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CD19-targeted chimeric antigen receptors (CAR) T cells are one of the most remarkable cellular therapies for managing B cell malignancies. However, long-term disease-free survival is still a challenge to overcome. Here, we evaluated the influence of different hinge, transmembrane (TM), and costimulatory CAR domains, as well as manufacturing conditions, cellular product type, doses, patient's age, and tumor types on the clinical outcomes of patients with B cell cancers treated with CD19 CAR T cells. The primary outcome was defined as the best complete response (BCR), and the secondary outcomes were the best objective response (BOR) and 12-month overall survival (OS). The covariates considered were the type of hinge, TM, and costimulatory domains in the CAR, CAR T cell manufacturing conditions, cell population transduced with the CAR, the number of CAR T cell infusions, amount of CAR T cells injected/Kg, CD19 CAR type (name), tumor type, and age. Fifty-six studies (3493 patients) were included in the systematic review and 46 (3421 patients) in the meta-analysis. The overall BCR rate was 56%, with 60% OS and 75% BOR. Younger patients displayed remarkably higher BCR prevalence without differences in OS. The presence of CD28 in the CAR's hinge, TM, and costimulatory domains improved all outcomes evaluated. Doses from one to 4.9 million cells/kg resulted in better clinical outcomes. Our data also suggest that regardless of whether patients have had high objective responses, they might have survival benefits from CD19 CAR T therapy. This meta-analysis is a critical hypothesis-generating instrument, capturing effects in the CD19 CAR T cells literature lacking randomized clinical trials and large observational studies., (© 2024. The Author(s).)

Chimeric antigen receptor (CAR)-T cell therapy is undeniably a promising tool in combating various types of hematological malignancies. However, it is not yet optimal and a significant number of patients experience a lack of response or relapse after the treatment. Therapy improvement requires careful analysis of the occurring problems and a deeper understanding of the reasons that stand behind them. In this review, we summarize the recent knowledge about CAR-T products' clinical performance and discuss diversified approaches taken to improve the major shortcomings of this therapy. Especially, we prioritize the challenges faced by CD19 CAR-T cell-based treatment of B cell-derived malignancies and revise the latest insights about mechanisms mediating therapy resistance. Since the loss of CD19 is one of the major obstacles to the success of CAR-T cell therapy, we present antigens that could be alternatively used for the treatment of various types of B cell-derived cancers.

Chimeric antigen receptor (CAR)-expressing T-cells are without a doubt a breakthrough therapy for hematological malignancies. Despite their success, clinical experience has revealed several challenges, which include relapse after targeting single antigens such as CD19 in the case of B-cell acute lymphoblastic leukemia (B-ALL), and the occurrence of side effects that could be severe in some cases. Therefore, it became clear that improved safety approaches, and targeting multiple antigens, should be considered to further improve CAR T-cell therapy for B-ALL. In this paper, we address both issues by investigating the use of CD10 as a therapeutic target for B-ALL with our switchable UniCAR system. The UniCAR platform is a modular platform that depends on the presence of two elements to function. These include UniCAR T-cells and the target modules (TMs), which cross-link the T-cells to their respective targets on tumor cells. The TMs function as keys that control the switchability of UniCAR T-cells. Here, we demonstrate that UniCAR T-cells, armed with anti-CD10 TM, can efficiently kill B-ALL cell lines, as well as patient-derived B-ALL blasts, thereby highlighting the exciting possibility for using CD10 as an emerging therapeutic target for B-cell malignancies.

Despite the high rates of complete remission following chimeric antigen receptor (CAR) T cell therapy, its full capacity is currently limited by the generation of dysfunctional CAR T cells. Senescent or exhausted CAR T cells possess poor targeting and effector functions, as well as impaired cell proliferation and persistence in vivo. Strategies to detect, prevent or reverse T cell exhaustion are therefore required in order to enhance the effectiveness of CAR T immunotherapy. Here we report that CD19 CAR T cells from non-responding patients with B cell malignancies show enrichment of CD8 + cells with exhausted/senescent phenotype and display a distinct transcriptional signature with dysregulation of genes associated with terminal exhaustion. Furthermore, CAR T cells from non-responding patients exhibit reduced proliferative capacity and decreased IL-2 production in vitro, indicating functional impairment. Overall, our work reveals potential mediators of resistance, paving the way to studies that will enhance the efficacy and durability of CAR T therapy in B cell malignancies.

