CD19 Chimeric Antigen Receptor T-cell Research Articles

CD19 chimeric antigen receptor (CAR) engineered epstein-barr virus (EBV) specific T cells – an off-the-shelf, allogeneic CAR T-cell immunotherapy platform

Background & Aim Chimeric antigen receptor (CAR) T cells have demonstrated potent antitumor efficacy in treating a range of B-cell malignancies, yet technical and commercial challenges using an autologous approach hinder widespread development. Virus-specific T cells present a potential strategy towards an off-the-shelf, allogeneic platform due to the volume of supporting clinical data and additional advantages associated with native TCR engagement to latent virus antigens. Tabelecleucel (tab-cel®) is an investigational off-the-shelf, allogeneic T-cell immunotherapy utilizing native TCR specificity against Epstein-Barr virus (EBV) antigens present in lymphomas and other cancers. With low incidence of GvHD and no cytokine release syndrome, tab-cel® has been shown to be efficacious and well tolerated. Transfer of CAR transgenes into EBV-specific T cells represents an attractive approach to generate off-the-shelf, allogeneic CAR T therapies. Methods, Results & Conclusion We have generated EBV-specific T cells engineered with second generation CD19 CARs as tool compositions to evaluate the potential of this off-the-shelf CAR T approach. Allo-EBV.CD19.CAR T cells express high levels of CD19 CAR and maintain native EBV TCR expression. Allo-EBV.CD19 CAR T cells are predominantly central memory and show CD25, 4-1BB, and IFN-γ activation markers. Functional studies also demonstrate potent antitumor activity against CD19-positive NALM/6 and Raji tumor targets, but no activity against CD19-negative K562 cells. Allo-EBV.CD19.CAR T cells exhibit rapid and comparable killing kinetics compared to CD19.CAR T cells. Allo-CAR T cells also demosntarted antigen-specific proliferation against EBV and CD19-positive targets. Like unmodified EBV-specific T cells, allo.EBV.CD19.CAR T cells maintain the ability to kill B-lymphoblastoid cell lines (BLCL) and little reactivity or inflammatory cytokine secretion in response to HLA mismatched and antigen-negative targets. CD19 CAR engineered EBV T cells demonstrate efficient and potent killing of antigen-positive target cells, while exhibiting little alloreactivity. This platform provides a framework from which we are developing ATA3219, a next generation off-the-shelf, CD19-targeted CAR T utilizing a novel optimized stimulatory domain.

The response to lymphodepletion impacts PFS in patients with aggressive non-Hodgkin lymphoma treated with CD19 CAR T cells

AbstractFactors associated with durable remission after CD19 chimeric antigen receptor (CAR)-modified T-cell immunotherapy for aggressive B-cell non-Hodgkin lymphoma (NHL) have not been identified. We report multivariable analyses of factors affecting response and progression-free survival (PFS) in patients with aggressive NHL treated with cyclophosphamide and fludarabine lymphodepletion followed by 2 × 106 CD19-directed CAR T cells/kg. The best overall response rate was 51%, with 40% of patients achieving complete remission. The median PFS of patients with aggressive NHL who achieved complete remission was 20.0 months (median follow-up, 26.9 months). Multivariable analysis of clinical and treatment characteristics, serum biomarkers, and CAR T-cell manufacturing and pharmacokinetic data showed that a lower pre-lymphodepletion serum lactate dehydrogenase (LDH) level and a favorable cytokine profile, defined as serum day 0 monocyte chemoattractant protein-1 (MCP-1) and peak interleukin-7 (IL-7) concentrations above the median, were associated with better PFS. MCP-1 and IL-7 concentrations increased after lymphodepletion, and higher intensity of cyclophosphamide and fludarabine lymphodepletion was associated with higher probability of a favorable cytokine profile. PFS was superior in patients who received high-intensity lymphodepletion and achieved a favorable cytokine profile compared with those who received the same intensity of lymphodepletion without achieving a favorable cytokine profile. Even in high-risk patients with pre-lymphodepletion serum LDH levels above normal, a favorable cytokine profile after lymphodepletion was associated with a low risk of a PFS event. Strategies to augment the cytokine response to lymphodepletion could be tested in future studies of CD19 CAR T-cell immunotherapy for aggressive B-cell NHL. This trial was registered at www.clinicaltrials.gov as #NCT01865617.

