractive target to modulate Teff activity while preserving Treg activity. Early studies by Soiffer and colleagues demonstrated using T12, an anti-CD6 monoclonal antibody (mAb) that ex-vivo depletion of CD6+ donor cells prior to transplantation decreased the incidence of both acute and chronic GVHD, highlighting the importance of CD6+ cells in GVHD pathogenesis and validating it as a therapeutic target. However, it remains to be shown whether modulating the CD6-ALCAM pathway in vivo can attenuate GVHD. We investigated the use of itolizumab, a humanized anti-CD6 mAb that has demonstrated clinical efficacy in other autoimmune diseases, as both a preventive and therapeutic treatment for GVHD, using a humanized xenograft mouse model. Methods: Humanized xenograft mice were generated by intravenous transfer of 2x10^7 human PBMCs into 6-8 weeks old NOD/SCID IL2rγ-null (NSG). To investigate the ability of itolizumab to prevent GVHD, mice were dosed with either 60μg or 300μg of itolizumab, 150μg of abatacept (CTLA4-Ig), or vehicle, starting one day prior to PBMC transplantation. To investigate the therapeutic effect of itolizumab, mice were dosed with either 150μg of itolizumab or vehicle, starting at Day 5 post-PBMC transfer, when transplanted T cells are already activated. All treatments were administered IP every other day. Weight and disease scores were monitored throughout the study. At Days 18 and 35, peripheral blood was evaluated by flow cytometry to examine T cell prevalence, and tissues were collected for histological examination of pathology and T cell infiltration. Results: When administered as prevention (Day -1), treatment with either 60μg or 300μg of itolizumab significantly decreased mortality compared to the vehicle control (100% vs. 10%); this decrease was similar to the positive control group treated with abatacept (Figure 1). At 60μg, itolizumab-treated mice demonstrated significant reductions in the prevalence of human T cells in peripheral blood vs. vehicle-treated mice at Day 18 (<0.2% vs. 74.5%; p < 0.001). The reduction in peripheral T cells was accompanied by reductions in tissue-infiltrating T cells in lung (85-fold) and gut (9.5-fold), as well as reductions in disease scores and weight loss. When administered therapeutically, treatment with itolizumab was associated with a survival rate of 50% compared to 10% in the control group (Figure 2). Similarly, peripheral T cell prevalence (34.3% vs. 65.1%; p < 0.001), weight loss, and disease scores were inhibited by itolizumab compared to vehicle control mice. Conclusions: These data suggest that systemic treatment with itolizumab can modulate pathogenic Teff cell activity, establishing this antibody as a potential therapeutic for patents with GvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Ng: Equillium: Employment, Equity Ownership. Ampudia:Equillium: Employment. Soiffer:Mana therapeutic: Consultancy; Kiadis: Other: supervisory board; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Jazz: Consultancy. Ritz:Equillium: Research Funding; Merck: Research Funding; Avrobio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy; LifeVault Bio: Consultancy; Celgene: Consultancy; Aleta Biotherapeutics: Consultancy; Kite Pharma: Research Funding. Connelly:Equillium: Employment, Equity Ownership.

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NOTCH and CAR Signaling Control T Cell Lineage Commitment from Pluripotent Stem Cells

Blood ◽

10.1182/blood-2020-142894 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 30-30

Author(s):

Sjoukje van der Stegen ◽

Pieter Lindenbergh ◽

Roseanna Petrovic ◽

Benjamin Whitlock ◽

Raedun Clarke ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Cell Lineage ◽