Cytokine release syndrome (CRS) is the most common on-target toxicity of chimeric antigen receptor (CAR) T cell therapy. However, the prognostic significance of CRS has not been well elucidated. The aim of our study was to evaluate the association between CRS and efficacy after anti-CD19 CAR-T therapy in a retrospective cohort of 22 patients with relapsed/refractory B cell hematological malignancies. The complete remission (CR) rates after CAR-T therapy were 68%, and median value for progression-free survival (PFS) was 6.8 months. Eight of 22 (36.4%) patients showed ≥ grade 2 CRS. Statistical analysis found that patients with ≥ grade 2 CRS had higher CR rates and longer PFS than those with < grade 2 CRS. Moreover, bridging hematopoietic stem cell transplantation was another independent predictor for PFS. These data suggested that appropriate CRS may be beneficial to the efficacy of CAR-T therapy. The Clinical Trial Registration number is NCT03110640, NCT03302403.

Early response could be obtained in most patients with relapsed or refractory B cell lymphoblastic leukemia (R/R B-ALL) treated with chimeric antigen receptor T-cell (CAR-T) therapy, but relapse occurs in some patients. There is no consensus on treatment strategy post CAR-T cell therapy. In this retrospective study of humanized CD19-targeted CAR-T cell (hCART19s) therapy for R/R B-ALL, we analyzed the patients treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT) or received a second hCART19s infusion, and summarized their efficacy and safety. We retrospectively studied 28 R/R B-ALL patients treated with hCART19s in the Affiliated Hospital of Xuzhou Medical University from 2016 to 2020. After the first hCART19s infusion, 10 patients received allo-HSCT (CART+HSCT group), 7 patients received a second hCART19s infusion (CART2 group), and 11 patients did not receive HSCT or a second hCART19s infusion (CART1 group). The safety, efficacy, and long-term survival were analyzed. Of the 28 patients who received hCART19s treatment, 1 patient could not be evaluated for efficacy, and 25 (92.6%) achieved complete remission (CR) with 20 (74.7%) achieving minimal residual disease (MRD) negativity. Seven (25%) patients experienced grade 3-4 CRS, and one died from grade 5 CRS. No patient experienced ≥3 grade ICANS. The incidence of second CR is higher in the CART+HSCT group compared to the CART2 group (100% vs. 42.9%, p = 0.015). The median follow-up time was 1,240 days (range: 709-1,770). Significantly longer overall survival (OS) and leukemia-free survival (LFS) were achieved in the CART+HSCT group (median OS and LFS: not reached, p = 0.006 and 0.001, respectively) compared to the CART2 group (median OS: 482; median LFS: 189) and the CART1 group (median OS: 236; median LFS: 35). In the CART+HSCT group, the incidence of acute graft- versus -host disease (aGVHD) was 30% (3/10), and transplantation-related mortality was 30% (3/10). No chronic GVHD occurred. Multivariate analysis results showed that blasts ≥ 20% in the bone marrow and MRD ≥ 65.6% are independent factors for inferior OS and LFS, respectively, while receiving allo-HSCT is an independent factor associated with both longer OS and LFS. In conclusion, early allo-HSCT after CAR-T therapy can achieve long-term efficacy, and the adverse events are controllable., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Ma, Shen, Chen, Ma, Yan, Shi, Wang, Song, Sun, Cao, Cheng, Zhu, Sun, Li, Li, Zheng, Xu and Sang.)

