CAR T-cells in acute lymphoblastic leukemia: Current results.

The marketing authorization of tisagenlecleucel, a 2nd generation of CD19-directed CAR T-cells, containing the 4-1 BB co-stimulatory domain, in 2017 in USA and in 2018 in EU, has revolutionized the therapeutic strategy in advanced B-cell acute lymphoblastic leukemia (B-ALL) in children, adolescents and young adults (AYAs) with relapsed or refractory disease. This innovative treatment, based on a "living drug", has shown very impressive short-term responses. However, safety profile and complex logistics require high expertise centers and tight collaborations between addressing and treating centers. Current research is exploring the possibility to move to first line ALL with high-risk features and/or first high-risk relapse. More efficient CAR T-cells products, are still lacking to counteract the escape mechanisms already described. Moreover, to define the bridge-to-CAR time for each patient remains a challenge to obtain optimal disease burden allowing expansion and persistence of CAR T-cells. Also difficult is to identify patients who will benefit from further therapy after infusion, such as allogeneic HSCT or may be immuno-modulatory treatment. Finally, CAR T-cells directed against T-ALL are only in their beginning but require more complex engineering process to avoid T- cell immune-deficiency or fratricide.

Mucosal-associated invariant T (MAIT) cells, a new source of universal immune cells for chimeric antigen receptor (CAR)-cell therapy.

Treatment of hematological malignancies by autologous T cells expressing a chimeric antigen receptor (CAR) is a breakthrough in the field of cancer immunotherapy. As CAR-T cells are entering advanced phases of clinical development, there is a need to develop universal, ready-to-use products using immune cells from healthy donors, to reduce time to treatment, improve response rate and finally reduce the cost of production. Mucosal-associated invariant T cells (MAIT) are unconventional T cells which recognize microbial-derived riboflavin derivatives presented by the conserved MR1 molecule and are endowed with potent effector functions. Because they are not selected by classical MHC/peptide complexes and express a semi-invariant T cell receptor, MAIT cells do not mediate alloreactivity, prompting their use as a new source of universal effector cells for allogeneic CAR-T cell therapy without the need to inactivate their endogenous TCR. We produced CD19-CAR MAIT cells as proof-of-concept allowing subsequent head-to-head comparison with currently used CD19-CAR T cells. We demonstrated their anti-tumor efficacy in vitro and their capacity to engraft without mediating GVHD in preclinical immunodeficient mouse models. Universal, off-the-shelf CAR-MAIT cells could provide a suitable alternative to current autologous CAR-T cells to treat patients regardless of HLA disparity, without production delay, enabling a cost-effective manufacturing model for large-scale clinical application.

Perspectives on outpatient administration of CAR-T cell therapy in aggressive B-cell lymphoma and acute lymphoblastic leukemia.

Chimeric antigen receptor (CAR) T-cell therapies that specifically target the CD19 antigen have emerged as a highly effective treatment option in patients with refractory B-cell hematological malignancies. Safety and efficacy outcomes from the pivotal prospective clinical trials of axicabtagene ciloleucel, tisagenlecleucel and lisocabtagene maraleucel and the retrospective, postmarketing, real-world analyses have confirmed high response rates and durable remissions in patients who had failed multiple lines of therapy and had no meaningful treatment options. Although initially administered in the inpatient setting, there has been a growing interest in delivering CAR-T cell therapy in the outpatient setting; however, this has not been adopted as standard clinical practice for multiple reasons, including logistic and reimbursement issues. CAR-T cell therapy requires a multidisciplinary approach and coordination, particularly if given in an outpatient setting. The ability to monitor patients closely is necessary and proper protocols must be established to respond to clinical changes to ensure efficient, effective and rapid evaluation either in the clinic or emergency department for management decisions regarding fever, sepsis, cytokine release syndrome and neurological events, specifically immune effector cell-associated neurotoxicity syndrome. This review presents the authors' institutional experience with the preparation and delivery of outpatient CD19-directed CAR-T cell therapy.

Immunophenotypic shift in the B-cell precursors from regenerating bone marrow samples: A critical consideration for measurable residual disease assessment in B-lymphoblastic leukemia.

