CD19 CD19 Chimeric Antigen Receptor Research Articles

Mapping variant effects on anti-tumor hallmarks of primary human T cells with base-editing screens.

Single-nucleotide variants (SNVs) in key T cell genes can drive clinical pathologies and could be repurposed to improve cellular cancer immunotherapies. Here, we perform massively parallel base-editing screens to generate thousands of variants at gene loci annotated with known or potential clinical relevance. We discover a broad landscape of putative gain-of-function (GOF) and loss-of-function (LOF) mutations, including in PIK3CD and the gene encoding its regulatory subunit, PIK3R1, LCK, SOS1, AKT1 and RHOA. Base editing of PIK3CD and PIK3R1 variants in T cells with an engineered T cell receptor specific to a melanoma epitope or in different generations of CD19 chimeric antigen receptor (CAR) T cells demonstrates that discovered GOF variants, but not LOF or silent mutation controls, enhanced signaling, cytokine production and lysis of cognate melanoma and leukemia cell models, respectively. Additionally, we show that generations of CD19 CAR T cells engineered with PIK3CD GOF mutations demonstrate enhanced antigen-specific signaling, cytokine production and leukemia cell killing, including when benchmarked against other recent strategies.

CD19 CAR T-Cell Therapy in Autoimmune Disease — A Case Series with Follow-up

BackgroundTreatment for autoimmune diseases such as systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis often involves long-term immune suppression. Resetting aberrant autoimmunity in these diseases through deep depletion of B cells is a potential strategy for achieving sustained drug-free remission.MethodsWe evaluated 15 patients with severe SLE (8 patients), idiopathic inflammatory myositis (3 patients), or systemic sclerosis (4 patients) who received a single infusion of CD19 chimeric antigen receptor (CAR) T cells after preconditioning with fludarabine and cyclophosphamide. Efficacy up to 2 years after CAR T-cell infusion was assessed by means of Definition of Remission in SLE (DORIS) remission criteria, American College of Rheumatology–European League against Rheumatism (ACR–EULAR) major clinical response, and the score on the European Scleroderma Trials and Research Group (EUSTAR) activity index (with higher scores indicating greater disease activity), among others. Safety variables, including cytokine release syndrome and infections, were recorded.ResultsThe median follow-up was 15 months (range, 4 to 29). The mean (±SD) duration of B-cell aplasia was 112±47 days. All the patients with SLE had DORIS remission, all the patients with idiopathic inflammatory myositis had an ACR–EULAR major clinical response, and all the patients with systemic sclerosis had a decrease in the score on the EUSTAR activity index. Immunosuppressive therapy was completely stopped in all the patients. Grade 1 cytokine release syndrome occurred in 10 patients. One patient each had grade 2 cytokine release syndrome, grade 1 immune effector cell–associated neurotoxicity syndrome, and pneumonia that resulted in hospitalization.ConclusionsIn this case series, CD19 CAR T-cell transfer appeared to be feasible, safe, and efficacious in three different autoimmune diseases, providing rationale for further controlled clinical trials. (Funded by Deutsche Forschungsgemeinschaft and others.)

Combination of Ibrutinib with CD19 Chimeric Antigen Receptor (CAR) T Cells Suggests Greater Contribution to CAR T-Cell Efficacy Than Other Approved BTK Inhibitors in the Preclinical Setting

Background: CD19-directed CAR T-cell therapy has demonstrated remarkable clinical benefits for patients with B-cell malignancies, but impaired CAR T-cell fitness can result in lack of response or relapse. Rational combinations that enhance T-cell fitness may overcome primary and secondary resistance. Bruton tyrosine kinase inhibitors (BTKi), such as Ibrutinib (Ibr) have been shown to enhance CAR T-cell efficacy (Fraietta et al, Blood 2016;Liu et al, Cancer Sci 2020), but the precise mechanism remains unclear. In addition to BTK, Ibr potently inhibits IL-2-inducible tyrosine kinase (ITK), an important mediator of T-cell signaling. Ibr has been shown to enhance Th1 polarization through suppression of the Th2 differentiation pathway, thereby potentiating a cytotoxic effector response (Qin et al, J Immunother 2020). Consistent with this finding, the Th1 phenotype was more frequent in peripheral blood mononuclear cells of Ibr-exposed patients than in acalabrutinib (Acala)-exposed patients (Scarfò et al, Blood 2021). Therefore, we aimed to assess the potential impact of ITK inhibition (ITKi) on CD19 CAR T-cell function by comparing Ibr to other approved BTKi that display low or no ITKi activity (Estupiñán et al, Front Cell Dev Biol 2021). Methods: CD19-41bb CAR T cells (CAR19) were generated by lentiviral transduction of healthy donor T cells. BTKi additivity to CAR19 function was evaluated in a Luciferase (Luc)+ Ramos, BTKi resistant systemic tumor model in NSG mice co-treated with covalent BTKis Ibr, Acala, zanubrutinib (Zanu), and CAR T cells. Next, to evaluate the impact of ITKi on CAR19 fitness and cytotoxicity, repeated stimulation in an in vitro cytotoxicity assay was performed at low CAR19 effector-to-tumor ratio (1:20) to mimic higher tumor burden stress conditions in the presence of each BTKi, including non-covalent BTKi pirtobrutinib (Pirto) (500 nM). Tumor cells were replenished every 3 days and cytotoxicity was measured on day 7 by luciferase assay. For phenotypic characterization, CAR T cells were repeatedly stimulated by plate-bound anti-Whitlow linker every 3 days. Viability and proliferation were measured by cell counter. Flow cytometry (FACS) was performed on surface and intracellular T-cell phenotypic, activation, and exhaustion markers. Metabolic fitness (glycolysis and ATP production) was characterized using Seahorse (Van der Windt, 2012). Impact of Ibr on signal strength was evaluated in CD19-expressing Jurkat reporter cells (CAR-J19) with knock-in of mKate in frame with Nur77. Ibr was added to coculture of CAR-J19 and tumor cells and upregulation of Nur77 was measured by FACS. Downstream ITK signaling pathway was measured by Western blot. Results: Preliminary in vivo experiments demonstrated that co-administration of each BTKi with CD19 CAR T cells improved tumor control at day 26 (N=5 per group; P≤0.0004 vs CAR T cells alone). In serial killing experiments, addition of Ibr to the coculture increased proliferation (n=2; P<0.05), viability (n=2; P=0.01), and the cytotoxicity of CAR T cells against Ramos cells ( P=0.02 vs CAR19 alone) to a greater extent than other BTKis tested (Fig. 1A). Prolonged stimulation of CAR19 with Ibr led to downregulation of activation and inhibitory markers on CAR19 and maintenance of a naive T-cell phenotype (Fig. 1B) that was confirmed by increased metabolic fitness (n=5; P<0.05). Addition of Ibr to CAR-J19 resulted in a 72% reduction in CAR activation and reduced phosphorylation of ITK, PLCG, and Zap70. Additional data including the impact of ITK knockout on CAR-T cells will be presented. Conclusions: Our study expands on the hypothesized mechanism of action underlying the clinical benefit observed with Ibr in CD19 CAR T-cell-treated patients with chronic lymphocytic leukemia or mantle cell lymphoma (Liu et al, Front Immunol 2022; Wang et al, NEJM 2020). Our data suggest that Ibr enhances CAR T-cell fitness to a greater extent than both covalent and noncovalent BTKi that display reduced or no ITKi activity. Through ITKi, Ibr tempers CAR T-cell activation induced cell death, maintains a naive state and prevents premature exhaustion, thereby enabling more efficacious tumor cell killing over time. Together, these data support the further exploration of Ibr to enhance CD19 CAR T-cell efficacy.

