Characterization of CAR T cell expansion and cytotoxic potential during Ex Vivo manufacturing using image-based cytometry

CAR T-cell toxicities resembling hemophagocytic lymphohistiocytosis (HLH) occur in a subset of patients with cytokine release syndrome (CRS). As a variant of conventional CRS, a comprehensive characterization of CAR T-cell associated HLH (carHLH) and investigations into associated risk factors are lacking. In the context of 59 patients infused with CD22 CAR T-cells where a substantial proportion developed carHLH, we comprehensively describe the manifestations and timing of carHLH as a CRS variant and explore factors associated with this clinical profile. Amongst 52 subjects with CRS, 21 (40.4%) developed carHLH. Clinical features of carHLH included hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, coagulopathy, hepatic transaminitis, hyperbilirubinemia, severe neutropenia, elevated lactate dehydrogenase and occasionally hemophagocytosis. Development of carHLH was associated with pre-infusion NK-cell lymphopenia and higher bone marrow T/NK-cell ratio, which was further amplified with CAR T-cell expansion. Following CRS, more robust CAR T-cell and CD8 T-cell expansion in concert with pronounced NK-cell lymphopenia amplified pre-infusion differences in those with carHLH without evidence for defects in NK-cell mediated cytotoxicity. CarHLH was further characterized by persistent elevation of HLH-associated inflammatory cytokines, which contrasted with declining levels in those without carHLH. In the setting of CAR T-cell mediated expansion, clinical manifestations and immunophenotypic profiling in those with carHLH overlap with features of secondary HLH, prompting consideration of an alternative framework for identification and management of this toxicity profile to optimize outcomes following CAR T-cell infusion.

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

Blood ◽

10.1182/blood.v128.22.58.58 ◽

2016 ◽

Vol 128 (22) ◽

pp. 58-58 ◽

Cited By ~ 11

Author(s):

Mark Blaine Geyer ◽

Jae H. Park ◽

Isabelle Riviere ◽

Brigitte Senechal ◽

Xiuyan Wang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Expansion ◽

Research Funding ◽

Ex Vivo ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Equity Ownership ◽

T Cell Expansion

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

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830 Targeting cellular senescence to increase CAR-T cell fitness

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0830 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A882-A882

Author(s):

Rashmi Bankoti ◽

Hazal Pektas Akbal ◽

Maddalena Adorno ◽

Benedetta Di Robilant

Keyword(s):

T Cells ◽

T Cell ◽

Cellular Senescence ◽

Cell Expansion ◽

Ex Vivo ◽

Cell Aging ◽

Mrna Levels ◽

Car T Cell ◽

Car T ◽

T Cell Expansion

BackgroundImmunosenescence refers to the age-associated decline of the adaptive immune system, which results in increased incidence and severity of infections, cancers, and autoimmunity. The elderly show reduced numbers of naïve T cells, skewed CD4:CD8 ratio, reduced proliferative and functional capabilities, and increased expression of senescence markers. These phenomena have strong repercussion in adoptive immunotherapy.Notably, the ex vivo manufacturing process of CAR-T cells per se induces senescence extremely quickly; 15 days of T cell expansion age cells 30 years, as measured by telomere length, T cells differentiation and CDKN2a mRNA levels.To circumvent this problem, we here propose the modulation of USP16, an epigenetic regulator of stem cells and senescence in multiple tissues. Downregulation of USP16 rejuvenates T cells, offering a powerful tool to dramatically improve the efficacy of CAR-T treatments.MethodsDuring ex vivo CAR-T cell manufacture, cells age very rapidly, strongly decreasing T cell fitness. Importantly, we observed that cellular senescence is an early event that precedes T cell exhaustion upon CD3/CD28 T cell stimulation, making it a very interesting pathway to target. In line with this hypothesis, we demonstrated that reducing cellular senescence increases CAR-T cell functions both in vitro and in vivo.ResultsWe identified an epigenetic regulator, USP16, whose mRNA levels increase during T cell expansion and correlate with the expression of the aging marker par excellence, CDKN2a. Genetic modulation of USP16 in CD19 and GD2 CAR-expressing T cells not only reduces senescence markers but also expands the naive (CD45RA+CD62L+) population and enhances cell self-renewal, without negative effects on T cell expansion. USP16 modulation also results in increased killing, polyfunctionality, and expansion upon in vitro stimulation with tumor cells. Notably, the delay of cellular senescence induces long-lasting cellular fitness (figure 1) as T cells are less exhausted upon multiple tumor challenges. In vivo, T cells rejuvenated by USP16 modulation, are 60% more efficient in controlling tumor growth in a mouse model of leukemia (NALM-6) and neuroblastoma (CHLA-255).Abstract 830 Figure 1Effect of USP16 modulation in T cell agingThe schematic shows the relation between cell functionality, exhaustion and cellular senescence in normal T cell aging (top) and when USP16 is inhibited (bottom). USP16 modulation reduces T cell aging, increasing cell functionality and delaying exhaustion and cellular senescence.ConclusionsWe demonstrated that modulation of USP16 prevents cellular senescence and increases self-renewal in T cells. This approach can significantly improve CAR-T therapy in multiple diseases, including leukemias and solid tumors. Development of small molecules against USP16 could offer a viable solution to improve T cell fitness during manufacturing.Ethics ApprovalThe study was approved by Institutional Animal Care and Use Committees (IACUC), approval number CR-0104.

