Generation of CD19-Targeted Chimeric Antigen Receptor T Cells.
Kiani Jafar,Naderi Mahmood,Torabi-Rahvar Monireh,Ranjbar Azam,Aghayan Hamid-Reza,Janzamin Ehsan,Ahmadbeigi Naser
Archives of Iranian medicine
BACKGROUND:Current advancements in the field of chimeric antigen receptor (CAR) therapy, particularly U.S. FDA approval of Kymriah and Yescarta, heralds a new era of cancer treatment. This rapid progress in technology has urged more countries and institutions to keep pace with the fast-growing and developing technology of producing CAR T cell-based therapies in the race to develop new cancer-targeting drugs. Hence, for stepping in line with global advances and to pave the way for subsequent preclinical and clinical studies, we have established a development protocol for a cancer-targeting CAR T cell; we have chosen CD19 CAR T cell as a well-defined model to set-up T cell expansion, activation, and viral transduction as the prerequisites for diverse CAR T cell therapies. METHODS:T cells from peripheral blood mononuclear cells (PBMCs) were activated and expanded. CD19 CAR lentiviral particles were produced in the Lenti-X™ 293T Cell Line using PolyFect Transfection Reagent. RESULTS:Activation protocol resulted in (65 ± 4%; P = 0.046) increase in the rate of activated T cells 24 hours after the initiation of the procedure. The expansion methodology resulted in a high purity of the T cell population (96 ± 3%) in the pool of PBMCs within 14 days of the procedure. Finally, 35 ± 6% of T cells were transduced with CD19 lentivirus with MOI of 3. CONCLUSION:Collectively, the results of this study prove that we have successfully overcome the first hurdle on the road to reach CAR T cell technology which is the prerequisite for developing preclinical and clinical phases of CAR therapy in settings with basic resources.
[CARs, CRS and neurotoxicity: severe complications after administration of immunotherapy : Essentials for intensivists].
Prinz J,d'Hargues Y,Gödel P,Shimabukuro-Vornhagen A,Kochanek M,Böll B
Medizinische Klinik, Intensivmedizin und Notfallmedizin
BACKGROUND:The development of chimeric antigen receptor (CAR) T‑cells has shown promising results in relapsed/refractory B‑cell acute lymphoblastic leukemia/lymphoma (B-ALL) and diffuse large cell B‑cell lymphoma. Complications, especially cytokine release syndrome (CRS) and CAR T‑cell related encephalopathy syndrome (CRES), can be life threatening. The management of both plays a key role in CAR T‑cell therapy. OBJECTIVES:Diagnosis, clinical presentation and development of complications in the treatment with CAR T‑cells. MATERIALS AND METHODS:Summary of incidence, mortality and treatment of severe complications after administration of CAR T‑cells referring to current studies and therapy recommendations. RESULTS:Complications after administration of CAR T‑cells, especially CRS and CRES, can be life threatening. The timely identification of side effects and their appropriate treatment usually leads to complete recovery. CONCLUSIONS:Using a therapy algorithm in the treatment with CAR T‑cells allows safe management of toxicities and can be helpful in recognizing them in time.
Increasing the safety and efficacy of chimeric antigen receptor T cell therapy.
Li Hua,Zhao Yangbing
Protein & cell
Chimeric antigen receptor (CAR) T cell therapy is a promising cancer treatment that has recently been undergoing rapid development. However, there are still some major challenges, including precise tumor targeting to avoid off-target or "on-target/off-tumor" toxicity, adequate T cell infiltration and migration to solid tumors and T cell proliferation and persistence across the physical and biochemical barriers of solid tumors. In this review, we focus on the primary challenges and strategies to design safe and effective CAR T cells, including using novel cutting-edge technologies for CAR and vector designs to increase both the safety and efficacy, further T cell modification to overcome the tumor-associated immune suppression, and using gene editing technologies to generate universal CAR T cells. All these efforts promote the development and evolution of CAR T cell therapy and move toward our ultimate goal-curing cancer with high safety, high efficacy, and low cost.
Tisagenlecleucel, an approved anti-CD19 chimeric antigen receptor T-cell therapy for the treatment of leukemia.
Liu Y,Chen X,Han W,Zhang Y
Drugs of today (Barcelona, Spain : 1998)
On August 30, 2017, the U.S. Food and Drug Administration (FDA) approved Novartis' tisagenlecleucel (CTL-019, Kymriah), which is a synthetic bioimmune product of anti-CD19 chimeric antigen receptor (CAR) T cells, for the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). This was a milestone in tumor immunology on account of the significant antitumor effect of tisagenlecleucel for the treatment of relapsed/refractory B-ALL patients. Conventional standard therapies for B-ALL have high failure rates, thus developing new therapies is crucial for patients with B-ALL. Results from clinical trials indicate that anti-CD19 CAR T-cell therapies could successfully induce high response rates in B-ALL patients. However, related toxicities, such as cytokine release syndrome and CAR T-cell-related encephalopathy syndrome, may be severe or even fatal, and the management of such toxicities is therefore vital. This review will focus on the clinical application of anti-CD19 CAR T-cell therapy in B-ALL treatment, including design features of CAR constructs, therapeutic use of tisagenlecleucel, CAR T-cell therapy clinical trials and related toxicity, and prospects for cancer immunotherapy.
Acute lymphoblastic leukemia relapse after CD19-targeted chimeric antigen receptor T cell therapy.
Wang Jiasheng,Hu Yongxian,Huang He
Journal of leukocyte biology
CART19 therapy has revolutionized the treatment of CD19 acute lymphoblastic leukemia, demonstrating an unprecedented complete remission rate; however, as follow-up prolongs, a high relapse rate after CART19 therapy has emerged as one of the major problems. Relapse can be attributed to the loss of leukemic cell immunogenicity, diminished function and amount of CART19 cells, and the inhibitory bone marrow microenvironment. Although studies to prevent and treat relapse have begun, some encouraging results have demonstrated the possibility of decreasing the relapse rate. In this review, we focus on the possible mechanisms behind relapse. We will summarize and propose strategies to prevent and manage relapse on the basis of these potential mechanisms.
[Chimeric antigen receptor T cell (CAR-T) therapy for refractory/relapsed hematological malignancy].