We performed a phase 1 clinical trial to evaluate outcomes in patients receiving donor-derived CD19-specific chimeric antigen receptor (CAR) T cells for B-cell malignancy that relapsed or persisted after matched related allogeneic hemopoietic stem cell transplant. To overcome the cost and transgene-capacity limitations of traditional viral vectors, CAR T cells were produced using the piggyBac transposon system of genetic modification. Following CAR T-cell infusion, 1 patient developed a gradually enlarging retroperitoneal tumor due to a CAR-expressing CD4+ T-cell lymphoma. Screening of other patients led to the detection, in an asymptomatic patient, of a second CAR T-cell tumor in thoracic para-aortic lymph nodes. Analysis of the first lymphoma showed a high transgene copy number, but no insertion into typical oncogenes. There were also structural changes such as altered genomic copy number and point mutations unrelated to the insertion sites. Transcriptome analysis showed transgene promoter-driven upregulation of transcription of surrounding regions despite insulator sequences surrounding the transgene. However, marked global changes in transcription predominantly correlated with gene copy number rather than insertion sites. In both patients, the CAR T-cell-derived lymphoma progressed and 1 patient died. We describe the first 2 cases of malignant lymphoma derived from CAR gene-modified T cells. Although CAR T cells have an enviable record of safety to date, our results emphasize the need for caution and regular follow-up of CAR T recipients, especially when novel methods of gene transfer are used to create genetically modified immune therapies. This trial was registered at www.anzctr.org.au as ACTRN12617001579381., (© 2021 by The American Society of Hematology.)

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., (Copyright © 2021 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

CAR-T Cells have opened new doors for cellular immunotherapies and provides new therapeutic options for patients with refractory B-cell malignancies, B-cell acute lymphoblastic leukemia and diffuse large B-cel lymphoma. CAR-T Cells have benefited from an accelerated approval procedure in many countries. Indeed, The French health authorities have approved the specialties Tisacel ® and Axicel ®, but additional data including the use of CAR-T Cells in real life were also mandatory. In regard to the scientific interest of the project, LYSA-LYSARC committed itself to prospectively and retrospectively collect information on patients eligible for CAR-T Cells as required by French health authorities. Other academic cooperating groups (GRAALL, IFM, SFCE, FILO and the scientific society SFGM-TC) were associated to this initiative which aims to build a nationwide CAR-T Cells devoted registry, so-called DESCART (Dispositif d'Enregistrement et Suivi des patients traités par CAR-T cells). DESCAR-T is a real-life multicentric registry set up in French sites qualified for CAR-T Cells treatment. DESCAR-T objective is to describe the use of CAR-T Cells in real life. All paediatric and adult patients with hematological malignancy fulfilling CAR-T Cells approval criteria and for whom a CAR-T Cells therapy has been discussed are included from 1 July 2018. Clinical data are directly collected from medical records and patients are treated according to the centers' routine practices. One of the distinctive features of DESCAR-T is its link with HTA for CAR-T Cells s reimbursement by the French public health system. DESCAR-T is the first national registry promoted by an academic group allowing centralized data collection for both academic and HTA/health authorities' purposes., (Copyright © 2021 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Spearheaded by the therapeutic use of chimeric antigen receptors (CARs) targeting CD19, synthetic immunology has entered the clinical arena. CARs are recombinant receptors for antigen that engage cell surface molecules through the variable region of an antibody and signal through arrayed T-cell activating and costimulatory domains. CARs allow redirection of T-cell cytotoxicity against any antigen of choice, independent of MHC expression. Patient T cells engineered to express CARs specific for CD19 have yielded remarkable outcomes in subjects with relapsed/refractory B- cell malignancies, setting off unprecedented interest in T-cell engineering and cell-based cancer immunotherapy. In this review, we present the challenges to extend the use of CAR T cells to solid tumors and other pathologies. We further highlight progress in CAR design, cell manufacturing, and genome editing, which in aggregate hold the promise of generating safer and more effective genetically instructed immunity. Novel engineered cell types, including innate T-cell types, natural killer (NK) cells, macrophages, and induced pluripotent stem cell-derived immune cells, are on the horizon, as are applications of CAR T cells to treat autoimmunity, severe infections, and senescence-associated pathologies., (© 2021 Wiley-VCH GmbH.)