Efficacy and safety of CD19 chimeric antigen receptor T cells for the treatment of 22 patients with B-cell lymphoma

目的探讨CD19 CAR-T治疗B细胞淋巴瘤的疗效及安全性。方法评估2017年2月1日至2018年7月1日CD19 CAR-T治疗22例B细胞淋巴瘤患者的疗效及不良反应情况。结果22例患者输注CD19 CAR-T后，总体完全缓解（CR）率为45.5%，部分缓解（PR）率为31.8%，总有效率为77.3%。其中12例复发难治患者9例有效，2例达CR，7例PR；10例微小残留病（MRD）阳性患者，8例MRD转阴。全部患者外周血中均检测到CD19 CAR-T细胞在体内增殖，复发难治患者与MRD阳性患者T细胞增殖的达峰时间分别为治疗后第4.5（1~12）天和治疗后第12（5~19）天，外周血CAR-T细胞分别占总的T淋巴细胞的4.02%（2.23%~28.60%）和10.10%（3.55%~24.74%）。MRD转阴患者持续缓解，中位随访8（3~18）个月均未复发，且此组患者有3例联合PD-1抗体治疗，均达CR。复发难治患者中，7例CAR-T治疗后达PR患者疗效保持时间为1.5~6.0个月，PD-1表达率为25.7%~55.3%，5例CAR-T治疗无效患者PD-1均高表达；共有3例患者联合应用PD-1抗体，其中2例有效；2例CAR-T治疗后达CR患者中1例行异基因造血干细胞移植，另1例随访12个月仍持续缓解。22例患者输注CAR-T细胞后14例发生不同程度的细胞因子释放综合征（CRS），其中9例为1级CRS，4例为2级CRS，其中1例复发难治患者发生3级CRS，经糖皮质激素、IL-6抗体治疗后CRS得到控制。治疗有效的17例患者中14例发生CRS，治疗无效的5例患者均未发生CRS。难治复发患者发生CRS的严重程度高于MRD阳性患者。结论CD19 CAR-T在CD19+ B细胞淋巴瘤中取得了疗效。CAR-T联合免疫检查点抑制剂的应用能够更好地提高疗效，CAR-T细胞治疗可作为复发难治患者的挽救治疗，清除B细胞淋巴瘤的MRD效果更好且不良反应小。

Chimeric Antigen Receptor T-Cell Therapy for Diffuse Large B-Cell Lymphoma

CD19 chimeric antigen receptor T-cell (CAR-T) therapy, a genetically engineered cell therapy, showed unprecedented efficacy in the treatment of relapsed or refractory diffuse large B-cell lymphoma. Two agents, axicabtagene ciloleucel and tisagenlecleucel, were approved by the Food and Drug Administration in 2017. However, CAR-T therapy is a treatment with complex logistics and high costs, as well as inherent adverse events, including cytokine-release syndrome and neurotoxicity. In addition, predictive biomarkers for efficacy and toxicity are lacking. Industry-academy cooperation is urgently required to develop CAR-T therapy that is effective, safe, and affordable for patients in Korea. Keywords: B-cell non-Hodgkin lymphoma; Lymphoma, Large B-cell, Diffuse; Chimeric antigen receptor T-cell; CAR-T and immunotherapy 중심 단어: 비호지킨 B세포 ë¦¼í”„ì¢ ; 광범위큰Bì„¸í¬ë¦¼í”„ì¢ ; 키메릭항원수용체T세포; 면역치료

CAR T‐cells' Interaction with Artificial Antigen Presenting Cell Surface

Adoptive Cell Transfer (ACT) using Chimeric Antigen Receptor (CAR) T-cells shows promising results in treating metastatic melanoma and B cell malignancies. However, the detailed mechanism of CAR T-cell's interaction with tumors is still not well-understood. In this work, we created an artificial antigen presenting cell (APC) surface for 2nd generation (CD3 and CD28 signaling domains) CD19 and GD2 CAR T-cells and studied the interaction of CAR T-cells in terms of synapse formation. We created circular patterns of antibodies against CAR T-cells using micro-contact printing and measured the level of activation and degranulation in both CAR T-cells by quantifying the intensity of the phosphorylated CD3ζ chain (p-CD3ζ) and the Lysosome-Associated Membrane Protein 1 (LAMP-1) respectively. The results show that we can study the interaction of CAR T cell with APC surface in a reproducible manner and that different CAR T cells have a different level of activation and degranulation. Our findings suggest that the artificial APC allows for quantitative measurement of effectiveness for different CAR T cells with controlled APC density. Support or Funding Information This work was supported by the National Science Foundation through the Early-concept Grants for Exploratory Research (EAGER) (Award number: 1649243) and was performed in part at the Joint School of Nanoscience and Nanoengineering, a member of the Southeastern Nanotechnology Infrastructure Corridor (SENIC) and National Nanotechnology Coordinated Infrastructure (NNCI), which is supported by the National Science Foundation (Grant ECCS-1542174). The figure shows the interaction of GD2 CAR T-cell with 5 μm micropatterns of 1A7 (target). The aggregation of the phosphorylated CD3 zeta chain (p-CD3ζ) at the contact with the micropattern is clear. Micropatterns of 1A7 are labeled with Alexa Fluor 568 Goat anti-mouse secondary antibody and p-CD3ζ is labeled with Alexa Fluor 488 primary antibody. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

YIA19-003: Size Matters: Larger Size CD19-Positive Extracellular Vesicles in Chronic Lymphocytic Leukemia (CLL) Inhibit Chimeric Antigen Receptor T-Cell Function