Lineage Commitment ◽

Publicly Traded ◽

Car T Cell ◽

Car T ◽

Single Positive

Chimeric Antigen Receptor (CAR) T cells are a new treatment paradigm for relapsed/refractory hematopoietic malignancies. However, their autologous nature imposes manufacturing constraints that can delay CAR T cell availability and increase their cost. We previously established proof of principle that αβ T cell-derived induced pluripotent stem cells (TiPSCs) can provide a self-renewing source for in vitro CAR T cell production (Themeli, Nat Biotechnol, 2013). The use of cloned TiPSC further enhances the feasibility of verifying genome integrity of the genetically engineered stem cells and should in principle yield highly homogenous cell products. Using αβ T cell-derived TiPSCs transduced with a well-defined CD19-specific CAR (1928z; Park, NEJM, 2018), we previously demonstrated that TiPSCs can be differentiated into CAR T cells. These T cells retained their endogenous T cell receptor (TCR) and also displayed characteristics of innate lymphoid cells. We have now examined how the timing of CAR expression as well as the CAR signaling strength influence T cell lineage commitment, enabling better control towards αβ T cell lineage commitment. αβ T cell lineage development depends in part on a precisely orchestrated interactions between NOTCH and (pre)TCR signaling, the timing and strength of which are crucial for αβ lineage commitment. Because TiPSCs harbor rearranged TCRα and TCRβ genes, mature TCR expression occurs earlier than if it required VDJ recombination, skewing differentiation towards acquiring innate features including CD4-CD8- double-negative or CD8αα single-positive phenotypes. We show that providing strong NOTCH stimulation counteracts the effects of early antigen receptor expression, facilitating CD4+CD8αβ+ double positive (DP) formation. We hypothesized that CAR signaling in the absence of ligand binding (tonic signaling) may mimic a TCR signal, the strength and timing of which could re-direct lineage commitment. We therefore investigated CARs providing different levels of signaling strength and the impact of delaying the onset of CAR expression. Tonic CAR signaling was measured in peripheral blood T cells expressing 1928z or 1928z-1XX, a construct in which the second and third ITAM in the CD3ζ domain have been mutated to be non-functional (Feucht, Nat Med, 2019), following either retroviral transduction (SFG vector) orTRAC-targeted cDNA integration, placing CAR expression under the transcriptional control of the TCRα promoter (Eyquem, Nature, 2017). CAR signaling in the absence of antigen exposure, measured by phosphorylation of ITAM3, ERK1/2 and ZAP70, was reduced by bothTRAC-targeting and reduction of functional ITAMs, with additive effects when combined inTRAC-1928z-1XX. Three of these engineering strategies (virally expressed 1928z,TRAC-1928z andTRAC-1928z-1XX) were evaluated in the context of TiPSC-derived T cell differentiation. Virally expressed 1928z (resulting in constitutive CAR expression throughout differentiation) resulted in the predominant generation of innate-like CD8αα T cells, associated with the absence of early T cell lineage markers such as CD5, CD2 and CD1a. Delayed expression of 1928z throughTRACtargeting resulted in increased CD5, CD2 and CD1a, but did not yield any more CD4+CD8αβ+ DP cells. In TiPSC expressingTRAClocus-encoded 1928z-1XX, a greater DP population emerged, from which CD8αβ single-positive T cells could be induced. Phenotypic analyses of clonal TRAC-1928z-1XX TiPSC lines further establish the interplay between CAR and NOTCH1 in determining αβ lineage commitment. Together these data show that early TCR and CAR expression skew T cell lineage commitment towards an innate-like T cell fate, which can be overcome by controlling the strength and timing of NOTCH, TCR and CAR signaling. These studies pave the way for the predetermined generation of a variety of CAR T cell types endowed with different functional attributes. Disclosures Whitlock: Fate Therapeutics Inc.:Current Employment, Current equity holder in publicly-traded company.Clarke:Fate Therapeutics Inc.:Current Employment, Current equity holder in publicly-traded company.Valamehr:Fate Therapeutics, Inc:Current Employment, Current equity holder in publicly-traded company.Riviere:Juno Therapeutics:Other: Ownership interest, Research Funding;Takeda:Research Funding;Fate Therapeutics Inc.:Consultancy, Other: Ownership interest , Research Funding;FloDesign Sonics:Consultancy, Other: Ownership interest;Atara:Research Funding.Sadelain:Atara:Patents & Royalties, Research Funding;Fate Therapeutics:Patents & Royalties, Research Funding;Mnemo:Patents & Royalties;Takeda:Patents & Royalties, Research Funding;Minerva:Other: Biotechnologies , Patents & Royalties.