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A human orthogonal IL-2 and IL-2Rβ system enhances CAR T cell expansion and antitumor activity in a murine model of leukemia

Science Translational Medicine ◽

10.1126/scitranslmed.abg6986 ◽

2021 ◽

Vol 13 (625) ◽

Author(s):

Qian Zhang ◽

Morgan E. Hresko ◽

Lora K. Picton ◽

Leon Su ◽

Michael J. Hollander ◽

...

Keyword(s):

T Cell ◽

Antitumor Activity ◽

Murine Model ◽

Cell Expansion ◽

Car T Cell ◽

Car T ◽

T Cell Expansion

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TCR engagement negatively affects CD8 but not CD4 CAR T cell expansion and leukemic clearance

Science Translational Medicine ◽

10.1126/scitranslmed.aag1209 ◽

2017 ◽

Vol 9 (417) ◽

pp. eaag1209 ◽

Cited By ~ 60

Author(s):

Yinmeng Yang ◽

M. Eric Kohler ◽

Christopher D. Chien ◽

Christopher T. Sauter ◽

Elad Jacoby ◽

...

Keyword(s):

T Cell ◽

Cell Expansion ◽

Car T Cell ◽

Car T ◽

T Cell Expansion

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T Cell Maturation Stage Prior to and During GMP Processing Informs on CAR T Cell Expansion in Patients

Frontiers in Immunology ◽

10.3389/fimmu.2016.00648 ◽

2016 ◽

Vol 7 ◽

Cited By ~ 10

Author(s):

Yarne Klaver ◽

Sabine C. L. van Steenbergen ◽

Stefan Sleijfer ◽

Reno Debets ◽

Cor H. J. Lamers

Keyword(s):

T Cell ◽

Cell Expansion ◽

Cell Maturation ◽

Maturation Stage ◽

Car T Cell ◽

Car T ◽

T Cell Expansion ◽

T Cell Maturation

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Clinical Predictors of T Cell Fitness for CAR T Cell Manufacturing and Efficacy in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-115319 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1886-1886 ◽

Cited By ~ 5

Author(s):

Ehren Dancy ◽

Alfred L. Garfall ◽

Adam D. Cohen ◽

Joseph A Fraietta ◽

Megan Davis ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cell Expansion ◽