Background: Unprecedented clinical outcomes were reported after CD19 chimeric antigen receptor T-cell (CART19) therapy. However, the complete response rate in chronic lymphocytic leukemia (CLL) is much lower, at approximately 20%–30%. Several immune defects have been identified in CLL that result from the complex interaction between CLL cells and the microenvironment. This leukemic microenvironment is rich with extracellular vesicles (EVs) secreted by B-CLL cells. Here, we aimed to investigate the role of EVs play in the diminished CART response seen in some CLL patients. Methods: EVs were isolated from peripheral blood of 16 patients with untreated CLL at different Rai stages and risk profile by FISH. Cytometry was used to determine size, number of particles per μl, and CD19 expression. To investigate the impact of EVs on CAR T-cell functions, CART19 cells were stimulated with either CLL B cells or the CD19-positive cell line JEKO and different effector functions were analyzed. Results: Two patterns of EVs in CLL patients were identified; a single versus 2 distinct EV populations, characterized by size (small [EVssmall] and large [EVslarge], Fig 1A). In 25% of patients, EVs were CD19 positive (EVCD19+). CD19 positivity was detected only in patients with the EVslarge (Fig 1B). The EVs concentration, CD19 expression (EVsCD19+ vs EVsCD19-), or the size (EVssmall vs EVslarge) did not correlate with disease stage (early vs advanced Rai stage) or risk profile of CLL (low vs high risk). To investigate our hypothesis that EVs could modulate CART19 function, CART19 cell effector functions were examined in the presence of EVsCD19+, EVsCD19-, EVssmall, or EVslarge. EVs alone were insufficient to stimulate CART19 cells. However when CART19 cells were stimulated with the CD19-positive cell line JEKO, their effector functions were reduced only in the presence of EVsCD19+ but not EVsCD19-. This included a significant reduction in CART-specific killing (Fig 1C) and a reduction in cytokine production. The impairment of CART cell functions was independent of the size of EVs, ie, there was no impairment of CART functions with large or small size EVCD19- in co-culture. Conclusion: We identify CD19-positive large size EVs from patients with CLL and demonstrate that these EVs play a role in the leukemic microenvironment by reducing CAR T-cell activity. Studies are ongoing to define the mechanism(s).

Risks and Benefits of Chimeric Antigen Receptor T-Cell (CAR-T) Therapy in Cancer: A Systematic Review and Meta-Analysis.

Promising efficacy results of chimeric antigen receptor (CAR) T-cell therapy have been tempered by safety considerations. Our objective was to comprehensively summarize the efficacy and safety of CAR-T cell therapy in patients with relapsed or refractory hematologic or solid malignancies. MEDLINE, Embase, and the Cochrane Register of Controlled Trials (inception - November 21, 2017). Interventional studies investigating CAR-T cell therapy in patients with malignancies were included. Our primary outcome of interest was complete response (defined as the absence of detectable cancer). Two independent reviewers extracted relevant data, assessed risk of bias, and graded the quality of evidence using established methods. A total of 42 hematological malignancy studies and 18 solid tumor studies met were included (913 participants). Of 486 evaluable hematologic patients, 54.4% [95% CI, 42.5%-65.9%] experienced complete response in 27 CD19 CAR-T cell therapy studies. Of 65 evaluable hematologic patients, 24.4% [95% CI, 9.4%-50.3%] experienced complete response in seven non-CD19 CAR-T cell therapy studies. Cytokine release syndrome was experienced by 55.3% [95% CI, 40.3%-69.4%] of patients and neurotoxicity 37.2% [95% CI, 28.6%-46.8%] of patients with hematologic malignancies. Of 86 evaluable solid tumor patients, 4.1% [95% CI, 1.6%-10.6%] experienced complete response in eight CAR-T cell therapy studies. Limitations include heterogeneity of study populations, as well as high risk of bias of included studies. There was a strong signal for efficacy of CAR-T cell therapy in patients with CD19+ hematologic malignancies and no overall signal in solid tumor trials published to date. These results will help inform patients, physicians, and other stakeholders of the benefits and risks associated with CAR-T cell therapy.

Abstract A035: Combinatorial IGK-CD19 CAR primarily targets IgK+ malignant B-cells and is less prone to serum IgG inhibition