[Rinsho ketsueki] The Japanese journal of clinical hematology
Anti-tumor drugs have been the mainstay of cancer treatment; however, immunotherapy has been considered as supplementary, often less effective, and associated with toxicity. However, over the recent years, new types of immunotherapies have been developed, some of which are proving pivotal in current cancer treatment. Of these, chimeric antigen receptor T (CAR-T) cell therapy represents one of the promising types of immunotherapy, particularly for B-lineage malignancy treatment. Notably, many researchers are utilizing this technology for other types of cancer as well. Although it has some drawbacks such as lethal side-effects and extremely high costs, CAR-T cell therapy is estimated to be a potential game changer in the field of cancer treatment. This review provides an overview of the current status and future prospects of CAR-T therapy.
Insights into cytokine release syndrome and neurotoxicity after CD19-specific CAR-T cell therapy.
Gauthier Jordan,Turtle Cameron J
Current research in translational medicine
T-cells engineered to express CD19-specific chimeric antigen receptors (CD19 CAR-T cells) can achieve high response rates in patients with refractory/relapsed (R/R) CD19+ hematologic malignancies. Nonetheless, the efficacy of CD19-specific CAR-T cell therapy can be offset by significant toxicities, such as cytokine release syndrome (CRS) and neurotoxicity. In this report of our presentation at the 2018 Second French International Symposium on CAR-T cells (CAR-T day), we describe the clinical presentations of CRS and neurotoxicity in a cohort of 133 adults treated with CD19 CAR-T cells at the Fred Hutchinson Cancer Research Center, and provide insights into the mechanisms contributing to these toxicities.
Tisagenlecleucel: The First CAR on the Highway to Remission for Acute Lymphoblastic Leukemia.
Freyer Craig W
Journal of the advanced practitioner in oncology
Tisagenlecleucel is a first-in-class chimeric antigen receptor (CAR) T-cell therapy approved by the US Food and Drug Administration in 2017 for relapsed/refractory (RR) acute lymphoblastic leukemia (ALL) in patients up to 25 years of age. Tisagenlecleucel is an autologous T-cell therapy that is genetically engineered with a lentiviral vector to seek and eliminate CD19-expressing B cells throughout the patient's body and retain antitumor immune surveillance following remission. This groundbreaking cellular therapy brings unprecedented single-agent efficacy to patients with RR ALL, citing complete response rates of greater than 80% and 6-month relapse-free survivals exceeding 60% in a patient population with poor prognosis and few treatment options. Patients receiving CAR T-cell therapy are at risk for cytokine release syndrome (CRS), neurotoxicity, and infections, along with other toxicities that may be severe or life-threatening. The cornerstone of the management of moderate to severe CRS is treatment with the interleukin-6 antagonist tocilizumab, with dramatic responses often occurring within 24 hours. The optimal management of neurotoxicity following tisagenlecleucel remains undefined. It is critical that providers caring for patients receiving tisagenlecleucel understand the multistep process to prepare a patient for therapy, how to closely monitor patients for toxicity, and how to manage emergent adverse events following cell infusion.
Cytokine Release Syndrome Grade as a Predictive Marker for Infections in Patients With Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia Treated With Chimeric Antigen Receptor T Cells.
Park Jae H,Romero F Andres,Taur Ying,Sadelain Michel,Brentjens Renier J,Hohl Tobias M,Seo Susan K
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
Background:Chimeric antigen receptor (CAR)-modified T cells that target the CD19 antigen present a novel promising therapy for the treatment of relapsed B-cell acute lymphoblastic leukemia (B-ALL). Although cytokine release syndrome (CRS) and neurotoxicity have emerged as predominant noninfectious complications of CD19 CAR T-cell therapy, infections associated with this treatment modality have not been well documented. Methods:We analyzed infectious complications that followed CD19 CAR T-cell therapy in 53 adult patients with relapsed B-ALL enrolled in a phase I clinical trial at Memorial Sloan Kettering Cancer Center (NCT01044069). Results:Overall, 22 patients (42%) experienced 26 infections (17 bacterial, 4 fungal, and 5 viral) within the first 30 days of CAR T-cell infusion. In 10 of 32 (31%) patients in whom complete remission was achieved, 15 infections developed between days 31 and 180; the majority of these late infections were due to respiratory viruses. In general, bacterial, fungal, and viral infections were detected at a median of 18, 23, and 48 days, respectively, after CAR T-cell infusion. CRS grade 3 or higher was independently associated with increased risk of subsequent infection (adjusted hazard ratio [HR], 2.67; P = .05) and in particular with bloodstream infection (adjusted HR, 19.97; P < .001). Three of 53 patients (6%) died of an infection-related cause. Conclusions:Infections in adult patients with relapsed B-ALL are common after CD19 CAR T-cell therapy. Understanding the infectious complications that are temporally coincident with CD19 CAR T-cell therapy is critical for developing effective prophylactic and other supportive care measures to improve clinical outcomes. Clinical Trials Registration:NCT01044069.
The severe cytokine release syndrome in phase I trials of CD19-CAR-T cell therapy: a systematic review.
Jin Zhen,Xiang Rufang,Qing Kai,Li Xiaoyang,Zhang Yunxiang,Wang Lining,Zhu Hongming,Mao Yuanfei,Xu Zizhen,Li Junmin
Annals of hematology
CD19 chimeric antigen receptor (CAR) T cell therapy has shown impressive results in treating acute lymphoblastic leukemia (B-ALL), chronic lymphoblastic leukemia (B-CLL), and B-cell non-Hodgkin lymphoma (B-NHL) over the past few years. Meanwhile, the cytokine release syndrome (CRS), which could be moderate or even life-threatening, has emerged as the most significant adverse effect in the clinical course of this novel targeting immunotherapy. In this systematic review, we analyzed the incidence of severe CRS in 19 clinical trials selected from studies published between 2010 and 2017. The pooled severe CRS proportion was 29.3% (95% confidence interval [CI] 12.3-49.1%) in B-ALL, 38.8% (95%CI 12.9-67.6%) in B-CLL, and 19.8% (95%CI 4.2-40.8%) in B-NHL. In the univariate meta regression analysis, the proliferation of CD19-CAR-T cell in vivo was correlated with the severe CRS. Specifically, total infusion cell dose contributed to the severe CRS occurring in B-ALL patients but not in B-CLL or B-NHL patients. Tumor burden was strongly associated with the severity of CRS in B-ALL. Besides, post-HSCT CD19 CAR-T cell infusion represented lower severe CRS incidence. Further investigations into the risk factors of CRS in B-CLL and B-NHL are needed.