Accurate knowledge of expression patterns/levels of commonly used MRD markers in regenerative normal-B-cell-precursors (BCP) is highly desirable to distinguish leukemic-blasts from regenerative-BCP for multicolor flow cytometry (MFC)-based measurable residual disease (MRD) assessment in B-lymphoblastic leukemia (B-ALL). However, the data highlighting therapy-related immunophenotypic-shift in regenerative-BCPs is scarce and limited to small cohort. Herein, we report the in-depth evaluation of immunophenotypic shift in regenerative-BCPs from a large cohort of BALL-MRD samples. Ten-color MFC-MRD analysis was performed in pediatric-BALL at the end-of-induction (EOI), end-of-consolidation (EOC), and subsequent-follow-up (SFU) time-points. We studied normalized-mean fluorescent intensity (nMFI) and coefficient-of-variation of immunofluorescence (CVIF) of CD10, CD19, CD20, CD34, CD38, and CD45 expression in regenerative-BCP (early, BCP1 and late, BCP2) from 200 BALL-MRD samples, and compared them with BCP from 15 regenerating control (RC) TALL-MRD samples and 20 treatment-naïve bone-marrow control (TNSC) samples. Regenerative-BCP1 showed downregulation in CD10 and CD34 expression with increased CVIF and reduced nMFI (p < 0.001), upregulation of CD20 with increased nMFI (p = 0.014) and heterogeneous CD45 expression with increased CVIF (p < 0.001). Immunophenotypic shift was less pronounced in the BCP2 compared to BCP1 compartment with increased CVIF in all but CD45 (p < 0.05) and reduced nMFI only in CD45 expression (p = 0.005). Downregulation of CD10/CD34 and upregulation of CD20 was higher at EOI than EOC and SFU time-points (p < 0.001). Regenerative-BCPs are characterized by the significant immunophenotypic shift in commonly used B-ALL-MRD markers, especially CD10 and CD34 expression, as compared to treatment-naïve BCPs. Therefore, the templates/database for BMRD analysis must be developed using regenerative-BCP.

Manufacturing and Management of CAR T-Cell Therapy in "COVID-19's Time": Central Versus Point of Care Proposals.

The COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), has generated a significant repercussion on the administration of adoptive cell therapies, including chimeric antigen receptor (CAR) T-cells. The closing of borders, the reduction of people transit and the confinement of the population has affected the supply chains of these life-saving medical products. The aim of this mini-review is to focus on how the COVID-19 pandemic has affected CAR T-cell therapy and taking into consideration the differences between the large-scale centralized productions for the pharmaceutical industry versus product manufacturing in the academic/hospital environment. We also review different aspects of CAR T-cell therapy and our managerial experience of patient selection, resource prioritization and some practical aspects to consider for safe administration. Although hospitals have been forced to change their usual workflows to cope with the saturation of health services by hospitalized patients, we recommend centers to continue offering this potentially curative treatment for patients with relapsed/refractory hematologic malignancies. Consequently, we propose appropriate selection criteria, early intervention to attenuate neurotoxicity or cytokine release syndrome with tocilizumab and prophylactic/preventive strategies to prevent infection. These considerations may apply to other emerging adoptive cell treatments and the corresponding manufacturing processes.

Concepts in immuno-oncology: tackling B cell malignancies with CD19-directed bispecific T cell engager therapies.

The B cell surface antigen CD19 is a target for treating B cell malignancies, such as B cell precursor acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma. The BiTE® immuno-oncology platform includes blinatumomab, which is approved for relapsed/refractory B cell precursor acute lymphoblastic leukemia and B cell precursor acute lymphoblastic leukemia with minimal residual disease. Blinatumomab is also being evaluated in combination with other agents (tyrosine kinase inhibitors, checkpoint inhibitors, and chemotherapy) in various treatment settings, including frontline protocols. An extended half-life BiTE molecule is also under investigation. Patients receiving blinatumomab may experience cytokine release syndrome and neurotoxicity; however, these events may be less frequent and severe than in patients receiving other CD19-targeted immunotherapies, such as chimeric antigen receptor T cell therapy. We review BiTE technology for treating malignancies that express CD19, analyzing the benefits and limitations of this bispecific T cell engager platform from clinical experience with blinatumomab.