Abstract The first chimeric antigen receptor (CAR) T-cell therapies have been approved for treatment of B-cell malignancies. This is mainly due to the success of CAR T-cells targeting B-lymphocyte antigen CD19, which has led to astonishing results in clinical trials. Since CD19 is a general B-cell antigen, CAR-T-cells eliminate all B-lineage cells, including nonmalignant B cells. Therefore, the patients suffer from the impaired humoral immune response, increasing susceptibility to severe infections. Since B-cell lymphoma and chronic lymphocytic leukemia cells have a clonally restricted expression of Immunoglobulin (Ig) light chains, either Ig-kappa or Ig-lambda, Ig-kappa positive tumor cells can be targeted while sparing normal Ig-lambda positive B-cells. In this respect, we isolated the sequence encoding the antigen-binding parts of an anti-Ig kappa antibody and designed a second-generation CAR construct (IGK CAR). Expression of IGK CAR in expanded peripheral blood T-cells and subsequent testing of the CAR T-cells in various in vitro assay with targeT-cells demonstrated cytokine production and potent killing of Ig-kappa expressing B-cell lines such as BL-41, whereas no response was observed against Ig lambda positive B-cell lines such as Granta-519. We compared IGK CAR with a clinical CD19 CAR (fmc63) and observed similar potency in targeT-cell killing. Previous reports have shown that the presence of free immunoglobulins present in human serum could inhibit IGK CAR T-cells, and our tests confirmed this. To improve IGK CAR T-cells in the presence of IgGs while maintaining the specificity, we utilized a combinatorial CAR system, where the signaling domains were split. Our design demonstrated efficient killing of Ig-kappa positive cells and was less sensitive to free IgG as compared to IGK CAR T-cells. Additionally, we observed a trade-off between specificity and cytotoxic potential. Increasing one individual component of the combinatorial system made the cells less prone to serum IgG inhibition but demonstrated somewhat higher cytotoxic activity against Ig-kappa negative targets. Our fully adjustable design, therefore, brings another perspective to the field by regulating the individual expression levels according to the treatment needs, hence enabling T-cells to be either more aggressive or specific depending on the treatment efficiency and on the on-target toxicity in patients. Taken together, our in vitro data demonstrate that IGK-CD19 CAR combination is as potent as IGK or CD19 CAR T-cells, and provides an alternative by combining their benefits into one design and thus reduces on-target toxicity. Citation Format: Hakan Köksal, Pierre Dillard, Sólrún Melkorka Maggadóttir, Gunnar Kvalheim, Erlend Bremertun Smeland, June Helen Myklebust, Else Marit Inderberg, Sébastien Wälchli. Combinatorial IGK-CD19 CAR primarily targets IgK+ malignant B-cells and is less prone to serum IgG inhibition [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A035.

CAR T-cell bioengineering: Single variable domain of heavy chain antibody targeted CARs.

Redirecting the recognition specificity of T lymphocytes to designated tumour cell surface antigens by transferring chimeric antigen receptor (CAR) genes is becoming an effective strategy to combat cancer. Today, CAR T-cell therapy has proven successful in the treatment of haematological malignancies and the first CD19 CAR T-cell products has already entered the market. This success is expanding CAR design for broader malignancies including solid tumours. Nevertheless, CARs such as those built on antigen-specific single chain antibody variable fragment (scFv) may induce some adverse effects. Here, we briefly review CAR T-cell bioengineering and discuss selected important initiatives for improved T-cell reprogramming, function and safety. In this respect, we further elaborate on unconventional CARs structured on single variable domain of heavy chain (VHH) antibodies (single-domain antibodies) as an alternative to scFv, because of their interesting immunological and physicochemical characteristics and unique structure, which shows a high degree of homology with human VH3 gene family.

Manufacturing chimeric antigen receptor T cells: issues and challenges

Clinical trials of adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have delivered unprecedented responses in patients with relapsed refractory B-cell malignancy. These results have prompted Food and Drug Administration (FDA) approval of two CAR T-cell products in this high-risk patient population. The widening range of indications for CAR T-cell therapy and increasing patient numbers present a significant logistical challenge to manufacturers aiming for reproducible delivery systems for high-quality clinical CAR T-cell products. This review discusses current and novel CAR T-cell processing methodologies and the quality control systems needed to meet the increasing clinical demand for these exciting new therapies.

Early Response Data for Pediatric Patients with Non-Hodgkin Lymphoma Treated with CD19 Chimeric Antigen Receptor (CAR) T-Cells