The biological basis and clinical symptoms of CAR-T therapy-associated toxicites.
Titov Aleksei,Petukhov Alexey,Staliarova Alena,Motorin Dmitriy,Bulatov Emil,Shuvalov Oleg,Soond Surinder M,Piacentini Mauro,Melino Gerry,Zaritskey Andrey,Barlev Nickolai A
Cell death & disease
Currently, immunotherapy is attracting a lot of attention and may potentially become a leading approach in the treatment of cancer. One emerging therapeutic, the chimeric-antigen receptor T-cell adoptive immunotherapy (CAR-T) is showing remarkable efficacy in the treatment of several B-cell malignancies. The popularity of CAR-T has been founded on two CAR T-cell products recently approved by FDA (during 2017) in the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia and B-cell lymphoma. However, their toxicities observed in clinical trials were extremely significant and in some cases even fatal with no approved algorithms for toxicity prediction being available to date. A deeper understanding of the biological basis of such complications is the key to prompt and comprehensive clinical management. Here we review the wide spectrum of effects associated with CAR T cell therapy with a major focus on the pathogenesis of cytokine release syndrome and neurotoxicity as the most common, potentially life-threatening effects of this treatment. We discuss the basis of clinical management and the existing models that predict the severity of toxicity, as well as the key factors that modulate this event. Finally, we will summarize the literature detailing universal allogenic CAR T-cells and their toxicity profile.
Tisagenlecleucel for the treatment of B-cell acute lymphoblastic leukemia.
Leahy Allison Barz,Elgarten Caitlin W,Grupp Stephan A,Maude Shannon L,Teachey David T
Expert review of anticancer therapy
INTRODUCTION:Cure rates for pediatric and young adult patients with refractory or recurrently relapsed acute lymphoblastic leukemia (ALL) are dismal. Survival from time of relapse is typically measured in weeks to months, and standard chemotherapy and currently approved targeted therapy achieve remission in less than a third of affected patients. To date, the only definitive curative therapy has been allogeneic hematopoietic stem cell transplant (HSCT). Advances in immunotherapy, with the introduction of chimeric antigen receptor T-cell therapies and the development of tisagenlecleucel, have changed the landscape. Areas covered: This review will describe the pharmacology of tisagenlecleucel and summarize the clinical evidence for its use in the treatment of multiple-relapsed or refractory B-cell ALL (B-ALL). Also discussed are other immunotherapies for B-ALL as well as the most commonly-encountered toxicities and corresponding management strategies. Expert commentary: Early phase trials indicate that tisagenlecleucel significantly improves survival for patients with B-ALL that is refractory or in second or later relapse. In responding patients, remissions have been reported on the order of years, and thus, tisagenlecleucel may herald a dramatic shift in the treatment paradigm of this largely fatal disease.
Hemofiltration Successfully Eliminates Severe Cytokine Release Syndrome Following CD19 CAR-T-Cell Therapy.
Liu Yanfen,Chen Xinfeng,Wang Dao,Li Hong,Huang Jianmin,Zhang Zhen,Qiao Yingjin,Zhang Hongling,Zeng Ying,Tang Chao,Yang Shuangning,Wan Xiaochun,Chen Youhai H,Zhang Yi
Journal of immunotherapy (Hagerstown, Md. : 1997)
Cytokine release syndrome (CRS) remains to be a major adverse effect of chimeric antigen receptor T (CAR-T) cell therapy in B-cell acute lymphoblastic leukemia (B-ALL) and lymphoma. It was urgent to explore novel strategy for managing severe CRS. We conducted a clinical trial to assess the safety and efficacy of CD19-targeting CAR-T-cells in the treatment of relapsed and chemotherapy-refractory B-ALL and lymphoma. A 10-year-old boy with B-ALL who never achieved minimal residual disease (MRD) negative status after 5 courses of chemotherapy was enrolled into our study and received a total of 3.19×10/kg autologous CD19 CAR-T-cells. Before CAR-T-cell infusion, naive lymphocytes made up 41.8% of bone marrow cells, which were reduced to 1% at the 14th day after transfusion, with MRD<10. However, this patient developed grade 4 CRS, multiple organ failure, hemophagocytic syndrome, neurotoxicity, and severe pulmonary infection after CAR-T-cell therapy. Tocilizumab and glucocorticoids treatment were ineffective for controlling the adverse effects and in contrast, hemofiltration immediately ameliorated the severe CRS and prevented the exacerbation of multiple organ dysfunction, pneumonia, and hydrosarca caused by CAR-T-cell therapy. All side effects disappeared within days following hemofiltration. Hemofiltration helped quickly clear cytokines, speeded up patient recover, and successfully resolved the severe CRS crisis. This was the first report, reporting the successful use of hemofiltration to eliminate adverse reactions of CAR-T-cell therapy.
Cytokine release syndrome and neurotoxicity after CD19 chimeric antigen receptor-modified (CAR-) T cell therapy.
Hay Kevin A
British journal of haematology
Chimeric antigen receptor-modified (CAR)-T cells have demonstrated impressive results in the treatment of haematological malignancies. However, cytokine release syndrome (CRS) and neurotoxicity are common toxicities which are potentially life-threatening in severe cases. Risk factors for CRS and neurotoxicity identified so far include disease burden, lymphodepletion intensity and CAR-T cell dose administered. Risk-adapted dosing, with lower CAR-T cell doses administered to B-cell acute lymphoblastic leukaemia patients with high marrow blast counts, has been successful at decreasing severe CRS rates in this population. Intervention with therapies, such as tocilizumab and corticosteroids, have been effective at ameliorating toxicity, enabling CAR-T cells to be administered safely to many patients without significantly compromising efficacy. Deeper understanding of the pathophysiology of underlying CRS and neurotoxicity will enable the development of novel approaches to reduce toxicity and improve outcomes.