Targeting T cell malignancies with universal CD7-targeting chimeric antigen receptor T cells (UCART7) can lead to profound immune deficiency due to loss of normal T and NK cells. While a small population of endogenous CD7- T cells exists, these cells are unlikely to be able to repopulate the entire immune repertoire after UCART7 treatment, as they are limited in number and proliferative capacity. To rescue T and NK cells after UCART7, we created hematopoietic stem cells genetically deleted for CD7 (CD7-KO HSCs). CD7-KO HSCs were able to engraft immunodeficient mice and differentiate into T and NK cells lacking CD7 expression. CD7-KO T and NK cells could perform effector functions as robustly as control T and NK cells. Furthermore, CD7-KO T cells were phenotypically and functionally distinct from endogenous CD7- T cells, indicating that CD7-KO T cells can supplement immune functions lacking in CD7- T cells. Mice engrafted with CD7-KO HSCs maintained T and NK cell numbers after UCART7 treatment, while these were significantly decreased in control mice. These studies support the development of CD7-KO HSCs to augment host immunity in patients with T cell malignancies after UCART7 treatment.

Rationale: Viruses are the most common pathogens that can cause infection-related non-recurrent death after transplantation, occurring mostly from the early stages of hematopoietic stem cell transplantation (HSCT) to within 1 year after transplantation. Human coronavirus (HCoV)-NL63 is a coronavirus that could cause mortality among patients with underlying disease complications. Serological tests are of limited diagnostic value in immunocompromised hosts and cases of latent infection reactivation. In contrast, macro-genomic high-throughput (DNA and RNA) sequencing allows for rapid and accurate diagnosis of infecting pathogens for targeted treatment., Patient Concerns: In this report, we describe a patient who exhibited acute B-lymphocytic leukemia and developed complicated pulmonary HCoV-NL63 infection after a second allogeneic HSCT (allo-HSCT). Six months after the second allo-HSCT, he developed sudden-onset hyperthermia and cough with decreased oxygen saturation. Chest computed tomography (CT) suggested bilateral multiple rounded ground-glass opacities with the pulmonary lobules as units., Diagnoses: HCoV-NL63 was detected by metagenomic next-generation sequencing (NGS), and HCoV-NL63 viral pneumonia was diagnosed., Interventions: The treatment was mainly based on the use of antiviral therapy, hormone administration, and gamma-globulin., Outcomes: After the therapy, the body temperature returned to normal, the chest CT findings had improved on review, and the viral copy number eventually became negative., Lessons: The latest NGS is an effective method for early infection diagnosis. The HCoV-NL63 virus can cause inflammatory factor storm and alter the neutrophil-to-lymphocyte ratio (NLR). This case suggests that the patient's NLR and cytokine levels could be monitored during the clinical treatment to assess the disease and its treatment outcome in a timely manner., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