Background:Pediatric patients with relapsed or refractory CD19+non-Hodgkin lymphoma (NHL) have poor outcomes despite use of chemotherapy and hematopoietic stem cell transplant (HSCT). Clinical trials of CD19 CAR T-cells have demonstrated efficacy in salvaging adult patients with relapsed and refractory NHL.Objectives:The objectives of this analysis is to assess the safety, toxicity, feasibility and efficacy of SCRI-CAR19v1 for pediatric patients with relapsed or refractory NHL.Design/Methods:The ongoing phase 2 trial (NCT02028455) has enrolled and treated 8 pediatric subjects with CD19+NHL. Subjects underwent apheresis, with their CD4 and CD8 T cell subsets prepared immunomagnetically. T cells were stimulated with anti-CD3xCD28 bead stimulation, and then transduced with a SIN lentiviral vector to direct co-expression of the FMC63scFv:IgG4hinge:CD28tm:4-1BB:ζ CAR and the selection/tracking/suicide construct EGFRt. The transduced cells were propagated using recombinant human cytokines to numbers suitable for clinical use. Subjects received lymphodepletion of fludarabine and cyclophosphamide followed by 1x106CD19 CAR T-cells/kg as a 1:1 ratio of CD4 and CD8 cells. Response was assessed at 3 and 9 weeks. Adverse events were graded according to CTCAEv4 except cytokine release syndrome (CRS) was graded according to Lee et al.Results: Treated subjects had relapsed or refractory diffuse large B cell lymphoma (DLBCL) (4/8), Burkitt's lymphoma (2/8), gray zone B cell lymphoma (1/8), primary mediastinal B cell lymphoma (PMBCL) (1/8), and ranged from 4-18 years old. Two subjects received prior hematopoietic stem cell transplant (HSCT); the subject with PMBCL received auto- and allogeneic HSCT and a subject with Burkitt's received autologous HSCT. Five subjects received prior immunotherapy with brentuximab, nivolumab, rituximab, and/or obinutuzumab. One subject had received ibrutinib. No subject had received prior CAR T-cells. CD4 and CD8 products were successfully manufactured and infused for all subjects. All subjects had expansion of CAR T-cells in the peripheral blood, bone marrow and CSF, with ongoing persistence at last check (range 14 days - 9 months). Toxicity information through day 30 revealed the occurrence of mild CRS in 4 subjects (grade 1 n=3, grade 2 n=1), and one case of severe CRS (grade 3). Mild neurotoxicity was observed in 2 subjects (grade 1 n=1, grade 2 n=1) with no occurrence of severe neurotoxicity. Response assessment at 3 weeks (n=6) revealed anti-tumor responses in 5 subjects, including complete response (CR) by week 9 (n=2, both DLBCL). CR was not sustained in either subject despite ongoing CAR T cell persistence. One of these subjects had a PET avid lesion proven by biopsy to be necrotic tissue but subsequently developed CD19+recurrence at that site. The other subject developed a new CD19+site of disease at six months; however, achieved a 2nd CR 3 weeks after receiving a second infusion of the originally manufactured CAR T-cells. One partial response (PR) subject experienced clearance of marrow disease with stable lymphoma but developed CD19 negative progression at 9 weeks. Updated enrollment, toxicity and response assessments will be presented.Conclusion:SCRI-CAR19v1 therapy demonstrates efficacy in pediatric patients with relapsed and refractory NHL and appears to be well tolerated with less severe toxicities than observed for pediatric patients with CD19+leukemia. Persistence of the CAR T-cells is excellent with no early loss of CAR T-cell engraftment reported to date. Although early responses were observed, these were not durable perhaps reflecting biologic/immunologic differences between B cell lymphomas in children in comparison to NHL in adults. DisclosuresPulsipher:Adaptive Biotech: Consultancy, Research Funding; Amgen: Honoraria; CSL Behring: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau. Park:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Jensen:Juno Therapeutics, Inc.: Consultancy, Patents & Royalties, Research Funding.

Piggybac CD19 CAR T Cells Eradicate CNS Leukemia By Direct Delivery into Cerebral Ventricle of Xenograft Mice Model

[Introduction]Despite the improvement in the treatment for childhood acute lymphoblastic leukemia (ALL), a central nervous system (CNS) involvement is still a risk factor for mortality. Moreover, CNS-directed treatments can cause acute and late adverse complications. Therefore, more effective and less toxic treatment strategy for CNS-ALL is needed.Recent clinical trials show that the anti-CD19 chimeric antigen receptor T-cell (CAR-T) therapy have an excellent effect against refractory and relapsed ALL. In these trials, some clinical effect of systemic intravenous (i.v.) delivery of CAR-T cells against CNS-ALL were reported and these reports implied clinical possibility and necessity for optimization of CAR-T therapy against CNS-ALL.The superior effect of direct delivery of CAR-T cells into solid tumors has been published, and we and others have shown the feasibility of intrathecal (IT) donor lymphocyte infusion for CNS-ALL without severe adverse events (Yanagisawa et al, IJH 2016). These reports inspired us to apply direct CAR-T cells infusion into CNS as a new therapeutic approach against CNS-ALL. However, as the lethally neurotoxicity has been reported in CAR-T clinical trials, preclinical study is a crucial step for clinical use of IT administration of CD19 CAR-T cells.We have previously reported ALL patient-derived xenograft (PDX) model with CNS infiltration in NOD/SCID/γc Null (NOG) mouse (Kato et al, ASH 2010). With this model, we have revealed the unique characteristics of CNS infiltrated leukemic cells and proposed new strategy targeting the CNS-ALL (Kato et al, Blood, 2018).In this study, we established an isolated CNS-ALL xenograft model with the intra-cerebroventricular (i.c.v.) injection of ALL cells and tested the feasibility and safety of the i.c.v. delivery of CAR-T cells into this model.[Method]CD19 CAR-T cells were generated with the piggyBac transposon system from isolated PBMCs donated from a healthy volunteer and expanded in the presence of IL-7 and IL-15 (Morita et al, Mol Ther Methdos Clin Dev 2018). SUSR cells, BCR-ABL fusion gene positive pre-B ALL cells, were genetically engineered to express GFP-FFLuc (SUSRGFP/luc).2×105 SUSRGFP/luc cells were injected by i.c.v. into 8-10 weeks old male NOG mice. Seven days after injection, confirming that SUSRGFP/luc cells were detected by IVIS imaging system only in CNS region, 2×106 piggyBac CD19 CAR-T cells injected by i.c.v. (CAR-T i.c.v.) (n=6), or i.v via tail-vein (CAR-T i.v) (n=5). As control groups, non-transduced T-cells from the same donor (Mock-T i.c.v.) (n=5) or vehicle (Medium i.c.v.) (n=5) were injected by i.c.v. No treatment group (n=4) received no i.c.v. injection. ALL invasion was followed by IVIS imaging. To assess the safety, we serially measured body weight, rectal temperature and scores of clinical symptoms of each mouse every morning after treatment.[Results]All mice in the Medium i.c.v. and no treatment group died by day 45. Both in the CAR-T i.v. and i.c.v. group, all mice were alive at day 45. Regardless of the route of injection, CAR-T administration prolonged the survival period. IVIS imaging revealed that 3/5 mice both in Mock-T i.c.v. and CAR-T i.v group failed to eliminate CNS-ALL at day 40. On the other hand, CAR-T i.c.v. completely eradicated ALL in all mice. Histopathological and flowcytometry analysis at day 4 after treatment also showed decrease of ALL cells and expansion of human CD3 positive T-cells in CAR-T i.c.v. mouse.Regarding clinical symptoms, weight loss, decrease in rectal temperature and elevation of clinical scores were observed only in SUSR transplanted CAR-T i.c.v. group during a few days after treatment. These changes were transient and not fatal. No neurological symptoms were observed in all mice. Histopathological analysis of CNS at day 4 showed slightly thickened epithelium of choroid plexus and edema localized around the lateral ventricle only in CAR-T i.c.v. mouse. Any other abnormalities associated neurotoxicity was not detected.[Discussion]In this preclinical study, we demonstrated that i.c.v. delivery of CD19 CAR-T cells had superior effect to i.v. delivery without any additional side effects. Our data suggests that IT administration of CD19 CAR-T cells would be an effective and feasible therapeutic approach against CNS-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effective Chimeric Antigen Receptor T Cell Therapy for Relapsed and Refractory Acute Lymphoblastic Leukemia in Children Can be Administered Safely Under the Real-Time Monitoring of Th1/Th2 Cytokine Patterns Measured By the Cytometric Bead Array Technology