Improved outcomes of large B-cell lymphoma patients treated with CD19 CAR T in the UK over time.

The success of CD19 Chimeric antigen receptor (CAR) T-cell therapy in large B-cell lymphoma (LBCL) has been partially offset by toxicity and logistical challenges, which off-the-shelf agents like CD20xCD3 bispecific antibodies might potentially overcome. However, when using CAR T outcomes as the 'standard-of-care comparator̕ for relapsed/refractory (r/r) LBCL, a potential learning curve with implementing a novel, complex therapy like CAR T needs to be considered. To address this, we analysed 726 UK patients intended to be treated with CD19 CAR T for r/r LBCL and compared outcomes between the first year of the national CAR T programme (Era 1; 2019) and the more recent treatment era (Era 2; 2020-2022). We identified significant improvements for Era 2 versus Era 1 in dropout rate (17% vs. 27%, p = 0.001), progression-free survival (1-year PFS 50% vs. 32%, p < 0.001) and overall survival (1-year OS 60% vs. 40%, p < 0.001). We also observed increased use of bridging therapy, improvement in bridging outcomes, more tocilizumab/corticosteroid use, reduced high-grade cytokine release syndrome (4% vs. 9%, p = 0.01) and intensive care unit admissions (20% vs. 32%, p = 0.001). Our results demonstrate significant improvement in CAR T outcomes over time, highlighting the importance of using up-to-date clinical data when comparing CAR T against new treatment options for r/r LBCL.

Low toxicity and excellent outcomes in patients with DLBCL without residual lymphoma at the time of CD19 CAR T-cell therapy.

CD19 chimeric antigen receptor (CAR) T-cell therapy represents a breakthrough for patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), inducing sustained remissions in these patients. However, CAR T cells can result in significant toxicities. Preinfusion disease burden is associated with toxicities and outcomes after CAR T-cell therapy. We identified 33 patients with R/R DLBCL treated at 8 academic centers who had no detectable disease at the time of CAR T-cell therapy. The median time from leukapheresis to CAR T-cell infusion was 48 (19-193) days. Nine patients received axicabtagene ciloleucel, and 24 received tisagenlecleucel. There was no severe (grade ≥3) cytokine release syndrome, and only 1 patient developed severe neurotoxicity (grade 4). After a median follow-up of 16 months, 13 patients relapsed (39.4%) and 6 died (18.1%). One-year event-free survival and overall survival were 59.6% and 81.3%, respectively. Our findings suggest that, in patients with R/R DLBCL who have an indication for CAR T-cell therapy, treating patients in complete remission at the time of infusion is feasible, safe, and associated with favorable disease control. Further exploration in a larger clinical trial setting is warranted.

Outcomes among adult recipients of CD19 CAR T-cell therapy for Burkitt lymphoma.

7571 Background: Burkitt lymphoma (BL) is a rare, but highly aggressive B-cell lymphoma associated with especially poor outcomes in patients with relapsed/refractory disease. Multiple CD19 chimeric antigen receptor (CAR) T-cell products have been FDA approved to treat relapsed/refractory B-cell non-Hodgkin lymphoma in the second-line setting, however little is known about CAR T-cell responses in patients with BL. This multicenter study aims to assess real-world outcomes among patients with BL treated with CAR T-cell therapy across the United States. Methods: This multicenter, retrospective chart review was conducted by abstracting data from the medical charts of patients with BL who received CAR T-cell therapy. Demographic characteristics were assessed along with pre-CAR treatments, bridging therapies, infusion-related complications, CAR-T responses, and the use of post-CAR systemic therapy and/or hematopoietic stem cell transplant. Results: A total of 13 patients with relapsed/refractory BL received CAR T-cell therapy after a median of 3 prior therapies (range 1-6). Most patients received axi-cel (n = 8), although tisa-cel (n = 3) and liso-cel (n = 2) were also represented. The median age of these patients was 38 (range 24-68). A total of 6 (46.2%) had evidence of central nervous system involvement at diagnosis and 5 (38.5%) had a prior history of autologous stem cell transplant. The overall response rate 1 month after infusion was 69.2% with a complete response rate of 53.8%. All patients experienced grade 1-2 cytokine release syndrome (CRS). No one experienced grade ≥3 CRS, but 2 patients developed grade ≥3 immune effector cell-associated neurotoxicity syndrome (ICANS). Twenty-eight-day mortality was 23%, including one patient who died from grade 4 ICANS. Regarding long term data, only 4 (30.8%) patients sustained a complete response for more than 6 months. Median progression-free survival was 4.2 months (95% CI 28.2-257.6), and the 1-year overall survival rate was 61.5% (95% CI 0.56-0.67). Two (15.4%) patients received consolidative allogeneic stem cell transplants after CAR T-cell therapy, but both ultimately developed progressive disease. Conclusions: Relapsed/refractory BL is a rare and aggressive disease that remains challenging to treat despite the advent of CAR T-cell therapy. Further investigation is needed to determine the most effective management of these patients. We expect to include additional patients in this analysis over the coming months.