CAR-T bridging to allo-HSCT as a treatment strategy for relapsed adult acute B-lymphoblastic leukemia: a case report.
Wen Shupeng,Niu Zhiyun,Xing Lina,Wang Ying,Li Hang,Kuang Na,Luo Jianmin,Zhang Xuejun,Wang Fuxu
BACKGROUND:Adults with relapsed acute lymphoblastic leukemia (ALL) have a poor prognosis, especially in patients who relapsed within 6 months of complete remission 1 (CR1). Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the treatment of choice. However, this can only be considered after complete remission 2 (CR2) is achieved. Therefore, bridging treatment is urgently needed. CASE PRESENTATION:In the present study, we report a relapsed adult B-cell ALL case that achieved CR2 after treatment with CD19-directed chimeric antigen receptor (CAR)-modified T cell (CAR-T) therapy. After subsequent allo-HSCT, the patient acquired 21 months of disease-free survival. CONCLUSION:The present results confirm that both CAR-T and allo-HSCT are effective for treating refractory or relapsed B-ALL. However, a novel sequential treatment strategy with these two therapeutic methods may achieve longer disease-free survival time.
Long-term Survival and Value of Chimeric Antigen Receptor T-Cell Therapy for Pediatric Patients With Relapsed or Refractory Leukemia.
Whittington Melanie D,McQueen R Brett,Ollendorf Daniel A,Kumar Varun M,Chapman Richard H,Tice Jeffrey A,Pearson Steven D,Campbell Jonathan D
Importance:Among children and young adults with relapsed or refractory B-cell acute lymphoblastic leukemia, the rate of 5-year disease-free survival is 10% to 20%. Approval of tisagenlecleucel, a chimeric antigen receptor T-cell therapy, represents a new and potentially curative treatment option. However, tisagenlecleucel is expensive, with a current list price of $475 000 per one-time administration. Objective:To estimate the long-term survival and value of tisagenlecleucel for children and young adults with B-cell acute lymphoblastic leukemia. Design, Setting, and Participants:In this cost-effectiveness analysis, a decision analytic model was designed to extrapolate trial evidence to a patient lifetime horizon. The survival evidence for the model was extracted from 3 studies: B2202 (enrolled patients from April 8, 2015, to November 23, 2016), B2205J (enrolled patients from August 14, 2014, to February 1, 2016), and B2101J (enrolled patients from March 15, 2012, to November 30, 2015). Long-term survival and outcomes of patients younger than 25 years with B-cell acute lymphoblastic leukemia that is refractory or in second or later relapse were derived using flexible parametric modeling from the direct extrapolation of event-free survival and overall survival curves. The published Kaplan-Meier curves were digitized from November 1, 2017, to November 30, 2017, using an algorithm to impute patient-level time-to-event data. Sensitivity and scenario analyses assessed uncertainty in the evidence and model assumptions to further bound the range of cost-effectiveness. Data were analyzed from December 1, 2017, to March 31, 2018. Interventions:The primary intervention of interest was tisagenlecleucel. The comparator of interest was the chemoimmunotherapeutic agent clofarabine. Main Outcomes and Measures:Model outcomes included life-years gained, quality-adjusted life-years (QALYs) gained, and incremental costs per life-year and QALY gained. Results:Forty percent of patients initiating treatment with tisagenlecleucel are expected to be long-term survivors, or alive and responding to treatment after 5 years. Tisagenlecleucel had a total discounted cost of $667 000, with discounted life-years gained of 10.34 years and 9.28 QALYs gained. The clofarabine comparator had a total discounted cost of approximately $337 000, with discounted life-years gained of 2.43 years and 2.10 QALYs gained. This difference resulted in an incremental cost-effectiveness ratio of approximately $42 000 per life-year gained and approximately $46 000 per QALY gained for tisagenlecleucel vs clofarabine. These results were robust to probabilistic sensitivity analyses. Across scenario analyses that included more conservative assumptions regarding long-term relapse and survival, the incremental cost-effectiveness ratio ranged from $37 000 to $78 000 per QALY gained. Conclusions and Relevance:Tisagenlecleucel likely provides gains in survival and seems to be priced in alignment with these benefits. This study suggests that payers and innovators should develop novel payment models that reduce the risk and uncertainty around long-term value and provide safeguards to ensure high-value care.
Outcome of patients with relapsed/refractory acute lymphoblastic leukemia after blinatumomab failure: No change in the level of CD19 expression.
Jabbour Elias,Düll Johannes,Yilmaz Musa,Khoury Joseph D,Ravandi Farhad,Jain Nitin,Einsele Hermann,Garcia-Manero Guillermo,Konopleva Marina,Short Nicholas J,Thompson Philip A,Wierda William,Daver Naval,Cortes Jorge,O'brien Susan,Kantarjian Hagop,Topp Max S
American journal of hematology
Blinatumomab, a bi-specific T-cell engaging CD3-CD19 antibody construct, has shown significant activity in patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (ALL). Despite this improvement, most patients relapse. Here, we describe the outcome of 68 patients with R/R ALL after failure of blinatumomab therapy: 38 (56%) blinatumomab refractory; 30 (44%) relapsing after initial response. After a median follow-up of 49 months, 9 (13%) patients remained alive. The median overall survival after blinatumomab failure was 5.2 months. At the time of failure, among 61 patients evaluated for immunophenotype, 56 (92%) had CD19-positive blasts; only five (8%) had ALL recurrence with CD19-negative disease. Two patients progressed with lower CD19 expression. In summary, the outcome of patients with R/R ALL after blinatumomab failure is poor and treatment of these patients remains an unmet medical need. Our findings indicate that blinatumomab therapy would not exclude a significant number of patients from the potential benefit of subsequent CD19-directed therapies such as chimeric antigen receptor T-cell therapy.
All systems go: converging synthetic biology and combinatorial treatment for CAR-T cell therapy.