Background and Objectives: Chimeric antigen receptor modified T (CAR-T) cell therapy has emerged as a promising strategy for the treatment of relapsed and refractory hematological malignancies recently. CD19 chimeric antigen receptor T (CD19CAR-T) cell therapy is a representative case showing promising results in treating relapsed and refractory B-lineage acute lymphoblastic leukemia (r/r B-ALL). However, challenges remain due to the concurrent severe side-effects such as cytokine release syndrome (CRS) or neurological toxicities which may cause death if they are not properly managed. So, how to safely use the CAR-T cell therapy in patients has drawn a lot of concern and attention worldwide. The objective of this study was to investigate the cytokine patterns to early identify and monitor the severe side-effects of cytokine release syndrome (CRS) and neurotoxicity caused by the therapy so that the therapy can be safely administered. Patients and Method: From December 2015 to March 2017, five patients with r/r B-ALL treated with CD19CAR-T cells were enrolled in this study. The Th1/Th2 cytokines including Interleukin (IL)-2, IL-4, IL6, IL-10, Tumor necrosis factor (TNF) and Interferon-gamma (IFN-γ ) in the serum and cerebral spinal fluid (CSF) were quickly measured by cytometric bead array (CBA) technology which allowed the result being available within 5 hours after the samples taken. The test were repeated more than once a day during the therapy. Results: 5 pediatric patients (2 boys and 3 girls) with r/r B-ALL aged 4 months -12 years old were treated by CD19CAR-T cell therapy. Four patients achieved complete remission (80% of CR rate) with minimal residual diseases (MRD) negative detected by flow cytometry within 2 weeks. One patient did not respond to the therapy due to the poor quality of his T cells. Five to 10 times of cytokine measurements were carried out for the five patients. Two types of cytokine patterns were identified: (1) very high levels of IL-6, IFN-γ with moderately elevated IL-10 indicated the CAR-T therapy effective pattern; (2) IL-6 slightly - severely elevated IL-6 level without concurrent elevation of IFN-γ represented a CAR-T cell therapy ineffective pattern but rather indicating a current infection. All the four responding patients presented the CAR-T therapy effective cytokine pattern, while the non-responding patient presented the CAR-T therapy ineffective cytokine pattern. We also analyzed the relationship between the cytokine levels and the severity of the side-effects and found that they were highly correlated. The cytokine levels in the CSF of two patients with seizure were also measured and we found that the elevation of the cytokine pattern in the CSF was paralleled to those in their serum but CNS-leukemia and CAR-T cells in their CSFs were not detected, indicating that the neurotoxicity concurrently presented in these two patients might be caused by the cytokines in the serum leaked into the CSF through the blood brain barrier. Extremely high level of cytokines occurred at 12 hours after CAR-T cell infusion in a patient was identified immediately and treated timely in onn-ICU setting to avoid the fatal consequence. Acute toxicities including fever, hypotension and other neurological toxicities occurred in responding patients within 2 weeks post infusion and managed properly with tocilizumab and/or steroids according to the “real-time” monitoring of this simple 6-Th1/Th2 cytokine patterns at the non-ICU setting. In conclusion, our study demonstrates that CD19CAR-T cell therapy can be administered safely for patients with relapsed and refractory leukemia under the “real-time” monitoring of a simple 6-cytokine pattern. DisclosuresNo relevant conflicts of interest to declare.