Cytokines induced memory-like NK cells engineered to express CD19 CAR exhibit enhanced responses against B cell malignancies.

CD19 chimeric antigen receptor (CAR) engineered NK cells have been used for treating patients with relapsed and/or refractory B cell malignancies and show encouraging outcomes and safety profile. However, the poor persistence of NK cells remains a major challenge for CAR NK cell therapy. Memory-like NK cells (MLNK) induced by IL-12, IL-15, and IL-18 have shown enhanced and prolonged responses to tumor re-stimulation, making them an attractive candidate for adoptive cellular immunotherapy. Here, we show efficient and stable gene delivery of CD19 CAR to memory-like NK cells using retroviral vectors with transduction efficiency comparable to those achieved with conventional NK cells. Analysis of surface molecules revealed a distinct phenotypic profile in CAR engineered memory-like NK cells (CAR MLNK), as evidenced by increased expression of CD94 and downregulation of NKp30 as well as KIR2DL1. Compared to conventional CAR NK cells, CAR MLNK cells exhibited significantly increased IFN-γ production and degranulation in response to CD19+ target cells, resulting in enhanced cytotoxic activity against CD19+ leukemia cells and lymphoma cells. Furthermore, memory properties induced by IL-12/-15/-18 improved the in vivo persistence of CAR MLNK cells and significantly suppressed tumor growth in a exnograft mouse model of lymphoma, leading to prolonged survival of CD19+ tumor-bearing mouse. Altogether, our data indicate that CD19 CAR engineered memory-like NK cells exhibited superior persistence and antitumor activity against CD19+ tumors, which might be an attractive approach for treating patient with relapse or refractory B cell malignancies.

Activation priming and cytokine polyfunctionality modulate the enhanced functionality of low-affinity CD19 CAR T cells.

We recently described a low-affinity second-generation CD19 chimeric antigen receptor (CAR) CAT that showed enhanced expansion, cytotoxicity, and antitumor efficacy compared with the high-affinity (FMC63-based) CAR used in tisagenlecleucel, in preclinical models. Furthermore, CAT demonstrated an excellent toxicity profile, enhanced invivo expansion, and long-term persistence in a phase 1 clinical study. To understand the molecular mechanisms behind these properties of CAT CAR T cells, we performed a systematic invitro characterization of the transcriptomic (RNA sequencing) and protein (cytometry by time of flight) changes occurring in T cells expressing low-affinity vs high-affinity CD19 CARs following stimulation with CD19-expressing cells. Our results show that CAT CAR T cells exhibit enhanced activation to CD19 stimulation and a distinct transcriptomic and protein profile, with increased activation and cytokine polyfunctionality compared with FMC63 CAR T cells. We demonstrate that the enhanced functionality of low-affinity CAT CAR T cells is a consequence of an antigen-dependent priming induced by residual CD19-expressing B cells present in the manufacture.

Which one is better for refractory/relapsed acute B-cell lymphoblastic leukemia: Single-target (CD19) or dual-target (tandem or sequential CD19/CD22) CAR T-cell therapy?

CD19 chimeric antigen receptor (CAR) T-cell therapy has shown great success against B-cell acute lymphoblastic leukemia (B-ALL). Tandem and sequential CD19/CD22 dual-target CAR T-cell therapies have been developed to reduce the possibility of CD19-negative relapse; however, the superior strategy is still uncertain. This study screened 219 patients with relapsed/refractory B-ALL who were enrolled in clinical trials of either CD19 (NCT03919240) or CD19/CD22 CAR T-cell therapy (NCT03614858). The complete remission (CR) rates in the single CD19, tandem CD19/CD22, and sequential CD19/CD22 groups were 83.0% (122/147), 98.0% (50/51), and 95.2% (20/21), respectively (single CD19 vs. tandem CD19/CD22, P = 0.006). Patients with high-risk factors achieved a higher rate of CR in the tandem CD19/CD22 group than in the single CD19 group (100.0% vs. 82.4%, P = 0.017). Tandem CD19/CD22 CAR T-cell therapy was one of the significant favorable factors in the multivariate analysis of the CR rate. The incidence of adverse events was similar among the three groups. Multivariable analysis in CR patients showed that a low frequency of relapse, a low tumor burden, minimal residual disease-negative CR and bridging to transplantation were independently associated with better leukemia-free survival. Our findings suggested that tandem CD19/CD22 CAR T-cell therapy obtains a better response than CD19 CAR T-cell therapy and a similar response to sequential CD19/CD22 CAR T-cell therapy.

OP0279 CAR-T CELL TREATMENT OF REFRACTORY SYSTEMIC LUPUS ERYTHEMATOSUS- SAFETY AND PRELIMINARY EFFICACY DATA FROM THE FIRST FOUR PATIENTS