Lanitis Evripidis,Coukos George,Irving Melita
Current opinion in biotechnology
Synthetic biology has been transformative to the treatment of advanced hematological malignancies by chimeric antigen receptor (CAR)-engineered T cells. A range of obstacles are now understood to limit the responses of solid epithelial-derived tumors to CAR therapy. For example, inefficient tumor homing and a fortified stroma can restrain the number of CAR-T cells reaching the tumor bed. Upon transendothelial migration across the tumor vasculature, CAR-T cells face a highly suppressive microenvironment that can quickly render them hypofunctional. Safety also remains a critical issue for advancing CAR therapy of solid tumors. Innovative CAR design as well as coengineering and combinatorial treatment strategies with oncolytic adenovirus, radiotherapy, vaccines, chemotherapy, small molecules and monoclonal antibodies hold tremendous potential to support CAR-T cell control of solid tumors, either by directly promoting CAR-T cell function, or/and by re-programming the TME and harnessing the endogenous immune system against the tumor.
Cell-based artificial APC resistant to lentiviral transduction for efficient generation of CAR-T cells from various cell sources.
Schmidts Andrea,Marsh Leah C,Srivastava Ambike A,Bouffard Amanda A,Boroughs Angela C,Scarfò Irene,Larson Rebecca C,Bedoya Felipe,Choi Bryan D,Frigault Matthew J,Bailey Stefanie R,Leick Mark B,Vatsa Sonika,Kann Michael C,Prew Michelle S,Kleinstiver Benjamin P,Joung J Keith,Maus Marcela V
Journal for immunotherapy of cancer
BACKGROUND:Adoptive cell therapy with chimeric antigen receptor T cells (CAR-T) has become a standard treatment for patients with certain aggressive B cell malignancies and holds promise to improve the care of patients suffering from numerous other cancers in the future. However, the high manufacturing cost of CAR-T cell therapies poses a major barrier to their broader clinical application. Among the key cost drivers of CAR-T production are single-use reagents for T cell activation and clinical-grade viral vector. The presence of variable amounts of contaminating monocytes in the starting material poses an additional challenge to CAR-T manufacturing, since they can impede T cell stimulation and transduction, resulting in manufacturing failure. METHODS:We created K562-based artificial antigen-presenting cells (aAPC) with genetically encoded T cell stimulation and costimulation that represent an inexhaustible source for T cell activation. We additionally disrupted endogenous expression of the low-density lipoprotein receptor (LDLR) on these aAPC (aAPC-ΔLDLR) using CRISPR-Cas9 gene editing nucleases to prevent inadvertent lentiviral transduction and avoid the sink effect on viral vector during transduction. Using various T cell sources, we produced CD19-directed CAR-T cells via aAPC-ΔLDLR-based activation and tested their in vitro and in vivo antitumor potency against B cell malignancies. RESULTS:We found that lack of LDLR expression on our aAPC-ΔLDLR conferred resistance to lentiviral transduction during CAR-T production. Using aAPC-ΔLDLR, we achieved efficient expansion of CAR-T cells even from unpurified starting material like peripheral blood mononuclear cells or unmanipulated leukapheresis product, containing substantial proportions of monocytes. CD19-directed CAR-T cells that we produced via aAPC-ΔLDLR-based expansion demonstrated potent antitumor responses in preclinical models of acute lymphoblastic leukemia and B-cell lymphoma. CONCLUSIONS:Our aAPC-ΔLDLR represent an attractive approach for manufacturing of lentivirally transduced T cells that may be simpler and more cost efficient than currently available methods.
A Genetic Screen to Identify Gain- and Loss-of-Function Modifications that Enhance T-cell Infiltration into Tumors.
Rogers Laura M,Wang Zhaoming,Mott Sarah L,Dupuy Adam J,Weiner George J
Cancer immunology research
T-cell-mediated cancer immunotherapies, including anti-PD-1 and T cells expressing chimeric antigen receptors (CAR-T cells), are becoming standard treatments for many cancer types. CAR-T therapy, in particular, has been successful in treating circulating, but not solid, tumors. One challenge limiting immunotherapy success is that tumors lacking T-cell infiltration do not respond to treatment. Therefore, one potential strategy to overcome resistance is to enhance the ability of T cells to traffic into tumors. Here, we describe an unbiased genetic screen approach utilizing the mutagenesis system to identify candidate genes in T cells that might be modified to drive intratumoral T-cell accumulation. This screen identified over 400 candidate genes in three tumor models. These results indicated substantial variation in gene candidate selection, depending on the tumor model and whether or not mice were treated with anti-PD-1, yet some candidate genes were identified in all tumor models and with anti-PD-1 therapy. Inhibition of the most frequently mutated gene, , affected chemokine receptor expression and enhanced T-cell trafficking and Screen candidates should be further validated as therapeutic targets, with particular relevance to enhancing infiltration of adoptively transferred T cells into solid tumors.
Determinants of response and resistance to CAR T cell therapy.
Lesch Stefanie,Benmebarek Mohamed-Reda,Cadilha Bruno L,Stoiber Stefan,Subklewe Marion,Endres Stefan,Kobold Sebastian
Seminars in cancer biology
The remarkable success of chimeric antigen receptor (CAR)-engineered T cells in pre-B cell acute lymphoblastic leukemia (ALL) and B cell lymphoma led to the approval of anti-CD19 CAR T cells as the first ever CAR T cell therapy in 2017. However, with the number of CAR T cell-treated patients increasing, observations of tumor escape and resistance to CAR T cell therapy with disease relapse are demonstrating the current limitations of this therapeutic modality. Mechanisms hampering CAR T cell efficiency include limited T cell persistence, caused for example by T cell exhaustion and activation-induced cell death (AICD), as well as therapy-related toxicity. Furthermore, the physical properties, antigen heterogeneity and immunosuppressive capacities of solid tumors have prevented the success of CAR T cells in these entities. Herein we review current obstacles of CAR T cell therapy and propose strategies in order to overcome these hurdles and expand CAR T cell therapy to a broader range of cancer patients.
[The evaluation of modified cell infusion method to reduce febrile non-hemolytic transfusion reaction in CD(19) chimeric antigen receptor T cell threapy].