BACKGROUNDLittle is known about pathogen-specific humoral immunity after chimeric antigen receptor-modified T (CAR-T) cell therapy for B cell malignancies.METHODSWe conducted a prospective cross-sectional study of CD19-targeted or B cell maturation antigen-targeted (BCMA-targeted) CAR-T cell therapy recipients at least 6 months posttreatment and in remission. We measured pathogen-specific IgG against 12 vaccine-preventable infections and the number of viral and bacterial epitopes to which IgG was detected ("epitope hits") using a serological profiling assay. The primary outcome was the proportion of participants with IgG levels above a threshold correlated with seroprotection for vaccine-preventable infections.RESULTSWe enrolled 65 children and adults a median of 20 months after CD19- (n = 54) or BCMA- (n = 11) CAR-T cell therapy. Among 30 adults without IgG replacement therapy (IGRT) in the prior 16 weeks, 27 (90%) had hypogammaglobulinemia. These individuals had seroprotection to a median of 67% (IQR, 59%-73%) of tested infections. Proportions of participants with seroprotection per pathogen were comparable to population-based studies, but most individuals lacked seroprotection to specific pathogens. Compared with CD19-CAR-T cell recipients, BCMA-CAR-T cell recipients were half as likely to have seroprotection (prevalence ratio, 0.47; 95% CI, 0.18-1.25) and had fewer pathogen-specific epitope hits (mean difference, -90 epitope hits; 95% CI, -157 to -22).CONCLUSIONSeroprotection for vaccine-preventable infections in adult CD19-CAR-T cell recipients was comparable to the general population. BCMA-CAR-T cell recipients had fewer pathogen-specific antibodies. Deficits in both groups support the need for vaccine and immunoglobulin replacement therapy studies.FUNDINGSwiss National Science Foundation (Early Postdoc Mobility grant P2BSP3&#95;188162), NIH/National Cancer Institute (NIH/NCI) (U01CA247548 and P01CA018029), NIH/NCI Cancer Center Support Grants (P30CA0087-48 and P30CA015704-44), American Society for Transplantation and Cellular Therapy, and Juno Therapeutics/BMS.

Chimeric antigen receptor (CAR)-T cell therapy is a genetically-modified cellular immunotherapy that has a current established role in the treatment of relapsed/refractory B-cell acute lymphoblastic leukaemia and diffuse large B-cell lymphoma, with emerging utility in a spectrum of other haematological and solid organ malignancies. It is associated with a number of characteristic toxicities, most notably cytokine release syndrome and neurotoxicity, for which laboratory testing can aid in the prediction of severity and in monitoring. Other toxicities, such as cytopenias/marrow hypoplasia, hypogammagloblinaemia and delayed immune reconstitution are recognised and require monitoring due to the implications for infection risk and prophylaxis. The detection or quantitation of circulating CAR-T can be clinically useful, and is achieved through both direct methods, if available, or indirect/surrogate methods. It is important that the laboratory is informed of the CAR-T therapy and target antigen whenever tissue is collected, both for response assessment and investigation of possible relapse, so that the expression of the relevant antigen can be assessed, in order to distinguish antigen-positive and -negative relapses. Finally, the measurement of circulating tumour DNA has an evolving role in the surveillance of malignancy, with evidence of its utility in the post-CAR-T setting, including predicting patients who will inevitably experience frank relapse, potentially allowing for pre-emptive therapy., (Copyright © 2021 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Chimeric antigen receptor (CAR) T-cells are a new class of cancer treatments manufactured through autologous or allogeneic T cells genetic engineering to induce CAR expression directed against a membrane antigen present at the surface of malignant cells. In Europe, tisagenlecleucel (Kymriah™) has a marketing authorization for the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia in children and young adults and for the relapsed/refractory diffuse large B-cell lymphoma (DLBCL). The marketing authorization for axicabtagene ciloleucel (Yescarta™) is the treatment of relapsed/refractory DLBCL and mediastinal B-cell lymphoma. Both products are "living drugs" and genetically modified autologous T cells directed against CD19 which is an antigen expressed throughout B lymphoid differentiation and on many B malignancies. This collaborative work - part of a series of expert works on the topic - aims to provide practical advice to assist collection facilities that procure the starting material i.e. blood mononuclear cells for autologous CAR T-cell manufacturing., (Copyright © 2021 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Background: The unprecedented efficacy of chimeric antigen receptor T (CAR-T) cell immunotherapy of CD19 + B-cell malignancies has opened a new and useful way for the treatment of malignant tumors. Nonetheless, there are still formidable challenges in the field of CAR-T cell therapy, such as the biodistribution of CAR-T cells in vivo., Methods: NALM-6, a human B-cell acute lymphoblastic leukemia (B-ALL) cell line, was used as target cells. CAR-T cells were injected into a mice model with or without target cells. Then we measured the distribution of CAR-T cells in mice. In addition, an exploratory clinical trial was conducted in 13 r/r B-cell non-Hodgkin lymphoma (B-NHL) patients, who received CAR-T cell infusion. The dynamic changes in patient blood parameters over time after infusion were detected by qPCR and flow cytometry., Results: CAR-T cells still proliferated over time after being infused into the mice without target cells within 2 weeks. However, CAR-T cells did not increase significantly in the presence of target cells within 2 weeks after infusion, but expanded at week 6. In the clinical trial, we found that CAR-T cells peaked at 7-21 days after infusion and lasted for 420 days in peripheral blood of patients. Simultaneously, mild side effects were observed, which could be effectively controlled within 2 months in these patients., Conclusions: CAR-T cells can expand themselves with or without target cells in mice, and persist for a long time in NHL patients without serious side effects., Trial Registration: The registration date of the clinical trial is May 17, 2018 and the trial registration numbers is NCT03528421 .