BackgroundWhile treatment of Systemic lupus erythematosus (SLE) has substantially improved, a subset of patients experiences severe progressive disease despite T- and B cell targeted therapy. Furthermore, drug-free remission and seroconversion is difficult to achieve in SLE to dateObjectivesTo study the safety, tolerability, and preliminary efficacy of deep B cell depletion using autologous CD19 chimeric antigen receptor (CAR) T cells in patients with severe and treatment-refractory SLEMethodsThe CAR product was manufactured by CliniMACS Prodigy system (Miltenyi Biotec, Bergisch Gladbach, Germany). T-cells were enriched from the patients’ peripheral blood apheresis product and 1x108 cells were used as starting cell population. The cells were transfected with a lentiviral vector encoding an anti-CD19 CAR is composed of the FMC63 scFv, a CD8- derived hinge region, TNFRSF19-derived transmembrane domain, CD3ζ intracellular domain, and 4-1BB co-stimulatory domain (Miltenyi Biotec) and expanded for 12 days. After conditioning with cyclophosphamide/ fludarabine patients received 1x106 CD19-CAR-T cells/kg body weight as a single infusion. All SLE treatments with the exception of low dose prednisolone were stopped before CAR-T cell administration. After CAR-T cell treatment, also prednisolone was stopped. Tolerability was assessed by monitoring for Cytokine-release syndrome (CRS), immune-related effector cell neurotoxicity syndrome (ICANS) and infections. Preliminary efficacy was assessed by reaching a Lupus Low Disease Activity State (LLDAS), seroconversion in dsDNA antibodies and ANA and cessation of all SLE-specific treatmentsResultsAs of January 22, 2022, our 4 SLE patients had been treated with CD19 CAR-T cells with a follow up of 10 months (patient 1, female aged 20, SLEDAI-2K: 16), 7 months (patient 2, male aged 22; SLEDAI-2K:8), 2 months (patient 3, female aged 22; SLEDAI 2K: 6), and 1 month (patient 4; female aged 24; SLEDAI-2K: 6), respectively. All patients had active severe SLE with failure of standard treatment including pulsed steroids, hydroxychloroquine, mycophenolate, cyclophosphamide, intravenous immunoglobulins, rituximab and belimumab before CD19 CAR-T cell administration. All patients had active kidney disease. No infections occurred. All four patients experienced fever without proof of infectious disease (CRS °I); only one patient was treated with a single dose of tocilizumab. No ICANS and no CRS of other organs occurred. In vivo, CAR-T cells rapidly expanded to a maximum of 27,6% (day 9, patient 1), 41,2% (day 9, patient 2), 11,5% (day 9, patient 3) and 59,1% (day 9, patient 4) of total circulating T cells followed by a typical decline, with circulating CAR-T cells being continuously detectable during the next months. Expansion of CAR T cells preceded the complete and sustained depletion of circulating B cells. Patient 1 experienced sustained drug-free remission (SLEDAI-2K=0) with complete loss of ANA and dsDNA antibodies despite reappearance of B cells at 6 months. Patient 2 also experienced complete loss of ANA and dsDNA antibodies with B cells not yet returned. Low-level proteinuria remained most likely due to previously accrued damage in glomerular filter function (SLEDAI-2K: 2). Patient 3 and patient 4 had a shorter observation period to date but also achieved clinical remission (both SLEDAI-2K 0). All patients met LLDAS and could successfully stop all SLE-specific medication, including glucocorticoids. No SLE flare occurred so far.ConclusionTaken together, these data show that CD19 CAR T-cell therapy is well tolerated and may induce rapid remission of severe refractory SLE.

Fludarabine exposure predicts outcome after CD19 CAR T-cell therapy in children and young adults with acute leukemia

AbstractThe addition of fludarabine to cyclophosphamide as a lymphodepleting regimen prior to CD19 chimeric antigen receptor (CAR) T-cell therapy significantly improved outcomes in patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). Fludarabine exposure, previously shown to be highly variable when dosing is based on body surface area (BSA), is a predictor for survival in allogeneic hematopoietic cell transplantation (allo-HCT). Hence, we hypothesized that an optimal exposure of fludarabine might be of clinical importance in CD19 CAR T-cell treatment. We examined the effect of cumulative fludarabine exposure during lymphodepletion, defined as concentration-time curve (AUC), on clinical outcome and lymphocyte kinetics. A retrospective analysis was conducted with data from 26 patients receiving tisagenlecleucel for r/r B-ALL. Exposure of fludarabine was shown to be a predictor for leukemia-free survival (LFS), B-cell aplasia, and CD19-positive relapse following CAR T-cell infusion. Minimal event probability was observed at a cumulative fludarabine AUCT0−∞ ≥14 mg*h/L, and underexposure was defined as an AUCT0−∞ <14 mg*h/L. In the underexposed group, the median LFS was 1.8 months, and the occurrence of CD19-positive relapse within 1 year was 100%, which was higher compared with the group with an AUCT0−∞ ≥14 mg*h/L (12.9 months; P < .001; and 27.4%; P = .0001, respectively). Furthermore, the duration of B-cell aplasia within 6 months was shorter in the underexposed group (77.3% vs 37.3%; P = .009). These results suggest that optimizing fludarabine exposure may have a relevant impact on LFS following CAR T-cell therapy, which needs to be validated in a prospective clinical trial.

Fludarabine Exposure Predicts Outcome after CD19 CAR T Cell Therapy in Children and Young Adults with Acute Leukemia; An Exploratory, Observational Study

The addition of fludarabine to cyclophosphamide as lymphodepleting regimen prior to adoptive transfer of CD19 chimeric antigen receptor (CAR) T cells significantly improves CAR T cell expansion and correlates with longer B cell aplasia and a decreased probability of developing a CD19+ relapse (Gardner, 2017). Dosing of fludarabine is currently based on body surface area. We previously showed that this leads to a highly variable plasma exposure that correlates with clinical outcome after allogeneic hematopoietic cell transplantation (Langenhorst, 2019). We therefore hypothesized that optimal exposure of fludarabine might be of clinical importance in the CD19 CAR T setting.An observational cohort analysis was conducted with data from 26 consecutive patients receiving tisagenlecleucel as treatment for refractory/relapsed B cell acute lymphoblastic leukemia (B-ALL; table 1). Prior to CAR T cell infusion, patients received fludarabine on 4 consecutive days at a daily dosage of 30 mg/m 2 and cyclophosphamide on 2 consecutive days at a daily dosage of 500 mg/m 2. Fludarabine concentrations were measured longitudinally after fludarabine infusion using a liquid chromatography mass spectrometry method. The total exposure (Area Under the Curve (AUC 0−∞)) was subsequently determined using a fludarabine population pharmacokinetic model (Langenhorst, 2019). The study was performed in accordance with the Declaration of Helsinki. The primary outcome parameter was leukemia free survival, defined as the time between CAR19 T cell infusion and the moment of measurable leukemic blasts (>5% or >0.01% by two subsequent measurements). The effect of fludarabine on leukemia free survival and the secondary outcome measures CD19+ relapse and B cell aplasia were explored using martingale residuals and further identified by fitting univariable Cox Proportional Hazards models. In addition, Kaplan Meier and cumulative incidence curves were plotted and compared with log-rank tests. To compare CAR T cell numbers over time in peripheral blood, the AUCs were computed and compared between exposure groups with the Mann-Whitney test. Analyses were performed using R4.03 with packages pknca, survival and survminer.The fludarabine AUC 0−∞ was highly variable, resulting in a large range of 8.7-21.8 mg*h/L. Exposure of fludarabine was shown to be a predictor for leukemia free survival, B cell aplasia, and CD19+ relapse following CAR T cell infusion. Minimal event probability was observed at a cumulative fludarabine exposure ≥14 mg*h/L and underexposure was therefore defined as an AUC 0−∞ <14 mg*h/L. In the underexposed group, leukemia free survival was lower (p<0.001; Figure 1A) and the occurrence of CD19+ relapse was higher (p<0.0001; figure 1B) compared to the group with an AUC 0−∞ ≥14 mg*h/L. Furthermore, the duration of B cell aplasia was shorter (p=0.009) and the AUCs of CAR T cell numbers lower (p=0.03) in the underexposed group. No significant differences in baseline characteristics were present between the two exposure groups.To our knowledge, this is the first study describing the effect of fludarabine exposure on outcome in a cohort of paediatric and young adults receiving CD19 CAR T cell therapy as treatment for B-ALL. These preliminary results suggest that optimizing fludarabine exposure may have a relevant impact on leukemia free survival following CAR T cell therapy. However, it should be noted that multivariate regression models are needed to show consistency of the relationship between fludarabine exposure and outcome. The limited number of patients did not allow for inclusion of potential covariates that may influence clinical outcome into the analysis. Therefore, our results need to be confirmed in a larger cohort. In conclusion, clinical outcome in patients receiving CAR19 T cell therapy might be improved by the optimization of fludarabine exposure in the lymphodepleting regimen.LD and FC contributed equally to this study. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Outcomes of Aggressive B Cell Lymphoma Patients with No Evidence of Measurable Disease at the Time of CD19 Chimeric Antigen Receptor T Cell Therapy: The Experience from the CAR T Cell Consortium