Preclinical Assessment of Efficacy and Safety Analysis of CAR-T Cells (ISIKOK-19) Targeting CD19-Expressing B-Cells for the First Turkish Academic Clinical Trial with Relapsed/Refractory ALL and NHL Patients

Objective: Relapsed and refractory CD19-positive B-cell acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL) are the focus of studies on hematological cancers. Treatment of these malignancies has undergone recent transformation with the development of new gene therapy and molecular biology techniques, which are safer and well-tolerated therapeutic approaches. The CD19 antigen is the most studied therapeutic target in these hematological cancers. This study reports the results of clinical-grade production, quality control, and in vivo efficacy processes of ISIKOK-19 cells as the first academic clinical trial of CAR-T cells targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey. Materials and Methods: We used a lentiviral vector encoding the CD19 antigen-specific antibody head (FMC63) conjugated with the CD8-CD28-CD3ζ sequence as a chimeric antigen receptor (CAR) along with a truncated form of EGFR (EGFRt) on human T-lymphocytes (CAR-T). We preclinically assessed the efficacy and safety of the manufactured CAR-T cells, namely ISIKOK-19, from both healthy donors' and ALL/NHL patients' peripheral blood mononuclear cells. Results: We showed significant enhancement of CAR lentivirus transduction efficacy in T-cells using BX-795, an inhibitor of the signaling molecule TBK1/IKKƐ, in order to cut the cost of CAR-T cell production. In addition, ISIKOK-19 cells demonstrated a significantly high level of cytotoxicity specifically against a CD19+ B-lymphocyte cancer model, RAJI cells, in NOD/SCID mice. Conclusion: This is the first report of preclinical assessment of efficacy and safety analysis of CAR-T cells (ISIKOK-19) targeting CD19-expressing B cells in relapsed/refractory ALL and NHL patients in Turkey.

Cost-effectiveness of Anti-CD19 chimeric antigen receptor T-Cell therapy in pediatric relapsed/refractory B-cell acute lymphoblastic leukemia. A societal view.

INTRODUCTION: In several studies, the chimeric antigen receptor T-cell therapy tisagenlecleucel demonstrated encouraging rates of remission and lasting survival benefits in pediatric patients with relapsed/refractory (r/r) acute lymphoblastic leukemia (ALL). We assessed the cost-effectiveness of tisagenlecleucel (list price: 320 000 EUR) among these patients when compared to clofarabine monotherapy (Clo-M), clofarabine combination therapy (Clo-C), and blinatumomab (Blina) from both a healthcare and a societal perspective. We also assessed future medical and future non-medical consumption costs. METHODS: A three-state partitioned survival model was used to simulate a cohort of pediatric patients (12 years of age) through different disease states until the end of life (lifetime horizon). Relevant outcomes were life years, quality-adjusted life years (QALYs), healthcare costs, societal costs, and the incremental cost-effectiveness ratio (ICER). Uncertainty was explored through deterministic and probabilistic sensitivity analyses as well as through several scenario analyzes. RESULTS: Total discounted costs for tisagenlecleucel were 552 679 EUR from a societal perspective, which was much higher than the total discounted costs from a healthcare perspective (ie, 409 563 EUR). Total discounted societal costs for the comparator regimens ranged between 160 803 EUR for Clo-M and 267 259 EUR for Blina. Highest QALYs were estimated for tisagenlecleucel (11.26), followed by Blina (2.25), Clo-C (1.70) and Clo-M (0.74). Discounted societal ICERs of tisagenlecleucel ranged between 31 682 EUR/QALY for Blina and 37 531 EUR/QALY for Clo-C and were considered cost-effective with a willingness-to-pay (WTP) threshold of 80 000 EUR/QALY. None of the scenarios exceeded this threshold, and more than 98% of the iterations in the probabilistic sensitivity analysis were cost-effective. DISCUSSION: At the current price and WTP threshold, tisagenlecleucel is cost-effective from both a healthcare and a societal perspective. Nevertheless, long-term effectiveness data are needed to validate the several assumptions that were necessary for this model.