Targeted Delivery of CpG-Mir-146a Mimic Oligonucleotides As a Therapeutic Strategy to Reduce NF-Κb-Mediated Pathogenic Inflammation and Myeloid Leukemia Progression

NF-kB signaling plays central role in the regulation of immune cell activity. The microRNA-146a-5p (miR-146a) provides negative feedback inhibition of the NF-kB pathway to prevent either excessive immunity, such as cytokine release syndrome. Low expression of miR-146a is also implicated in certain types of leukemia, especially in del(5q)-syndrome myelodysplastic and acute myeloid leukemia (MDS/AML). While miR-146a is a potential therapeutic target, the lack of efficient miRNA delivery methods limits clinical translation. We previously developed a strategy for targeted delivery of oligonucleotide therapeutics, such as siRNA, into human and mouse myeloid cells and B cells, using partly or completely phophorothioated (PS) single-stranded oligodeoxynucleotides containing CpG motif (CpG ODN). Here, we demonstrate that similar strategy, using CpG ODN optimized for targeting human monocytes, can be employed for the delivery of functional miR-146a mimic. The CpG-miR146a mimic conjugate, but not unconjugated miR-146a, was quickly internalized by target human and mouse myeloid cells, such as dendritic cells, macrophages and leukemic cells. In vitro CpG-miR146a mimic reduced protein levels of downstream targets, Irak1 and Traf6, as well as the inhibition of DNA binding and transcriptional activity of NF-kB in myeloid cells. We further verified functional activity of CpG-miR146a mimic against several models of acute inflammation. The intravenous injections of this oligonucleotide reduced myeloproliferation and excessive cytokine response to bacterial endotoxin or to live Listeria challenge in miR-146a-/- mice. Furthermore, CpG-miR146a mimic had similar anti-inflammatory effect also in a human model of cytokine release syndrome induced by CD19 chimeric antigen receptor (CAR) T-cell activity. CpG-miR146a effectively abrogated CAR T-cell induced IL-6 production by human monocytes in vitro. Finally, our studies indicated that CpG-miR146a mimic can target del(5q) MDS and AML cells in vitro and in vivo resulting in growth inhibition of MDSL and HL-60 acute myeloid leukemia. Our results suggest that CpG-miR146a strategy can be provide a new-in-class immunomodulatory therapeutics for treatment of acute inflammatory disorders, myeloproliferative diseases and myeloid leukemia. DisclosuresWang:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Forman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding. Kortylewski:N/A: Patents & Royalties: I am an inventor on the US patent 9,976,147.

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings. We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general. This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described. Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice. These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells.

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Abstract A071: PSMA-specific CARTyrin T-stem cell memory therapy eliminates solid tumor in subcutaneous prostate cancer model

Abstract Prostate-specific membrane antigen isoform 1 (PSMA) is a transmembrane glycoprotein overexpressed in >80% of primary and metastatic prostate cancers. PSMA is involved in folate uptake and confers a proliferative advantage to PSMA-expressing cells. Additionally, PSMA levels increase as cells become androgen-independent, a hallmark of advancing prostate disease. Although PSMA peptide and antibody drug conjugate therapies have shown promise, early first-generation chimeric antigen receptor (CAR) T-cell therapies have lacked clinical efficacy. Here we developed a novel CAR T-cell product (P-PSMA-101) via piggyBac™ transposition of a tri-cistronic transgene encoding a safety switch, a PSMA-specific Centyrin-based CAR (CARTyrin), and a selection gene—features that may improve safety and efficacy compared with previous anti-PSMA CAR T-cell therapies. We first developed and identified a lead anti-PSMA CARTyrin from over 250 available Centryin binders. We also tested the previously clinically applied anti-PSMA J591 scFv-based CAR for comparability. Initial assessment utilized mRNA delivery of candidate CARTyrins to confirm CAR surface expression and specific T-cell degranulation against PSMA+ prostate tumor cells or PSMA-engineered cells. We then used piggyBac to deliver our tri-cistronic vector system encoding the lead PSMA CARTyrin (P-PSMA-101), J591 scFv CAR, or a BCMA-specific CARTyrin to T cells, resulting in >95% CAR+ T cells after selection and expansion. Importantly, our unique production methodology leads to >60% T-stem cell memory (Tscm) cells, an early memory population that correlates with complete responses in CD19 CAR T-cell clinical trials. In vitro, P-PSMA-101 and J591 CAR T cells specifically proliferated, lysed, and secreted IFN-γ against PSMA+ LNCaP or PSMA-engineered K562s. No evidence of tonic signaling or exhaustion was detected. P-PSMA-101 demonstrated significantly enhanced antitumor efficacy and survival in comparison to J591 CAR T cell-treated mice (P-PSMA-101 >110 days versus J591 41 days) in a low “stress test” dose of T cells against established subcutaneous LNCaP(fLuc+) solid tumors in NSG mice. The >60% Tscm P-PSMA-101 expanded in vivo and gave rise to differentiated effector CAR+ T cells that were detected in the peripheral blood concomitant with a decrease in tumor burden below detectable caliper and bioluminescent imaging limits. P-PSMA-101 then contracted, yet persisted in the peripheral blood with >70% of T cells exhibiting a Tscm phenotype. On the contrary, J591 CAR T cells did not significantly control tumor burden or expand to appreciable levels in the peripheral blood. In a dose titration study, P-PSMA-101 eliminated established LNCaP tumor in 100% of animals for the duration of the studies (42 days post-treatment), while 2/3 low-dose animals remained tumor-free. Ongoing in vivo studies point towards potent antitumor effects in aggressive bone metastases models of prostate cancer. P-PSMA-101 is a first-in-class Centyrin-based CAR T-cell therapeutic that exhibits a persistently high frequency of Tscm and mediates durable anti-solid tumor efficacy that surpasses previously established anti-PSMA CAR T-cell therapy in our in vivo models. Future efforts will continue towards clinical application of P-PSMA-101 in patients with metastatic castrate-resistant prostate cancer. Citation Format: Jenessa Barbara Smith, Rebecca Codde, Yening Tan, Barnett E. Barnett, David Hermanson, Srinivas Rengarajan, Eric M. Ostertag, Devon J. Shedlock. PSMA-specific CARTyrin T-stem cell memory therapy eliminates solid tumor in subcutaneous prostate cancer model [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A071.