Outcomes of Aggressive B Cell Lymphoma Patients with No Evidence of Measurable Disease at the Time of CD19 Chimeric Antigen Receptor T Cell Therapy: The Experience from the CAR T Cell Consortium

Cytopenias After CD19 Chimeric Antigen Receptor T-Cells (CAR-T) Therapy for Diffuse Large B-Cell Lymphomas or Transformed Follicular Lymphoma: A Single Institution Experience.

IntroductionPatients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) have poor outcomes. Treatment with CD19 chimeric antigen receptor (CAR-T) cells, tisagenlecleucel and axicabtagene ciloleucel, has been associated with improved outcomes. Cytopenias were observed in clinical trials with both products; however, little is known regarding the patterns and outcomes of these cytopenias.Subjects and MethodsWe reviewed DLBCL patients (n=32) receiving either product between January and September 2018 at our institution.ResultsMedian duration of leukopenia, neutropenia, lymphopenia, anemia, and thrombocytopenia was 49, 9, 117.5, 125, and 95.5 days after CAR-T infusion, respectively. Filgrastim was used in 63% of patients, and 50% of patients received red cell or platelet transfusions. With the exception of neutropenia, increase in the duration of cytopenia of any lineage was associated with improvement in progression-free survival, and in overall survival in case of anemia. There was no association between the duration of cytopenias with either cytokine release syndrome or neurotoxicity.DiscussionOur data suggest a correlation between cytopenias and survival outcomes after CD19 CAR-T therapy. If validated, cytopenia may be proven useful as a biomarker of response and survival after CAR-T therapy.

Hematopoietic Cell Transplantation after CD19 Chimeric Antigen Receptor T Cell-Induced Acute Lymphoblastic Leukemia Remission Confers a Leukemia-Free Survival Advantage

Consolidative hematopoietic cell transplantation (HCT) after CD19 chimeric antigen receptor (CAR) T cell therapy is frequently performed for patients with refractory/ relapsed B cell acute lymphoblastic leukemia (B-ALL). However, there is controversy regarding the role of HCT following remission attainment. We evaluated the effect of consolidative HCT on leukemia-free survival (LFS) in pediatric and young adult subjects following CD19 CAR T cell induced remission. We evaluated the effect of consolidative HCT on LFS in pediatric and young adult subjects treated with a 41BB-CD19 CAR T cell product on a phase 1/2 trial, Pediatric and Young Adult Leukemia Adoptive Therapy (PLAT)-02 (ClinicalTrials.gov identifier NCT02028455), using a time-dependent Cox proportional hazards statistical model. Fifty of 64 subjects enrolled in PLAT-02 phase 1 and early phase 2 were evaluated, excluding 14 subjects who did not achieve remission, relapsed, or died before day 63 post-CAR T cell therapy. An improved LFS (P=.01) was observed in subjects who underwent consolidative HCT after CAR T cell therapy versus watchful waiting. Consolidative HCT improved LFS specifically in subjects who had no prior history of HCT, with a trend toward significance (P=.09). This benefit was not evident when restricted to the cohort of 34 subjects with a history of prior HCT (P=.45). However, for subjects who had CAR T cell functional persistence of 63 days or less, inclusive of those with a history of prior HCT, HCT significantly improved LFS outcomes (P=.01). These data support the use of consolidative HCT following CD19 CAR T cell-induced remission for patients with no prior history of HCT and those with short functional CAR T cell persistence.

Effects of glucocorticoids on the proliferation of CD19 CAR-T cells targeting B-cell tumor cell lines

目的评估糖皮质激素对CD19嵌合抗原受体T细胞（CAR-T细胞）靶向杀瘤过程中增殖的影响。方法健康志愿者的外周血单个核细胞（PBMC）作为T细胞来源，经CD3磁珠分选及CD19 CAR慢病毒转染制备CD19 CAR-T细胞；采用流式细胞术（FCM）测定CD19 CAR-T细胞转染率及其在培养体系中所占比例；采用CFDA SE细胞增殖示踪荧光探针试剂标记CD19 CAR-T细胞后测定其荧光强度；LDH细胞毒性检测方法检测不同浓度的糖皮质激素对B细胞肿瘤细胞株杀伤活性的影响。结果①CD19 CAR-T细胞CD19 CAR转染率为（51.34±5.28）％。②24 h后不同剂量甲泼尼龙对Nalm6、Pfeiffer及U2932细胞的杀伤活性高于地塞米松，48 h后低浓度（4 mg/ml）甲泼尼龙对Nalm6、Pfeiffer、U2932细胞杀伤活性高于低浓度（0.75 mg/ml）地塞米松，而高浓度（12 mg/ml）甲泼尼龙杀伤活性低于高浓度（2.25 mg/ml）地塞米松。但各个时间不同剂量甲泼尼龙组对EHEB细胞的杀伤活性均低于对应时间、剂量的地塞米松。③CD19 CAR-T细胞在不同浓度糖皮质激素作用下的比例及平均荧光强度比较，地塞米松对CD-19 CAR-T细胞的增殖抑制作用强于甲泼尼龙，两种糖皮质激素的高浓度组较低浓度组对CD19 CAR-T细胞的增殖抑制作用更明显。④CD19 CAR-T细胞与不同肿瘤细胞共培养体系中，其比例及平均荧光强度比较，地塞米松对CD19 CAR-T细胞的增殖抑制作用强于甲泼尼龙，两种糖皮质激素的高浓度组较低浓度组对CD19 CAR-T细胞的增殖抑制更明显。结论CD19 CAR-T细胞靶向不同肿瘤细胞株过程中地塞米松对CD19 CAR-T细胞的扩增和增殖抑制作用大于甲泼尼龙，高剂量组该抑制作用更加明显。