Purpose of Review: Chimeric antigen receptor T-cell (CAR-T) therapy is a form of adoptive cellular therapy that has revolutionized the treatment landscape in hematologic malignancies, especially B-cell lymphomas. In this review, we will discuss some of the landmark data behind these therapies and then lay out our approach to utilizing this new therapy., Recent Findings: CD19-directed CAR-Ts are the most common type currently used in treatment of relapsed B-cell lymphoid neoplasms. There are currently three FDA-approved products: axicabtagene ciluecel and tisagenlecleucel for the treatment of relapsed/refractory large B-cell lymphoma and pediatric B-cell acute lymphocytic leukemia (tisagenlecleucel only) and brexucabtagene autoleucel for the treatment of relapsed/refractory mantle cell lymphoma. These therapies are associated with distinctive acute toxicities such as cytokine release syndrome and neurotoxicity and chronic toxicities such as cytopenias and hypogammaglobulinemia. CAR-T therapy provides significant potential in the treatment of relapsed B-cell lymphomas despite current limitations. Several novel CAR cell designs are currently being studied in clinical trials which include tandem CAR-Ts, allogeneic CAR-Ts, and CAR-NK cells.

CD19-targeted chimeric antigen receptor-engineered (CD19 CAR) T-cell therapy has shown significant efficacy for relapsed or refractory (R/R) B-cell malignancies. Yet, CD19 CAR T cells fail to induce durable responses in most patients. Second infusions of CD19 CAR T cells (CART2) have been considered as a possible approach to improve outcomes. We analyzed data from 44 patients with R/R B-cell malignancies (acute lymphoblastic leukemia [ALL], n = 14; chronic lymphocytic leukemia [CLL], n = 9; non-Hodgkin lymphoma [NHL], n = 21) who received CART2 on a phase 1/2 trial (NCT01865617) at our institution. Despite a CART2 dose increase in 82% of patients, we observed a low incidence of severe toxicity after CART2 (grade ≥3 cytokine release syndrome, 9%; grade ≥3 neurotoxicity, 11%). After CART2, complete response (CR) was achieved in 22% of CLL, 19% of NHL, and 21% of ALL patients. The median durations of response after CART2 in CLL, NHL, and ALL patients were 33, 6, and 4 months, respectively. Addition of fludarabine to cyclophosphamide-based lymphodepletion before the first CAR T-cell infusion (CART1) and an increase in the CART2 dose compared with CART1 were independently associated with higher overall response rates and longer progression-free survival after CART2. We observed durable CAR T-cell persistence after CART2 in patients who received cyclophosphamide and fludarabine (Cy-Flu) lymphodepletion before CART1 and a higher CART2 compared with CART1 cell dose. The identification of 2 modifiable pretreatment factors independently associated with better outcomes after CART2 suggests strategies to improve in vivo CAR T-cell kinetics and responses after repeat CAR T-cell infusions, and has implications for the design of trials of novel CAR T-cell products after failure of prior CAR T-cell immunotherapies., (© 2021 by The American Society of Hematology.)

Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(-) disease. We report that CD19(-) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly, we reasoned that broadening the spectrum of CD19CAR T-cells to include both CD20 and CD22 would enable them to target CD19(-) escape BL-ALL while preserving their upfront efficacy. We created a CD19/20/22-targeting CAR T-cell by coexpressing individual CAR molecules on a single T-cell using one tricistronic transgene. CD19/20/22CAR T-cells killed CD19(-) blasts from patients who relapsed after CD19CAR T-cell therapy and CRISPR/Cas9 CD19 knockout primary BL-ALL both in vitro and in an animal model, while CD19CAR T-cells were ineffective. At the subcellular level, CD19/20/22CAR T-cells formed dense immune synapses with target cells that mediated effective cytolytic complex formation, were efficient serial killers in single-cell tracking studies, and were as efficacious as CD19CAR T-cells against primary CD19(+) disease. In conclusion, independent of CD19 expression, CD19/20/22CAR T-cells could be used as salvage or front-line CAR therapy for patients with recalcitrant disease.

Acute lymphoblastic leukemia (ALL) cases with e13a3 fusion transcripts are extremely rare. We report a 24-year-old male with Ph-positive (Ph+) ALL with an aberrant e13a3 fusion transcript treated with CD19-specific chimeric antigen receptor T-cell (CAR-T) therapy. He developed refractory disease post-chemotherapy induction, andreceived allogeneic hematopoietic stem cell transplantation (allo-HSCT) after salvage with imatinib in combination with chemotherapy regimen. Unfortunately, the patient relapsed after +90 days post-transplant. He was consented to CAR-T therapy trial and achieved complete remission, highlighting the efficacy of CAR-T treatment in relapsed-refractory B-ALL irrespective of the underlying genetic drivers in leukemia cells ., Competing Interests: Conflict of Competing Interest The authors declare no conflicts of interest associated with this manuscript., (Copyright © 2020. Published by Elsevier Inc.)

Chimeric antigen receptor (CAR)-T-cell is a safe and effective therapy of B-cell cancers but it is unknown if this is so in persons with prior hepatitis B virus (HBV) infection. We studied 70 subjects with advanced B-cell cancers receiving CAR-T-cell therapy, 12 of whom had chronic HBV-infection (HBsAg positive) and 29 with resolved HBV-infection (HBsAg negative and anti-HBc positive). Safety and efficacy were compared with 29 subjects without HBV-infection. HBV was reactivated in 2 subjects with chronic HBV-infection and 1 with resolved HBV-infection. There was no HBV-related hepatitis flare. Responses to CAR-T-cell therapy in the three cohorts were not significantly different. There was no significant difference in the incidence or severity of cytokine release syndrome (CRS) and neurologic toxicity between the cohorts. Our data suggest that chronic and resolved HBV-infection do not affect the safety and efficacy of CAR-T-cell therapy.

Chimeric antigen receptor (CAR) T cells targeting CD19 are a breakthrough treatment for relapsed, refractory B cell malignancies 1-5 . Despite impressive outcomes, relapse with CD19 - disease remains a challenge. We address this limitation through a first-in-human trial of bispecific anti-CD20, anti-CD19 (LV20.19) CAR T cells for relapsed, refractory B cell malignancies. Adult patients with B cell non-Hodgkin lymphoma or chronic lymphocytic leukemia were treated on a phase 1 dose escalation and expansion trial (NCT03019055) to evaluate the safety of 4-1BB-CD3ζ LV20.19 CAR T cells and the feasibility of on-site manufacturing using the CliniMACS Prodigy system. CAR T cell doses ranged from 2.5 × 10 5 -2.5 × 10 6 cells per kg. Cell manufacturing was set at 14 d with the goal of infusing non-cryopreserved LV20.19 CAR T cells. The target dose of LV20.19 CAR T cells was met in all CAR-naive patients, and 22 patients received LV20.19 CAR T cells on protocol. In the absence of dose-limiting toxicity, a dose of 2.5 × 10 6 cells per kg was chosen for expansion. Grade 3-4 cytokine release syndrome occurred in one (5%) patient, and grade 3-4 neurotoxicity occurred in three (14%) patients. Eighteen (82%) patients achieved an overall response at day 28, 14 (64%) had a complete response, and 4 (18%) had a partial response. The overall response rate to the dose of 2.5 × 10 6 cells per kg with non-cryopreserved infusion (n = 12) was 100% (complete response, 92%; partial response, 8%). Notably, loss of the CD19 antigen was not seen in patients who relapsed or experienced treatment failure. In conclusion, on-site manufacturing and infusion of non-cryopreserved LV20.19 CAR T cells were feasible and therapeutically safe, showing low toxicity and high efficacy. Bispecific CARs may improve clinical responses by mitigating target antigen downregulation as a mechanism of relapse.