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Evaluation Of CD33 Expression and Functional Analysis Of The CD33/CD3 Bispecific BiTE® Antibody AMG 330 In Primary AML Samples

Blood ◽

10.1182/blood.v122.21.239.239 ◽

2013 ◽

Vol 122 (21) ◽

pp. 239-239 ◽

Cited By ~ 2

Author(s):

Christina Krupka ◽

Peter Kufer ◽

Roman Kischel ◽

Gerhard Zugmaier ◽

Jan Boegeholz ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Ex Vivo ◽

Natural Setting ◽

Single Chain ◽

Activation Markers ◽

Expression Levels ◽

Equity Ownership

Abstract Antibody-based immunotherapy represents a promising strategy to specifically target and eliminate chemoresistant leukemic cells in acute myeloid leukemia (AML). We evaluated a single-chain bispecific CD33/CD3 BiTE® antibody (AMG 330) for its suitability as immunotherapy in AML. A prerequisite for successful immunotherapeutic approaches using this molecule is the expression of CD33 on AML blasts including leukemic stem cells (LSCs). Therefore, we quantified CD33 expression on AML blasts and LSCs by flow cytometry (mean fluorescence intensity, MFI) and correlated expression intensity with cytogenetic and molecular disease characteristics in order to identify patient cohorts possibly most suited for CD33-targeted therapies. CD33 expression was detected in >99% of patient samples (n=621, MFI ratio ≥ 1.5) although highly variable. A strong correlation between high CD33 expression levels and NPM1 mutations (p<0.001) was found. In contrast, low CD33 expression levels were significantly associated with complex karyotypes and t(8,21) translocations (p<0.001). Furthermore, LSCs within the CD34+/CD38- compartment displayed CD33 at higher levels than healthy donor stem cells (p=0.047). Importantly, colony formation of CD34+/Lin-cells from healthy donors was not affected after pre-incubation with AMG 330 and T-cells. A major hurdle for measuring cytotoxic effects on AML blasts has long been that primary AML patient samples show progressive cell death within a few days ex-vivo. To simulate the natural setting of target and T-cells in AML patients, we developed a long-term culture system for AML blasts that allowed us to observe these co-cultures for up to 5 weeks. Thus, we were able to show effective elimination of AML blasts within primary samples by AMG 330-activated and expanded residual CD3+/CD45RA-/CCR7+ memory T-lymphocytes. While the functional relevance of CD33 expression levels was shown by faster lysis kinetics of CD33BRIGHT vs. CD33DIM AML cell lines in an in-vitro cytotoxicity assay potent anti-leukemic activity on primary AML blasts was observed irrespective of CD33 expression level. At low effector to target ratios (up to 1:79), the recruited T-cells lysed autologous blasts completely in the majority of samples. Further T-cell analysis showed that naive T-cells (CD45RA+/CCR7+) were not expanded by AMG 330; neither were terminally differentiated T-cells (CD45RA+/CCR7-), probably due to their poor proliferative capacity. We did not observe an increase in percentage of CD3+/CD4+/CD25+/FoxP3+regulatory T-cells in the presence of AMG 330, suggesting that these cells may not have impacted AMG 330-mediated T-cell activity in our experiments. Compared to control cultures, T-cells were shown to up-regulate the activation markers CD25, PD-1, TIM3 and LAG3 upon response to AMG 330, which was partially reversible after complete target cell elimination. However, PD-1 up-regulation did not correlate with an up-regulation of PD-L1 on AML blasts despite substantial INFγ secretion by activated T-cells. This study provides the first correlation of CD33 expression levels to a comprehensive genotype analysis in adult AML patients. While CD33 expression may vary by AML biologic subgroup, AMG 330 exposure led to lysis of AML blasts even in samples with low levels of expression. Targeting CD33 using AMG 330 in primary AML samples led to efficient T-cell activation and expansion as expected from the mechanism of action of BiTE® antibodies. The remarkable ex-vivo activity of AMG 330 supports further development of AMG 330 as an immunotherapy for patients with AML. Disclosures: Kufer: AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Kischel:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Zugmaier:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Baeuerle:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Riethmüller:AMGEN Inc.: Equity Ownership.

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First Clinical Application of Talen Engineered Universal CAR19 T Cells in B-ALL

Blood ◽

10.1182/blood.v126.23.2046.2046 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2046-2046 ◽

Cited By ~ 54

Author(s):