Activity comparison of humanized CD19 CAR-T cells with murine CD19 CAR-T on Nalm-6 cells and xenograft tumor model

Objective: To compare the activity difference of the high affinity humanized CD19 chimeric antigen receptor (CAR)-T cells and murine CD19 CAR-T cells. Methods: Peripheral venous blood T cells from 8 healthy volunteers were collected and infected with humanized and murine CD19 CAR lentivirus. Human and murine CD19 CAR-T cells were prepared and cell proliferation was detected by cell counting kit-8 (CCK-8) method. The cytotoxicity of CD3(+) T cells, humanized and murine CD19 CAR-T cells to NALM-6 cells was detected by lactate dehydrogenase assay. Thirty BAL B/c nude mice transplanted with NALM-6 cells were randomly divided into 3 groups with 10 mice in each group and injected humanized CD19 CAR-T cells, mouse CD19 CAR-T cells and control CD3(+) T cell via tail vein, respectively. The proportion of NALM-6 cells in peripheral blood and the proportion of CD19 CAR-T cells in T cells from the vein of the inner canthus were detected by flow cytometry. The overall survival of BAL B/c nude mice was observed. Results: The proliferation of mouse and humanized CD19 CAR-T cells were (68.50±0.93)% and (80.63±1.41)%, respectively (t=20.353, P<0.001) after cultured in vitro for 24 hours, and were (91.38±1.41)% and (148.13±1.25)%, respectively (t=85.364, P<0.001) after cultured for 48 hours. When the effect to target ratio was 1∶1, there was no difference between the humanized and murine CD19 CAR-T cell group after co-culture for 24 hours (P=0.169), while the killing activity of humanized CD19 CAR-T cells against NALM-6 cells was higher than that of murine CD19 CAR-T cells (P<0.01) after 48 hours of co-culture. When the effect to target ratio was 4∶1, the cytotoxicity of humanized CD19 CAR-T cells against NALM-6 cells was higher than that of murine CD19 CAR-T cells in co-culture for 24 and 48 hours (P<0.01). On the seventh day of CD19 CAR-T cell therapy, the proportion of NALM-6 cells in the peripheral blood of BAL B/c nude mice decreased to the lowest level in the humanized CD19 CAR-T cell group and the murine CD19 CAR-T cell group. After 21 days, the proportion of NALM-6 cells in the murine CD19 CAR-T cell group was higher than that in the humanized CD19 CAR-T cell group (P(21 d)=0.001, P(28 d)<0.001, P(35 d)<0.001). The proportion of humanized and murine CD19 CAR-T cells in the peripheral blood reached the peaks after 7 days of therapy, and the proportion of humanized CD19 CAR-T cells was higher than that of murine CAR-T cells (P(7 d)=0.002). The CD19 CAR-T cells disappeared in the peripheral blood in the murine CD19 CAR-T cell group after 14 days of therapy, while in the humanized CD19 CAR-T cell group it disappeared after 21 days of therapy. The median survival of BAL B/c nude mice in the murine CD19 CAR-T cell group and the humanized CD19 CAR-T cell group was 42 days and 63 days, respectively (χ(2)=15.382, P<0.001). Conclusions: High affinity humanized CD19 CAR-T cells have stronger proliferation, higher cytotoxicity and longer survival time compared with those of murine CD19 CAR-T cells. The results indicate that the clinical efficacy of humanized CD19 CAR-T cells would be better than that of murine CD19 CAR-T cells.

Automated Manufacture of CAR19 Donor Lymphocytes Incorporated into an Escalated Dose DLI Protocol: Preliminary Results from the CARD Study