Wang J,Deng Q,Mu J,Jiang Y Y,Meng J X,Li Y M
Zhonghua nei ke za zhi
To retrospectively analyze the efficacy and safety of modified cell infusion method in reducing the incidence of febrile non-hemolytic transfusion reaction (FNHTR). A total of 69 patients were enrolled in the clinical trial of CD(19) chimeric antigen receptor T (CAR-T) cell treatment from February 2017 to October 2018. Study group received the modified cell infusion method, that 1×10(6) CAR-T cells were re-suspended in 2 mg human serum albumin with total volume of 20 ml and injected intravenously. The control group was intravenously administrated with CAR-T cell in 100 ml normal saline. The incidence of FNHTR, cytokine releasing syndrome (CRS) grade, cytokine level and efficacy were compared. (1)The incidence of FNHTR in the study group was 21.1%, significantly lower than that in the control group (71%)(=0.000). (2)There was no statistical difference in cell proliferation between the study group and the control group on day 4, 7, 14 and 21 after CAR-T cell infusion (=10.223, 3.254, 5.551, 7.605). (3)There was no statistical difference in CRS grading between the study group and the control group (=0.767). There was no statistical difference in the levels of interleukin 2 receptor (IL-2R), IL-6, tumor necrosis factor (TNF)-α between the two groups. (4)The C-reaction protein (CRP) level of the study group was lower than that of the control group on day 4 and 7 (=0.026, 0.007). (5)There was no statistical difference of response rates in acute lymphocytic leukemia (ALL) and non-Hodgkin lymphoma (NHL) patients between the two groups ((ALL)=0.842; (NHL)=0.866). The modified cell infusion method in CD(19) CAR-T cell treatment reduces the incidence of treatment-related FNHTR. It does not affect the proliferation of CAR-T cells , the grading of CRS and the response rates.
Chimeric Antigen Receptor T-Cell Therapies for Aggressive B-Cell Lymphomas: Current and Future State of the Art.
Abramson Jeremy S,Lunning Matthew,Palomba M Lia
American Society of Clinical Oncology educational book. American Society of Clinical Oncology. Annual Meeting
Aggressive B-cell lymphomas that are primary refractory to, or relapse after, frontline chemoimmunotherapy have a low cure rate with conventional therapies. Although high-dose chemotherapy remains the standard of care at first relapse for sufficiently young and fit patients, fewer than one-quarter of patients with relapsed/refractory disease are cured with this approach. Anti-CD19 chimeric antigen receptor (CAR) T cells have emerged as an effective therapy in patients with multiple relapsed/refractory disease, capable of inducing durable remissions in patients with chemotherapy-refractory disease. Three anti-CD19 CAR T cells for aggressive B-cell lymphoma (axicabtagene ciloleucel, tisagenlecleucel, and lisocabtagene ciloleucel) are either U.S. Food and Drug Administration approved or in late-stage development. All three CAR T cells produce durable remissions in 33%-40% of treated patients. Differences among these products include the specific CAR constructs, costimulatory domains, manufacturing process, dose, and eligibility criteria for their pivotal trials. Notable toxicities include cytokine release syndrome and neurologic toxicities, which are usually treatable and reversible, as well as cytopenias and hypogammaglobulinemia. Incidences of cytokine release syndrome and neurotoxicity differ across CAR T-cell products, related in part to the type of costimulatory domain. Potential mechanisms of resistance include CAR T-cell exhaustion and immune evasion, CD19 antigen loss, and a lack of persistence. Rational combination strategies with CAR T cells are under evaluation, including immune checkpoint inhibitors, immunomodulators, and tyrosine kinase inhibitors. Novel cell products are also being developed and include CAR T cells that target multiple tumor antigens, cytokine-secreting CAR T cells, and gene-edited CAR T cells, among others.
Killing Mechanisms of Chimeric Antigen Receptor (CAR) T Cells.
Benmebarek Mohamed-Reda,Karches Clara Helke,Cadilha Bruno Loureiro,Lesch Stefanie,Endres Stefan,Kobold Sebastian
International journal of molecular sciences
Effective adoptive T cell therapy (ACT) comprises the killing of cancer cells through the therapeutic use of transferred T cells. One of the main ACT approaches is chimeric antigen receptor (CAR) T cell therapy. CAR T cells mediate MHC-unrestricted tumor cell killing by enabling T cells to bind target cell surface antigens through a single-chain variable fragment (scFv) recognition domain. Upon engagement, CAR T cells form a non-classical immune synapse (IS), required for their effector function. These cells then mediate their anti-tumoral effects through the perforin and granzyme axis, the Fas and Fas ligand axis, as well as the release of cytokines to sensitize the tumor stroma. Their persistence in the host and functional outputs are tightly dependent on the receptor's individual components-scFv, spacer domain, and costimulatory domains-and how said component functions converge to augment CAR T cell performance. In this review, we bring forth the successes and limitations of CAR T cell therapy. We delve further into the current understanding of how CAR T cells are designed to function, survive, and ultimately mediate their anti-tumoral effects.
Construction and functional characterization of a fully human anti-CD19 chimeric antigen receptor (huCAR)-expressing primary human T cells.
Mirzaei Hamid Reza,Jamali Arezoo,Jafarzadeh Leila,Masoumi Elham,Alishah Khadijeh,Fallah Mehrjardi Keyvan,Emami Seyed Amir Hossein,Noorbakhsh Farshid,Till Brian G,Hadjati Jamshid
Journal of cellular physiology
Although remarkable results have been attained by adoptively transferring T cells expressing fully murine and/or humanized anti-CD19 chimeric antigen receptors (CARs) to treat B cell malignancies, evidence of human anti-mouse immune responses against CARs provides a rationale for the development of less immunogenic CARs. By developing a fully human CAR (huCAR), these human anti-mouse immune responses are likely eliminated. This, perhaps, not only increases the persistence of anti-CD19 CAR T cells-thereby reducing the risk of tumor relapse-but also facilitates administration of multiple, temporally separated doses of CAR T cells to the same recipient. To these ends, we have designed and constructed a second-generation fully human anti-CD19 CAR (or huCAR19) containing a fully human single-chain variable fragment (ScFv) fused with a CD8a hinge, a 4-1BB transmembrane domain and intracellular T cell signaling domains of 4-1BB and CD3z. T cells expressing this CAR specifically recognized and lysed CD19 target cells produced cytokines and proliferated in vitro. Moreover, cell volume data revealed that our huCAR construct cannot induce antigen-independent tonic signaling in the absence of cognate antigen. Considering our results, our anti-CD19 huCAR may overcome issues of transgene immunogenicity that plague trials utilizing CARs containing mouse-derived ScFvs. These results suggest that this huCAR19 be safely and effectively applied for adaptive T cell immunotherapy in clinical practice.