Waseem Qasim ◽

Persis Jal Amrolia ◽

Sujith Samarasinghe ◽

Sara Ghorashian ◽

Hong Zhan ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

B Cell ◽

T Cell Receptor ◽

Research Funding ◽

Cell Receptor ◽

Receptor Expression ◽

Molecular Remission ◽

Equity Ownership

Abstract Chimeric antigen receptor (CAR)19 T-cells exhibit powerful anti-leukemic effects in patients with B cell malignancies. However, the complexity of production of patient bespoke T cell products is a major barrier to the broader application of this approach. We are investigating a novel strategy to enable "off-the-shelf"' therapy with mismatched donor CAR19 T cells. Transcription activator-like effector nucleases (TALEN)s can be used to overcome HLA barriers by eliminating the risk of graft-versus-host disease (GvHD) through disruption of T cell receptor expression, and by simultaneously targeting CD52, cells can be rendered insensitive to the lymphodepleting agent Alemtuzumab. Administration of Alemtuzumab can then be exploited to prevent host-mediated rejection of HLA mismatched CAR19 T cells. We manufactured a bank of such cells from volunteer donor T cells under GMP conditions on behalf of Cellectis S.A for final stage validation studies using a third generation self inactivating lentiviral vector encoding a 4g7 CAR19 (CD19 scFv- 41BB- CD3ζ) linked to RQR8, an abbreviated sort/suicide gene encoding both CD34 and CD20 epitopes. Cells were then electroporated with two pairs of TALEN mRNA for multiplex targeting of both the T cell receptor alpha constant chain locus, and the CD52 gene locus. Following ex-vivo expansion, cells still expressing TCR were depleted using CliniMacs alpha/beta TCR depletion, yielding a T cell product with <1% TCR expression, 85% of which expressed CAR19, and 64% becoming CD52 negative. This universal CAR19 (UCART19) cell bank has been characterized in detail, including sterility, molecular and cytometric analyses and human/murine functional studies ahead of submissions for regulatory approvals and Phase 1 testing in trials for relapsed B cell leukaemia. In the interim we received a request for therapy on a compassionate basis for an infant with refractory relapsed B-ALL, and with the agreement of Cellectis, we treated this first patient under UK special therapy regulations. An 11 month girl with high risk CD19+infant ALL (t(11;19) rearrangement) relapsed in bone marrow 3 months after a myeloablative 8/10 mismatched unrelated donor transplant. Leukaemic blasts expressed CD19 but were CD52negative. Her disease progressed despite treatment with Blinatumomab (70% blasts in marrow) and we were unable to generate donor-derived CAR19 T cells on an existing study. Following institutional ethics review, detailed counseling, and parental consent, the patient received cytoreduction with Vincristine, Dexamethasone and Asparaginase followed by lymphodepleting conditioning with Fludarabine 90mg/m2, Cyclophosphamide 1.5g/m2 and Alemtuzumab 1mg/kg. Immediately prior to infusion of UCART19 cells, the bone marrow showed persisting disease (0.5% FISH positive). She received a single dose (4.5x106/kg) of UCART19 T cells without any significant toxicity. To date there has been no significant perturbation of cytokine levels in peripheral blood, and no indication of cytokine release syndrome. Although profoundly lymphopenic, UCART19 T cells were detectable by qPCR in the circulation by day 14 and at increased levels in both blood (VCN 0.35) and marrow (VCN 0.22) on day 28. The patient exhibited signs of count recovery and the bone marrow, while hypoplastic, was in cytogenetic and molecular remission. Chimerism was 90% donor, and a clearly demarcated population (7%) of third party cells indicated persistence of UCART19. A residual persistence of 3% recipient cells in the marrow suggests that leukemic clearance was not mediated by transplant mediated alloreactivity. Within the short period of follow up available, our intervention comprising lymphodepletion and infusion of UCART19 T cells has induced molecular remission where all other treatments had failed. This first-in-man application of TALEN engineered cells provides early proof of concept evidence for a ready-made T cell strategy that will now be tested in early phase clinical trials. Disclosures Qasim: CATAPULT: Research Funding; CELLMEDICA: Research Funding; CALIMMUNE: Research Funding; MILTENYI: Research Funding; AUTOLUS: Consultancy, Equity Ownership, Research Funding; CELLECTIS: Research Funding. Off Label Use: UCART19 T Cells are an unlicensed investigational medicinal product and in this case were used under MHRA special licence arrangements. Stafford:CELLECTIS: Research Funding. Peggs:Cellectis: Research Funding; Autolus: Consultancy, Equity Ownership. Thrasher:CATAPULT: Patents & Royalties, Research Funding; MILTENYI: Research Funding; AUTOLUS: Consultancy, Equity Ownership, Research Funding. Pule:AUTOLUS: Employment, Equity Ownership, Research Funding; CELLECTIS: Research Funding; AMGEN: Honoraria; UCLB: Patents & Royalties.

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