Research Funding ◽

Advisory Committees ◽

Car T Cell ◽

Car T ◽

T Cell Expansion

Abstract Introduction: The optimal clinical setting and cell product characteristics for chimeric antigen receptor (CAR) T cell therapy in multiple myeloma (MM) are uncertain. In CLL patients treated with anti-CD19 CAR T cells (CART19), prevalence of an early memory (early-mem) T cell phenotype (CD27+ CD45RO- CD8+) at time of leukapheresis was predictive of clinical response independently of other patient- or disease-specific factors and was associated with enhanced capacity for in vitro T cell expansion and CD19-responsive activation (Fraietta et al. Nat Med 2018). T cell fitness is therefore a major determinant of response to CAR T cell therapy. In an accompanying abstract (Cohen et al.), we report that higher percentage of early-mem T cells and CD4/CD8 ratio within the leukapheresis product are associated with favorable clinical response to anti-BCMA CAR T cells (CART-BCMA) in relapsed/refractory MM. Here, we compare leukapheresis samples from MM patients obtained at completion of induction therapy (post-ind) with those obtained in relapsed/refractory (rel/ref) patients for frequency of early-mem T cells, CD4/CD8 ratio, and in vitro T cell expansion. Methods: Cryopreserved leukapheresis samples were analyzed for the percentage of early-mem T cells and CD4/CD8 ratio by flow cytometry and in vitro expansion kinetics during anti-CD3/anti-CD28 bead stimulation. Post-ind samples were obtained between 2007 and 2014 from previously reported MM trials in which ex-vivo-expanded autologous T cells were infused post-ASCT to facilitate immune reconstitution (NCT01245673, NCT01426828, NCT00046852); rel/ref samples were from MM patients treated in a phase-one study of CART-BCMA (NCT02546167). Results: The post-ind cohort includes 38 patients with median age 55y (range 41-68) and prior exposure to lenalidomide (22), bortezomib (21), dexamethasone (38), cyclophosphamide (8), vincristine (2), thalidomide (8), and doxorubicin (4); median time from first systemic therapy to leukapheresis was 152 days (range 53-1886) with a median of 1 prior line of therapy (range 1-4). The rel/ref cohort included 25 patients with median age 58y (range 44-75), median 7 prior lines of therapy (range 3-13), and previously exposed to lenalidomide (25), bortezomib (25), pomalidomide (23), carfilzomib/oprozomib (24), daratumumab (19), cyclophosphamide (25), autologous SCT (23), allogeneic SCT (1), and anti-PD1 (7). Median marrow plasma cell content at leukapheresis was lower in the post-ind cohort (12.5%, range 0-80, n=37) compared to the rel/ref cohort (65%, range 0-95%). Percentage of early-mem T cells was higher in the post-ind vs rel/ref cohort (median 43.9% vs 29.0%, p=0.001, left figure). Likewise, CD4/CD8 ratio was higher in the post-ind vs rel/ref cohort (median 2.6 vs 0.87, p<0.0001, mid figure). Magnitude of in vitro T cell expansion during manufacturing (measured as population doublings by day 9, or PDL9), which correlated with response to CART19 in CLL, was higher in post-ind vs rel/ref cohort (median PDL9 5.3 vs 4.5, p=0.0008, right figure). Pooling data from both cohorts, PDL9 correlated with both early-mem T cell percentage (Spearman's rho 0.38, multiplicity adjusted p=0.01) and CD4/CD8 ratio (Spearman's rho 0.42, multiplicity adjusted p=0.005). Within the post-ind cohort, there was no significant association between early-mem T cell percentage and time since MM diagnosis, duration of therapy, exposure to specific therapies (including cyclophosphamide, bortezomib, or lenalidomide), or bone marrow plasma cell content at time of apheresis. However, in the post-ind cohort, there was a trend of toward lower percentage early-mem phenotype (29% vs 49%, p=0.07) and lower CD4/CD8 ratio (median 1.4 vs 2.7, p=0.04) among patients who required >2 lines of therapy prior to apheresis (n=3) compared to the rest of the cohort (n=35). Conclusion: In MM patients, frequency of the early-mem T cell phenotype, a functionally validated biomarker of fitness for CAR T cell manufacturing, was significantly higher in leukapheresis products obtained after induction therapy compared to the relapsed/refractory setting, as was CD4/CD8 ratio and magnitude of in vitro T cell expansion. This result suggests that CAR T cells for MM would yield better clinical responses at early points in the disease course, at periods of relatively low disease burden and before exposure to multiple lines of therapy. Figure. Figure. Disclosures Garfall: Novartis: Research Funding; Kite Pharma: Consultancy; Amgen: Research Funding; Bioinvent: Research Funding. Cohen:GlaxoSmithKline: Consultancy, Research Funding; Kite Pharma: Consultancy; Oncopeptides: Consultancy; Celgene: Consultancy; Novartis: Research Funding; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Janssen: Consultancy; Seattle Genetics: Consultancy. Fraietta:Novartis: Patents & Royalties: WO/2015/157252, WO/2016/164580, WO/2017/049166. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Levine:CRC Oncology: Consultancy; Brammer Bio: Consultancy; Cure Genetics: Consultancy; Incysus: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Siegel:Novartis: Research Funding. Stadtmauer:Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; AbbVie, Inc: Research Funding. Vogl:Karyopharm Therapeutics: Consultancy. Milone:Novartis: Patents & Royalties. June:Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding; Incyte: Research Funding; Tmunity: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy; CASI Pharmaceuticals: Consultancy.

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The effect of pembrolizumab in combination with CD19-targeted chimeric antigen receptor (CAR) T cells in relapsed acute lymphoblastic leukemia (ALL).