Chimeric antigen receptor T-cell therapy for cancer: a basic research-oriented perspective.
Han Chungyong,Kwon Byoung S
Chimeric antigen receptor (CAR) T cells have outstanding therapeutic potential for treating blood cancers. The prospects for this technology have accelerated basic research, clinical translation and Big Pharma's investment in the field of T-cell therapeutics. This interest has led to the discovery of key factors that affect CAR T-cell efficacy and play pivotal roles in T-cell immunology. Herein, we introduce advances in adoptive immunotherapy and the birth of CAR T cells, and review CAR T-cell studies that focus on three important features: CAR constructs, target antigens and T-cell phenotypes. At last, we highlight novel strategies that overcome the tumor microenvironment and circumvent CAR T-cell side effects, and consider the future direction of CAR T-cell development.
[Efficacy and safety of CD19 chimeric antigen receptor T cells for the treatment of 22 patients with B-cell lymphoma].
Xiao X,Jiang Y Y,Cao Y Q,Li Q,Jin X,Meng J X,Sui T,Li Y M,Zhao M F
Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi
To investigate the efficacy and safety of CD19 chimeric antigen receptor T (CAR-T) lymphocytes for the treatment of B cell lymphoma. A total of 22 patients with B-cell lymphoma from February 1, 2017 to July 1, 2018 were reviewed to evaluate the efficacy and adverse reactions of CD19 CAR-T. Of 22 patients with B-cell lymphoma received CD19 CAR-T cells, the median dose of CAR-T cells was 7.2 (2.0-12.0) ×10/kg. Nine of 12 cases of relapse refractory patients were overall response. Complete remission (CR) occurred in 2 of 12 patients, partial remission (PR) in 7 of 12 patients. The overall response in minor residual disease positive (MRD) group was 8 of 10 patients. CD19 CAR-T cells proliferated in vivo and were detectable in the blood of patients. The peak timepoints of CAR-T cells proliferated in the relapsed refractory and MRD positive groups were 12 (5-19) and 4.5 (1-12) days after treatment respectively, and among peripheral blood cells, CAR-T cells accounted for 10.10% (3.55%-24.74%) and 4.02% (2.23%-28.60%) of T lymphocytes respectively. The MRD positive patients achieved sustained remissions during a median follow-up of 8 months (rang 3-18 months) . None of all the patients relapsed during a median follow-up time of 10 months (3-18 months) . However, 7 PR responders of the relapsed refractory patients maintained a good condition for 1.5-6.0 months. One patient bridged to hematopoietic stem cell transplantation, another one sustained remission for 12 months. Cytokine-release syndrome (CRS) occurred in 14 patients with grade 1-2 CRS in MRD positive group and grade 3 CRS in relapsed refractory group. CAR-T cell therapy not only played a role in the rescue treatment of relapsed and refractory patients, but also produced a surprising effect in the consolidation and maintenance of B-cell lymphoma. CD19 CAR-T cells might be more effective in the treatment of MRD positive B-cell lymphoma patients than in the refractory or relapsed cases. High response rate was observed with fewer adverse reactions.
Evidence of long-lasting anti-CD19 activity of engrafted CD19 chimeric antigen receptor-modified T cells in a phase I study targeting pediatrics with acute lymphoblastic leukemia.
Ma Futian,Ho Jin-Yuan,Du Huan,Xuan Fan,Wu Xiaoli,Wang Qinglong,Wang Lin,Liu Ying,Ba Min,Wang Yizhuo,Luo Jianmin,Li Jianqiang
Ninety percent of relapse/refractory B-cell acute lymphatic leukemia (R/R B-ALL) patients can achieve complete remission (CR) after CD19-targeting chimeric antigen receptor T (CAR-T) cell therapy. However, around 50% of them relapse in 1 year. Persistent CAR-T cell engraftment is considered as the key to remain durable remission. Here, we initiated a phase I study to treat 10 pediatric B-ALL patients using a CD19-targeted second generation CAR with a 4-1BB intracellular costimulatory domain. All patients received a standard fludarabine and cyclophosphamide (FC) preconditioning regiment, followed by a CAR-T infusion with a median number of 0.5 (0.3-1.58) × 10 CAR+ T cells/kg. The pretreatment tumor burdens were high with a median bone marrow (BM) blasts percentage of 59.2% (7.31%-86.2%), excluding one patient only with brain infiltration of leukemia cells (0% BM blasts). The initial CR rate was 80% (n = 8/10). Four patients (40%) experienced serious (grade > 2) cytokine release syndrome (CRS) and three patients (30%) with obvious neurotoxicity. Monthly assessments of CD19+ minimal residual disease (MRD) and CAR-T engraftment demonstrated the anti-CD19 activity of long-term engrafted CAR-T cell clones in one patient for more than 2 years.
Comparative analysis and optimization of protocols for producing recombinant lentivirus carrying the anti-Her2 chimeric antigen receptor gene.
Yuan Weihua,Chen Jie,Cao Ying,Yang Lingcong,Shen Luxi,Bian Qi,Bin Shufang,Li Panyuan,Cao Jiawei,Fang Hezhi,Gu Haihua,Li Hongzhi
The journal of gene medicine
BACKGROUND:The production of anti-Her2 chimeric antigen receptor (CAR) T cells needs to be optimized to make it a reliable therapy. METHODS:Three types of lentiviral vectors expressing anti-Her2 CAR together with packaging plasmids were co-transfected into 293 T-17 cells. The vector with the best packaging efficiency was selected, and the packaging cell culture system and packaging plasmid system were optimized. Centrifugation speed was optimized for the concentration of lentivirus stock. The various purification methods used included membrane filtration, centrifugation with a sucrose cushion and the novelly-designed instantaneous high-speed centrifugation. The recombinant lentiviruses were transduced into human peripheral T cells with an optimized multiplicity of infection (MOI). CAR expression levels by three vectors and the efficacy of CAR-T cells were compared. RESULTS:When co-transfected, packaging cells in suspension were better than the commonly used adherent culture condition, with the packaging system psPAX2/pMD2.G being better than pCMV-dR8.91/pVSV-G. The optimal centrifugation speed for concentration was 20 000 g, rather than the generally used ultra-speed. Importantly, adding instantaneous centrifugation for purification significantly increased human peripheral T cell viability (from 13.25% to 62.80%), which is a technical breakthrough for CAR-T cell preparation. The best MOI value for transducing human peripheral T cells was 40. pLVX-EF1a-CAR-IRES-ZsGreen1 expressed the highest level of CAR in human peripheral T cells and the cytotoxicity of CAR-T cells reached 63.56%. CONCLUSIONS:We optimized the preparation of recombinant lentivirus that can express third-generation anti-Her2 CAR in T cells, which should lay the foundation for improving the efficacy of CAR-T cells with respect to killing target cells.