Introduction:Clinical studies of 2nd generation CD19 Chimeric Antigen Receptor (CAR) T-cells have delivered unprecedented clinical responses in patients with B-ALL. Increasing demand for CAR T-cells places huge pressure on the small number of laboratories with expertise in GMP CAR T-cell manufacture. Automation of CAR T-cell production using the CliniMACS Prodigy (Miltenyi Biotec) has enormous potential to simplify manufacture and to enhance CAR T-cell accessibility to patients.We have optimized and successfully validated GMP-grade CD19CAR T-cell manufacture on the CliniMACS Prodigy and have delivered the first in man Prodigy-manufactured CD19CAR T-cells in the Phase I CARD study (NCT02893189). Recruitment to the CARD study is open to adult patients with relapsed CD19+ disease following allogeneic stem cell transplantation (allo-SCT). The study is designed to assess the feasibility of automated CD19CAR T-cell manufacture and to assess the engraftment and safety profile of allogeneic CD19CAR T-cells generated using the CliniMACS Prodigy.Methods:To validate automated CAR T-cell manufacture on the CliniMACS Prodigy, large-scale experiments using cryopreserved or fresh healthy donor leukapheresis were performed and assessed for T-cell expansion, transduction efficiency, viability, purity, immunophenotype and functionality. T-cells were activated with TransAct, a polymeric nanomatrix incorporating CD3/CD28-specific antibodies, transduced with a SIN lentiviral vector encoding a second generation CD19 CAR (4G7-41BBz CAR) and underwent expansion in a closed system with automated feeding and media exchange prior to cryopreservation. Small-scale controls with TransAct / TexMACS or Dynabeads / X-vivo were run in parallel.The CARD study is designed to mirror escalated dose DLI protocols, allowing up to 3 consecutive doses of CD19CAR T cells at intervals of no shorter than 8 weeks, titrated to clinical response. Cell doses are based on CD3+ T-cell numbers rather than on absolute CD19CAR T-cell numbers (dose 1 = 1x106/kg CD3+ T-cells; dose 2 = 3x106/kg CD3+ T-cells and dose 3 = 1x107/kg CD3+ T-cells). A minimum transduction efficiency of 10% is stipulated as a release criterion for all trial products.Results:Preclinical scale up studies of the automated CAR T-cell manufacturing process with healthy donor leucapheresate confirmed equivalence of CAR T-cell specification and yield when compared to products manufactured using established WAVE-Bioreactor-based GMP T-cell processes in our facility (Qasim et al, Cytotherapy, 2016). Based on this data, automated CliniMACS Prodigy-based CD19CAR T-cell manufacture was subsequently approved by the MHRA, the UK regulatory body, for use in the CARD clinical trial.To date, we have enrolled 3 patients on study. Transduction efficiency was on average 46.6% (range 40.7 - 55.3%), with successful manufacture of 3 CD19CAR T-cell doses per patient. All products met sterility release criteria. GMP capacity, staffing and quality assurance systems permit manufacture of one CliniMACS Prodigy product per month using a team of 2 GMP staff with the possibility for further upscaling. Two patients have been treated at the initial T cell dose, with evidence of engraftment and expansion despite the absence of preconditioning.Conclusions:We have optimized and successfully validated GMP-grade CD19CAR T-cell manufacture using the CliniMACS Prodigy and have delivered the first in man Prodigy-manufactured CD19CAR T-cell products to adult patients with CD19+ disease relapse following allo-SCT. Preliminary results of CAR T-cell manufacture and clinical outcomes in initial patients will be presented. DisclosuresNickolay:Miltenyi Biotec GmbH: Research Funding. Mock:Miltenyi Biotec GmbH: Research Funding. Himoudi:Autolus: Research Funding. Thrasher:Orchard Therapeutics: Consultancy; Torus Therapeutics, Inc: Other: Advisory Board; Rocket Pharmaceuticals Ltd: Consultancy; 4bio Ventures Management Ltd: Other: Advisory Board. Pule:UCL: Patents & Royalties: UCL Business; Autolus Ltd: Employment, Equity Ownership, Research Funding. Qasim:Miltenyi: Research Funding; Servier: Research Funding; NIHR: Research Funding; Autolus Ltd: Consultancy, Equity Ownership; Bellicum: Research Funding; Cellectis: Research Funding; Orchard Therapeutics: Consultancy, Equity Ownership.

The Synergistic T-Cell Signal By CD79A/CD40 Costimulatory Endodomain Enhances CD19 Chimeric Antigen Receptor T-Cell Proliferation and Survival

Introduction: Adoptive T-cell therapy of gene modified to express CD19 chimeric antigen receptor (CAR) unveiled promising clinical outcomes, particularly in B-cell hematologic malignancies. Among several studies, CAR T-cell persistence is considered to be a surrogate marker of long-term clinical efficacy of CAR T-cell therapy. Consequently, various CD19 CAR T-cell costimulatory domains have been developed to improve T-cell signal, ultimately enhanced T-cell function and persistence. However, in the context of T-cell activation signaling, most of costimulatory molecules have already investigated, and no novel signaling domains have been reported to be useful recently. Here, we adopted B-cell signal moiety, CD79A, and dendritic cell signal moiety, CD40, and generated a composite costimulatory domain CD79A/CD40 to synergize T-cell signaling in order to improve T cell proliferation and persistence.Methods: The CD79A/CD40 costimulatory domain was developed and inserted in the middle of anti-CD19-CD28TM-CD3z-tEGFR construct. Then the novel CD19.79a40z CAR gene was subsequently cloned into retroviral vector and transduced into human CD3+ cells. The CAR expression was assessed and enriched by anti-EGFR mAb, which resulted in achieving more than 90% purity. Then we expanded CD19 CAR T-cell by coculture with irradiated EBV-LCL. These expanded cells were further used for downstream experiments including 51Cr release assay, coculture assay, intracellular cytokine staining assay (IC-CS), T cell proliferation assay, and T cell phenotype, in order to compare with conventional CD19.28z CAR T-cells. In addition, CD19 CAR-transduced Jurkat-Dual luciferase reporter cells were also used to assess NF-κB pathway activity.Results: After one course of viral transduction and LCL expansion, we obtained similar number of CAR-positive cells between CD19.79a40z and CD19.28z. The CD19.79a40z exhibited similar cytotoxic activities to CD19.28z, against K562-CD19 and two primary B-ALL samples (51Cr release assay). Also with coculture assay, both CD19.79a40z and CD19.28z showed robust cytotoxicity against K562-CD19 at E:T ratio of 1:1 and 1:8 after coculture for 120 hours without IL-2 supplementation. Interestingly, after extended coculture time to 168 hours, a tendency of target cell regrowth in CD19.28z was observed, whereas such regrowth was not seen in CD19.79a40z.Regarding IC-CS assay against K562-CD19, the proportions of IFN-γ+ and IL-2+ responders were comparable between CD19.79a40z and CD19.28z. Similarly IC-CS assay, ELISA revealed that CD19.79a40z and CD19.28z evenly produced IFN-γ, IL-2, and TNF-α upon exposure to K562-CD19.Furthermore, we also assessed the ability of CD19 CAR T-cell proliferation by coculture with K562-CD19, without IL-2 supplementation. Surprisingly, the novel CD19.79a40z unveiled robust T-cell proliferation and persistence throughout 2 weeks of culture when compared to CD19.28z (Figure).We next determined the plausible factors owing to T-cell survival advantage. Firstly, we assessed the surface markers of T-cell subset as well as exhaustion phenotypes, after coculture with K562-CD19 for 10 days. Again, CD19.79a40z showed similar T-cell subset composition and exhaustion markers to CD19.28z. Secondly, we examined the NF-κB activity of CD19 CAR after exposure to target antigens that the novel CD19.79a40z exhibited significantly higher NF-κB activity upon exposure to K562-CD19 and Raji when compared to CD19.28z. [Luciferase activity (units) of CD19.28z vs. CD19.79a.40z: K562-CD19 11306.65±747.63 vs. 21291.93±924.06; Raji 5524.93±321.68 vs. 7537.96±336.16, respectively (p= 0.0001)].Conclusions: The novel CD19.79a40z was successfully transduced into CD3+ cells with exhibited comparable cytotoxicity, cytokine secretion activities, T-cell subset composition, as well as T-cell exhaustion phenotypes to conventional CD19.28z CAR T-cells. On the contrary, the composite signaling domain, CD79A/CD40, astonishingly enhanced CD19 CAR T-cell proliferation and survival comparing to CD19.28z CAR T-cells. In addition, our construct presented higher NF-κB activity upon antigen exposure, which possibly synergized to T-cell signal and led to a proliferation advantage. Collectively, the greater CAR T-cell expansion might improve CAR T-cell persistence in vivo and ultimately improve the efficacy of adoptive CAR T-cell therapy. [Display omitted] DisclosuresKiyoi:FUJIFILM Corporation: Patents & Royalties, Research Funding; MSD K.K.: Research Funding; Eisai Co., Ltd.: Research Funding; Phizer Japan Inc.: Honoraria, Research Funding; ONO Pharmaceutical Co., Ltd.: Research Funding; Celgene Corporation: Consultancy, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Meiji Seika Pharma Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; JCR Pharmaceuticals Co.,Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis Pharma K.K.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