Assessment and management of cytokine release syndrome and neurotoxicity following CD19 CAR-T cell therapy.

Introduction: The success of CD19 chimeric antigen receptor (CAR)-T cell therapy for treatment of CD19 positive malignancies has led to the FDA approval of two CD19 CAR-T cell products, tisagenlecleucel and axicabtagene ciloleucel, and ongoing clinical trials of new products. Cytokine release syndrome (CRS) and neurotoxicity are common toxicities associated with CD19 CAR-T cell therapies.Areas covered: This review will discuss CRS and neurotoxicity associated with CD19 CAR-T cell therapies, including clinical presentation, risk factors, pathophysiology, and therapeutic or prophylactic interventions.Expert opinion: In conjunction with improved understanding of the pathophysiology of CRS and neurotoxicity, we expect that the recent development of consensus guidelines for the evaluation of these toxicities will enhance management of patients undergoing CD19 CAR-T cell therapies.

Chimeric Antigen Receptor T Cell Therapy for Acute Lymphoblastic Leukemia.

OPINION STATEMENT: Chimeric receptor antigen (CAR) T cells are an innovative cellular immunotherapeutic approach that involves genetic modification of T cells to express CAR targeting tumor antigen. Prior to the development of CAR-T, the only potential cure for patients with relapsed or refractory (RR) acute lymphoblastic leukemia (ALL) was allogeneic hematopoietic stem cell transplantation (HSCT). Several CAR-T cell products have been studied in prospective clinical trials which ultimately have resulted in the approval of one anti-CD19 CAR-T cell product in pediatric RR ALL: tisagenlecleucel (CD3ζ and 41BB). While some patients achieve durable responses with CAR-T, lack of response and relapse remains clinical challenges. Reasons for sub-optimal response include lack of CAR-T cell persistence and target antigen down-regulation. Future CARs are under development to improve long-term persistence and to be able to overcome resistance mechanisms associated with the disease and the hostile tumor microenvironment. With evolving understanding about CARs and new constructs under investigation, there is optimism that future products will improve the safety and efficacy from the current standard of care.

Development and Use of the Anti-CD19 Chimeric Antigen Receptor T-Cell Therapy Axicabtagene Ciloleucel in Large B-Cell Lymphoma: A Review.

Importance: Axicabtagene ciloleucel, an anti-CD19-CD28-CD3ζ chimeric antigen receptor T-cell therapy, was the first US Food and Drug Administration-approved, genetically engineered T-cell therapy for adults with relapsed or refractory large B-cell lymphoma (LBCL) after 2 or more lines of systemic therapy. There has not been a US Food and Drug Administration-approved product for these cancers in more than 4 decades. Observations: Unlike traditional anticancer therapies, axicabtagene ciloleucel is a patient-specific, live-cell product that has unique requirements for manufacturing, shipping, and storage, as well as for its administration and management of its adverse events. In addition, axicabtagene ciloleucel has demonstrated efficacy in patients with refractory LBCL. This review presents a timeline of the rapid clinical development of axicabtagene ciloleucel from bench to bedside, highlights how axicabtagene ciloleucel satisfies an unmet medical need for treatment of refractory LBCL, outlines the logistics of the production process and administration of axicabtagene ciloleucel, describes its mechanism of action, and summarizes the results of the pivotal study. This review also provides a survey of adverse events, with attention to the kinetics of their clinical presentation; discusses the management of adverse events; and offers suggestions for appropriate patient selection for safe administration of axicabtagene ciloleucel. Conclusions and Relevance: The integration of axicabtagene ciloleucel therapy into standard-of-care practice for relapsed/refractory LBCL is the beginning of a paradigm shift in the treatment of patients with LBCL and is likely to lead to improvements in their survival and curability. Timely referral to centers offering the therapy is necessary for optimal patient outcomes.

Autologous cryopreserved leukapheresis cellular material for chimeric antigen receptor-T cell manufacture.