Building upon the success of CART19: chimeric antigen receptor T cells for hematologic malignancies.
Rotolo Antonia,Karadimitris Anastasios,Ruella Marco
Leukemia & lymphoma
Chimeric antigen receptor T cell (CART) therapy has dramatically changed the therapeutic prospects for B cell malignancies. Over the last decade CD19-redirected CART have demonstrated the ability to induce deep, long-lasting remissions and possibly cure patients with relapsing B cell neoplasms. Such impressive results with CART19 fostered efforts to expand this technology to other incurable malignancies that naturally do not express CD19, such as acute myeloid leukemia (AML), Hodgkin lymphoma (HL) and multiple myeloma (MM). However, to reach this goal, several hurdles have to be overcome, in particular: (i) the apparent lack of suitable targets as effective as CD19; (ii) the immunosuppressive tumor microenvironment; (iii) intra-tumoral heterogeneity and antigen-negative relapses. Therefore, new strategies that allow safer and more potent CART platforms are under development and may provide grounds for new exciting breakthroughs in the field.
Chimeric antigen-receptor T-cell therapy for hematological malignancies and solid tumors: Clinical data to date, current limitations and perspectives.
Gauthier J,Yakoub-Agha I
Current research in translational medicine
Progress in our understanding of basic immunology along with the advent of bioengineering technologies have made possible the production of human T-cells expressing Chimeric Antigen Receptors (CAR T-cells). These CAR T-cells are designed to target specific antigens presented by cancer cells. Once CARs are bound to these antigens, CAR T-cells get activated and can initiate potent anti-tumor effects. We will here overview the bioengineering advances which made possible the clinical application of CAR T-cell therapy. We will review the data to date regarding anti-CD19 CAR T-cell therapy for acute lymphoblastic leukemia, non-Hodgkin lymphomas, and chronic lymphocytic leukemia. Besides CD19, CAR T-cells directed against the B-cell maturation antigen have also shown encouraging results to treat patients with refractory multiple myeloma. The more limited body of clinical research in the field of solid tumors will also be reviewed. Moreover, we will elaborate on the main toxicities of limitations of CAR T-cell therapy, namely cytokine release syndrome and neurotoxicity. While enjoying an undeniable hype, CAR T-cell therapy bears significant limitations. We will conclude by exposing the possible approaches to make CAR T-cells safer and more efficient beyond the CD19 target.
Retargeting CD19 Chimeric Antigen Receptor T Cells via Engineered CD19-Fusion Proteins.
Klesmith Justin R,Su Lihe,Wu Lan,Schrack Ian A,Dufort Fay J,Birt Alyssa,Ambrose Christine,Hackel Benjamin J,Lobb Roy R,Rennert Paul D
CD19-targeted chimeric antigen receptor (CAR) T-cells (CAR19s) show remarkable efficacy in the treatment of relapsed/refractory acute lymphocytic leukemia and Non-Hodgkin's lymphoma. However, the use of CAR T-cell therapy against CD19-negative hematological cancers and solid tumors has been challenging. We propose CD19-fusion proteins (CD19-FPs) to leverage the benefits of CAR19s while retargeting this validated cellular therapy to alternative tumor antigens. We demonstrate the ability of a fusion of CD19 extracellular domain (ECD) and a human epidermal growth factor receptor 2 (HER2) single-chain antibody fragment to retarget CAR19s to kill HER2 CD19 tumor cells. To enhance the modularity of this technology, we engineered a more robust CD19 ECD via deep mutational scanning with yeast display and flow cytometric selections for improved protease resistance and anti-CD19 antibody binding. These enhanced CD19 ECDs significantly increase, and in some cases recover, fusion protein expression while maintaining target antigen affinity. Importantly, CD19-FPs retarget CAR19s to kill tumor cells expressing multiple distinct antigens, including HER2, CD20, EGFR, BCMA, and Clec12A as N- or C-terminal fusions and linked to both antibody fragments and fibronectin ligands. This study provides fundamental insights into CD19 sequence-function relationships and defines a flexible and modular platform to retarget CAR19s to any tumor antigen.
Anti-CD19 chimeric antigen receptors T cells for the treatment of relapsed or refractory E2A-PBX1 positive acute lymphoblastic leukemia: report of three cases.
Zhang Jian,Yang Fei,Qiu Hui-Ying,Wu Qian,Kong Dan-Qing,Zhou Jin,Han Yue,Wu De-Pei
Leukemia & lymphoma
Patients with relapsed or refractory E2A-PBX1 positive acute B lymphoblastic leukemia (B-ALL) receiving anti-CD19 chimeric antigen receptor T cells (CAR-T) were retrospectively assessed to evaluate the efficacy and safety of disease burden on outcomes and to identify predictive variables. Of the three case patients, case 1 relapsed after hematopoietic stem cell transplantation. After being treated with anti-CD19 CAR-T, the patient showed minimal residual disease (MRD), and his fusion genes turned negative. Case 2, who suffered refractory leukemia, received anti-CD19 CAR-T treatment in an attempt to remove the MRD before transplantation. She showed MRD, and her fusion genes turned negative. Case 3 received anti-CD19 CAR-T treatment because of relapse after allo-SCT at the molecular level. After infusion, she developed severe pneumonia accompanied with the indication that the leukemia had progressed. Our findings suggest that anti-CD19 CAR-T cells therapy with a remarkable MRD eradicating ability might be an effective option for patients with relapsed and refractory E2A-PBX1 positive B-ALL.