CD19-Chimeric Antigen Receptor (CAR) Engineered Natural Killer (NK) Cell Therapy: Novel “Off the Shelf” Immunotherapy in CD20 Resistant B-Cell Non-Hodgkin Lymphoma (NHL) Cell Lines, Primary NHL Cells, and a Human Lymphoma Xenograft Model

Background: NK-92 is a continuously growing cell line consisting of “pure” (100%) activated NK cells. These cells were subsequently bioengineered to express human anti-CD19 CAR recognizing CD19 B cells (ie, CD19-CAR NK). The goal of this study was to establish the mechanistic rationale for NK-based therapy in B cell NHL (bNHL) and to determine the potency of this targeted “off the shelf” therapy in a host of in vitro bNHL cell lines (including anti-CD20 antibody resistant cells), primary NHL patient derived cells, and an in vivo NHL xenograft model.Methods: bNHL cell lines SUDHL10, SUDHL4, SUDHL2 (diffuse large B cell lymphoma, DLBCL), HF1 (follicular) and Raji (Burkitt) were used. Rituximab- (R) and obinutuzumab- (O) resistant bNHL cell lines (SUDHL2, SUDHL4, SUDHL10) were also established. Patient derived cell lines (EL-5 and KSC) were obtained from primary DLBCL patients via the Tufts Tissue Biorepository. CD19-CAR NK cellular therapy was supplied by NantKwest. In vitro NK cell mediated cytotoxicity was determined using G6PD release (aCella-tox assay), apoptosis by Annexin V & flow cytometry; 10,000 target bNHL cells were co-cultured with effector NK cells at clinically relevant effector to target ratios (E:T 1:1-10:1) for 4 hours. Further, the dynamic efficacy of CD19-CAR NK was determined utilzing droplet based microfluidics & live cell imaging. Global transcriptome analysis was used to establish the biological characteristics of R- & O- sensitive and -resistant bNHL cells & for expression of NK regulatory ligands. Key pathways and drivers were determined using established systems biology approaches (Beheshti A. Cancer Informatics 2015; Dashnamoorthy R. Cancer Res. 2016). Additional functional in vitro ELISA based assays (IFNγ & TNFα) were done. Finally, in vivo studies in SCID mice with a SUDHL10 xenograft model was studied with subcutaneous injections of CD19-CAR NK therapy.Results: We identified that untreated bNHL cell lines expressed high levels of NK activation ligands (CD70, CD86, ITGAL, MICB, etc) and expression of inhibitory ligands were minimal. Profiling analyses also showed that anti-CD20 resistant bNHL cell lines had unique biological properties suggestive of defective IFNγ & TNFα signaling components, while expression of NK regulatory ligands remained mostly intact in both sensitive & resistant bNHL cells. In microfluidics based analyses, we observed >90% death in SUDHL10 and SUDHL4 as well as in the corresponding anti-CD20 resistant cell lines. Global transcriptome analysis revealed that activation of IFNγ signaling was the key transcriptional response to CD19-CAR NK activity, confirmed by increased IFNγ secretion. Gene expression profiling showed activation of inflammatory responses via upregulated immunoregulatory interactions, IFNγ signaling and induction of apoptosis as conserved mechanisms in both parental and anti-CD20 resistant bNHL cells treated with CD19-CAR NK therapy (Fig 1A-B). Cytotoxicity studies showed significantly increased cytolytic activity at E:T ratios (1:1-10:1) via LDH release in all bNHL cell lines (Raji, SUDHL4 and SUDHL10) at 4 hours vs L428 (Hodgkin) or Jurkat (T cell lymphoma) cells (Fig 1C). Increased apoptosis 40-70% (E:T ratios 0.25:1-2:1) was also noted in Raji cells with CD19 CAR NK vs 20% cell death with E:T ratios (0.25:1-2:1) in parental NK-92. Furthermore, CD19-CAR NK co-culture E:T ratio (1:1) resulted in >75% death in O-resistant SUDHL4 (SUDHL4-OR) and 66% death in R-resistant HF1 (HF1-RR) cells demonstrating that anti-CD20 resistance did not appear to impact the effectiveness of cell kill with CD19-CAR NK therapy. Finally, results from studies in SCID mice using a SUDHL10 xenograft tumor model showed significant reduction in tumor volume with CD19-CAR NK therapy vs control cells (P <0.001) (Fig 1D).Conclusion: Altogether, we identified that most bNHL cell lines expressed high levels of NK activation ligands and that their biological properties are suggestive of IFNγ and TNFα signaling signaling suggesting a conserved mechanistic response to CD19-CAR NK cell therapy. Furthermore, CD19-CAR NK cell therapy induced prominent single-agent cytolytic activity against a spectrum of bNHL cells, including primary DLBCL cells, cells resistant to standard anti-CD20 antibody, and also a DLBCL in vivo xenograft model with cell death occurring via immunoregulatory pathways with prominent IFNγ signaling & induction of apoptosis. [Display omitted] DisclosuresBoissel:NantKwest, Inc.: Employment. Evens:Pharmacyclics: Consultancy; Merck: Consultancy; Affimed: Consultancy; Amgen: Consultancy; Seattle Genetics: Consultancy; Celgene: Consultancy; AbbVie: Consultancy; • Spectrum Pharmaceuticals: Consultancy; Kite Pharma: Consultancy; Millennium: Consultancy; Novartis: Consultancy.