Tisagenlecleucel, a CD19-specific autologous chimeric antigen receptor (CAR)-T cell therapy, is efficacious for the treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and diffuse large B-cell lymphoma. The tisagenlecleucel manufacturing process was initially developed in an academic setting and subsequently transferred to industry for qualification, validation and scaling up for global clinical trials and commercial distribution. Use of fresh leukapheresis material was recognized early on in the transfer process as a challenge with regard to establishing a global supply chain. To maximize manufacturing success rates and to overcome logistical challenges, cryopreservation was adapted into the Novartis manufacturing process from the beginning of clinical trials. Tisagenlecleucel manufactured in centralized facilities with cryopreserved leukapheresis material has been used successfully in global clinical trials at more than 50 clinical centers in 12 countries. Cryopreservation provides flexibility in scheduling leukapheresis when the patient's health is optimal to provide T cells; it also provides protection from external factors, such as shipping delays, and removes manufacturing time constraints. Several studies were performed to establish comparability of fresh versus cryopreserved leukapheresis material, to evaluate and optimize the cryopreservation process, to determine the optimal temperature and maximum hold time prior to cryopreservation and to determine the optimal temperature range for shipment and storage. Using the current validated industry manufacturing process, high success rates were achieved with regard to manufacturing tisagenlecleucel batches that met specifications and were released to patients. Consistent product quality and positive clinical outcomes support the use of cryopreserved non-mobilized peripheral mononuclear blood cells collected using leukapheresis for CAR-T cell manufacturing.

Low-cost generation of Good Manufacturing Practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials.

BACKGROUND AIMS: Protocols for the production of CD19-specific chimeric antigen receptor (CAR19) T cells are often complex and expensive because of the use of retroviral and lentiviral vectors or the need for CAR19 T-cell enrichment. We aimed to simplify the generation of CAR19 T cells from the peripheral blood of normal donors and patients using the piggyBac transposon system of gene modification. METHODS: We varied electroporation voltage, cytokines and stimulation conditions for the generation and expansion of CAR19 T cells over a 3-week culture period. RESULTS: Using optimized electroporation voltage, interleukin-15 alone and co-culturing CAR T cells with peripheral blood mononuclear cells, we were able to expand CAR19 T-cell cultures by up to 765-fold over 3 weeks in normal donors and 180-fold in patients with B-cell malignancies. Final median CAR19 expression of 72% was seen in normal donors, and 81% was seen in patients with acute lymphoblastic leukaemia, chronic lymphocytic leukemia or non-Hodgkin lymphoma. CAR19 T cells produced interferon gamma on stimulation with CD19(+) cell lines and efficiently lysed both CD19(+) cell lines and primary leukemia cells. In addition, combining CAR expression with an inducible caspase safety switch allowed elimination of CAR19 T cells by the application of a small molecule dimerizer. DISCUSSION: We have produced a simple, inexpensive and easily adoptable protocol for the generation of CAR19 T cells suitable for use in clinical trials using the piggyBac transposon system. This provides a robust platform for further enhancing the T-cell product and testing new CAR technologies.

Single-step purification of bispecific monoclonal antibodies for immunotherapeutic use by hydrophobic interaction chromatography.

A method for large scale production and single-step purification of bispecific antibodies is described. Hybrid-hybridomas were grown in hollow-fibre bioreactors with an average yield of 8 to 12 g of immunoglobulin per month. Bispecific antibodies were purified from the bioreactor supernatant by hydrophobic interaction chromatography which resolves bispecific antibodies, monospecific immunoglobulins, and culture medium supplements in one single chromatographic step. Proteins were analyzed by ELISA, SDS-PAGE, isoelectric focussing, indirect fluorescence staining, CTL-stimulation and T-cell proliferation assays. Finally, antibody preparations were checked for the presence of endotoxin and mouse DNA. Our results suggest that functional bispecific antibodies for use in therapeutic applications can be batch purified from bioreactor harvest by hydrophobic interaction chromatography in a single step. Compared to other methods such as affinity chromatography (protein A/G), ion-exchange or hydroxyapatite chromatography, our protocol offers a substantial reduction in labor time, cost, protein loss, and risk of contamination.