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.103 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 103-103 ◽

Cited By ~ 48

Author(s):

Shannon L. Maude ◽

George E Hucks ◽

Alix Eden Seif ◽

Mala Kiran Talekar ◽

David T. Teachey ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Expansion ◽

Lymphoblastic Leukemia ◽

Prior History ◽

Cell Detection ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

T Cell Expansion

103 Background: CD19-targeted CAR T cells show CR rates of 70-95% in B-ALL. Yet a subset of patients do not respond or relapse due to poor CAR T cell expansion and persistence. We hypothesized that PD-1 checkpoint pathway inhibition may improve CAR T cell expansion, function and persistence. Methods: Four children with relapsed B-ALL treated with murine (CTL019) or humanized (CTL119) anti-CD19 CAR T cells received 1-3 doses of the PD-1 inhibitor pembrolizumab (PEM) for partial/no response or prior history of poor CAR T cell persistence starting 14d-2mo post CAR T cell infusion. Results: PEM increased and/or prolonged detection of circulating CAR T cells in all 4 children, with objective responses in 2/4. It was well tolerated, with fever in 2 pts and no autoimmune toxicity. Pts 1-3 received CTL119 for CD19+ relapse after prior murine CD19 CAR T cells. Pt 1 had 1.2% CD19+ residual disease despite expansion with detectable CTL119 by D28 and received PEM at 2mo for progressive disease with decreasing circulating CTL119. CTL119 became detectable at 0.2% of CD3+ cells by flow cytometry, but disease progressed. Pt 2 had no response after initial CTL119 expansion with a rapid disappearance by D28. After CTL119 reinfusion with PEM added 14d later, circulating CAR T cells remained detectable at 4.4% by D28, but disease progressed with decreased CD19 expression. In Pt 3, prior treatment with both CTL019 and CTL119 produced CR with poor CAR T cell persistence followed by CD19+ relapse. CTL119 reinfusion combined with PEM at D14 resulted in CR with prolonged CTL119 persistence (detectable at D50 compared to loss by D36 after 1st CTL119 infusion). Pt 4 received PEM for widespread extramedullary (EM) involvement at D28 post CTL019 infusion despite marrow remission. Initial CTL019 expansion peaked at 63% at D10 and fell to 20% at D28. Resurgence of CTL019 expansion, with a 2nd peak of 70% 11d after PEM, was associated with dramatic reduction in PET-avid disease by 3mo post CTL019. Conclusions: PEM was safely combined with CAR T cells and increased or prolonged CAR T cell detection, with objective responses seen. Immune checkpoint pathways may impact response to CAR T cell treatments and warrant further investigation. Clinical trial information: NCT02374333, NCT02906371.

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Low IL-2 concentration favors generation of early memory T cells over terminal effectors during CAR T-cell expansion

Cytotherapy ◽

10.1016/j.jcyt.2017.02.008 ◽

2017 ◽

Vol 19 (5) ◽

pp. S8

Author(s):

A. Luostarinen ◽

T. Kaartinen ◽

P. Maliniemi ◽

J. Keto ◽

M. Arvas ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Expansion ◽

Memory T Cells ◽

Car T Cell ◽

Car T ◽

Early Memory ◽

T Cell Expansion

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Addition of Fludarabine to Cyclophosphamide Lymphodepletion Improves In Vivo Expansion of CD19 Chimeric Antigen Receptor-Modified T Cells and Clinical Outcome in Adults with B Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.3773.3773 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3773-3773 ◽

Cited By ~ 25

Author(s):