Media evaluation for production and expansion of anti-CD19 chimeric antigen receptor T cells

BackgroundThe use of CD19 chimeric antigen receptor (CAR) T cells to treat B-cell malignancies has proven beneficial. Several groups use serum to produce CD19 CAR T cells. Today, ready-to-use serum-free media that require no addition of serum are commercially available. Therefore, it becomes important to evaluate the production of CD19 CAR T cells with and without the addition of serum. MethodsT cells from buffy coats were cultured in AIM-V and TexMACS (TM) supplemented with 5% human serum (A5% and TM5%, respectively), and in TM without serum. Cells were activated with OKT3 and expanded in interleukin (IL)-2. Viral transduction was performed in RetroNectin-coated plates using the spinoculation method. CD19 CAR T cells were tested for their viability, expansion, transduction efficacy, phenotype and cytotoxicity. ResultsCD19 CAR T cells expanded in A5% and TM5% showed significantly better viability and higher fold expansion than cells expanded in TM. TM promoted the expansion of CD8+ T cells and effector phenotype of CD19 CAR T cells. The transduction efficacy and the cytotoxic function were comparable between the different media. Higher CD107a+ cells were detected in TM and TM5%, whereas higher IL-2+ and IL-17+ cells were detected in A5%. CD19 CAR exhibited co-expression of inhibitory receptors such as TIM-3+LAG-3+ and/or TIM-3+PD-1+. ConclusionOur results indicate that serum supplementation promotes better CD19 CAR T-cell expansion and viability in vitro. CD19 CAR T cells produced in TM medium showed lower CD4/CD8 ratio, which warrants further evaluation in clinical settings. Overall, the choice of culture medium impacts CD19 CAR T-cell end product.

Progress of chimeric antigen receptor T-cell for treatment of malignant lymphoma

Chimeric antigen receptor T-cell (CAR-T) is one of the effective methods for treatment of lymphoma. The way to improve the efficacy and control the reverse reactions still needs to be explored further. Several clinical trials have indicated CAR-T could have favorable effects on the B-cell lymphoma patients with controllable reverse reactions. However, antigen loss is a major factor for the acquired resistance to CD19 CAR-T therapy. Other clinical researches, including CD22 for treatment of B-cell lymphoma and CD30 for Hodgkin lymphoma, have increased the efficacy of CAR-T. Moreover, lots of trials have suggested that the patients who received cyclophosphamide or bendamustine plus fludarabine lymphodepletion can get a high effective rate. Key words: Lymphoma; Chimeric antigen receptor T-cell; Clinical protocols

NK-92 cell, another ideal carrier for chimeric antigen receptor.

The remarkable clinical outcomes of the treatment for B-cell malignancies through the application of CD19 chimeric antigen receptor T (CAR-T) cells have made adoptive immunotherapy with genetically modified immune effector cells a hotspot in the field of antitumor. However, numerous toxicities of CAR-T cells have been identified. Thus, some studies have resorted to another cytotoxic cell, NK-92 cell, to reach for better efficacy with minimal toxicity. Preclinical studies have confirmed the safety and feasibility of the genetically modified NK-92 cells with highly specific cytotoxicity in vitro and in vivo. Therefore, it is expected that NK-92 cell becomes another ideal carrier for CAR for its unique advantages over primary NK cells, parental NK-92 cells and autologous T cells.