Cameron J Turtle ◽

Laila-Aicha Hanafi ◽

Carolina Berger ◽

Daniel Sommermeyer ◽

Barbara Pender ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Expansion ◽

Research Funding ◽

Car T Cells ◽

Cell Dose ◽

Car T Cell ◽

Car T ◽

Ifn Γ ◽

T Cell Expansion

Abstract BACKGROUND: Chemotherapy followed by autologous T cells that are genetically modified to express a CD19-specific chimeric antigen receptor (CAR) has shown promise as a novel therapy for patients with relapsed or refractory B cell acute lymphoblastic leukemia (B-ALL); however, the risk of severe cytokine release syndrome (sCRS) and neurotoxicity has tempered enthusiasm for widespread application of this approach. The functional heterogeneity that is inherent in CAR-T cell products that are manufactured from undefined T cell subsets has hindered definition of dose-response relationships and identification of factors that may impact efficacy and toxicity. METHODS: We are conducting the first clinical trial that administers CD19 CAR-T cells manufactured from a defined composition of T cell subsets to adults with relapsed or refractory B-ALL. CD8+ and CD4+ T cells were enriched from each patient, transduced with a CD19 CAR lentivirus and separately expanded in vitro before formulation for infusion in a 1:1 ratio of CD8+:CD4+ CAR+ T cells at 2x105, 2x106 or 2x107 CAR-T cells/kg. Prior to CAR-T cell infusion, patients underwent lymphodepletion with a high-dose cyclophosphamide (Cy)-based regimen with or without fludarabine (Flu). RESULTS: Twenty-nine adults with B-ALL (median age 40, range 22 - 73 years; median 17% marrow blasts, range 0 - 97%), including 10 patients who had relapsed after allogeneic transplantation, received at least one CAR-T cell infusion. Twenty-four of 26 restaged patients (92%) achieved bone marrow (BM) complete remission (CR) by flow cytometry. CD4+ and CD8+ CAR-T cells expanded in vivo after infusion and their number in blood correlated with the infused CAR-T cell dose. Thirteen patients received lymphodepletion with Cy-based regimens without Flu. Ten of 12 restaged patients (83%) achieved BM CR by flow cytometry; however, 7 of these (70%) relapsed a median of 66 days after CAR-T cell infusion. Disease relapse correlated with a loss of CAR-T cell persistence in blood. We observed a CD8 cytotoxic T cell response to the murine scFv component of the CAR transgene that contributed to CAR-T cell rejection, and resulted in lack of CAR-T cell expansion after a second CAR-T cell infusion in 5 patients treated for persistent or relapsed disease. To minimize immune-mediated CAR-T cell rejection 14 patients were treated with Cy followed by Flu lymphodepletion (Cy/Flu, Cy 60 mg/kg x 1 and Flu 25 mg/m2 x 3-5) before CAR-T cell infusion. All patients (100%) who received Cy/Flu lymphodepletion achieved BM CR after CAR-T cell infusion. CAR-T cell expansion and persistence in blood was higher in Cy/Flu-lymphodepleted patients compared to their counterparts who received Cy alone (Day 28 after 2x106 CAR-T cells/kg: CD8+ CAR-T cells, mean 55.8/μL vs 0.10/μL, p<0.01; CD4+ CAR-T cells, 2.1/μL vs 0.02/μL, p<0.01), enabling reduction in CAR-T cell dose for Cy/Flu-treated patients. Patients who received Cy/Flu lymphodepletion appear to have longer disease-free survival (DFS) than those who received Cy alone (Cy/Flu, median, not reached; Cy alone, 150 days, p=0.09). CAR-T cell infusion was associated with sCRS, characterized by fever and hypotension requiring intensive care in 7 of 27 patients (26%) and neurotoxicity (≥ grade 3 CTCAE v4.03) in 13 of 27 patients (48%). Two patients died following complications of sCRS. Patients with sCRS or neurotoxicity had higher peak serum levels of IL-6, IFN-γ, ferritin and C-reactive protein compared to those without serious toxicity. Importantly IL-6, IFN-γ and TNF-α levels in serum collected on day 1 after CAR-T cell infusion from those who subsequently developed neurotoxicity were higher than those collected from their counterparts who did not develop neurotoxicity (IL-6, p<0.01; IFN-γ, p=0.05; TNF-α, p=0.04), providing potential biomarkers to test early intervention strategies to prevent neurotoxicity. The risks of sCRS and neurotoxicity correlated with higher leukemic marrow infiltration and increasing CAR-T cell dose. We have now adopted a risk-stratified approach to CAR-T cell dosing in which the CAR-T cell dose inversely correlates to the patient's bone marrow tumor burden. CONCLUSION: Risk-stratified dosing of CD19 CAR-T cells of defined subset composition is feasible and safe in a majority of patients with refractory B-ALL, and results in a CR rate of 92%. Addition of Flu to Cy-based lymphodepletion improves CAR-T cell expansion, persistence and DFS. Disclosures Turtle: Juno Therapeutics: Patents & Royalties, Research Funding. Berger:Juno Therapeutics: Patents & Royalties. Jensen:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Riddell:Adaptive Biotechnologies: Consultancy; Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Membership on an entity's Board of Directors or advisory committees. Maloney:Seattle Genetics: Honoraria; Janssen Scientific Affairs: Honoraria; Roche/Genentech: Honoraria; Juno Therapeutics: Research Funding.

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