Introduction: Though 85% of children and young adults with acute lymphoblastic leukemia (ALL) are cured, until recently, the prognosis of relapsed or refractory disease has been dismal. The advent of chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of relapsed/refractory ALL. The most well-studied, successful CARs are autologous, murine-based anti-CD19 CARs, but new constructs are currently under clinical investigation., Areas Covered: This review describes the history and design of CAR T cells, clinical trial outcomes of anti-CD19 and newer CARs, treatment-related toxicities including cytokine release syndrome and neurotoxicity, and issues with resistance and relapse. A search of PubMed and clinicaltrials.gov spanning from 2012-present was used to select original reports investigating the use of CAR T in pediatric patients., Expert Opinion: CD19-targeted CARs have demonstrated remarkable response rates and produced durable remissions in very high-risk pediatric patient populations. The therapies, however, are limited by unique treatment-related toxicities and considerable rates of antigen-positive and antigen-negative relapses. Current research efforts focused on elucidating mechanisms of resistance/relapse and on developing strategies to prevent and treat relapse are critical to optimizing the use of CAR-T. In addition, ongoing trials testing CARs earlier in therapy and for new indications are key to informing their widespread usage.

Leukemia, most commonly B-cell acute lymphoblastic leukemia (B-ALL), accounts for about 30% of childhood cancer diagnoses. While there have been dramatic improvements in childhood ALL outcomes, certain subgroups-particularly those who relapse-fare poorly. In addition, cure is associated with significant short- and long-term side effects. Given these challenges, there is great interest in novel, targeted approaches to therapy. A number of new immunotherapeutic agents have proven to be efficacious in relapsed or refractory disease and are now being investigated in frontline treatment regimens. Blinatumomab (a bispecific T-cell engager that targets cluster of differentiation [CD]-19) and inotuzumab ozogamicin (a humanized antibody-drug conjugate to CD22) have shown the most promise. Chimeric antigen receptor T (CAR-T) cells, a form of adoptive immunotherapy, rely on the transfer of genetically modified effector T cells that have the potential to persist in vivo for years, providing ongoing long-term disease control. In this article, we discuss the clinical biology and treatment of B-ALL with an emphasis on the role of immunotherapy in overcoming the challenges of conventional cytotoxic therapy. As immunotherapy continues to move into the frontline of pediatric B-ALL therapy, we also discuss strategies to address unique side effects associated with these agents and efforts to overcome mechanisms of resistance to immunotherapy.

A 22-year old male with a history of B-cell acute lymphoblastic leukemia with recent bone marrow transplantation and on immunosuppressive therapy presented with painless, subacute vision loss of two weeks duration. He exhibited a horizontal gaze palsy, nystagmus, and mildly swollen and hyperemic optic discs with peripapillary flame hemorrhage on retinal exam. He had bilateral cecocentral scotomas on visual field exam, and MRI of his brain/orbits demonstrated hyperintensities in the hypothalamus, periaqueductal gray, and dorsal rostral medullary regions. After continued progression of symptoms despite discontinuation of the patient's tacrolimus, an empiric trial of IV thiamine treatment was started before the patient's lab vitamin levels were available, given strong clinical suspicion for a nutritional etiology. The patient's clinical presentation improved dramatically, and he achieved a final visual acuity of 20/20, full visual fields bilaterally, and resolution of nystagmus. A final diagnosis of Wernicke's encephalopathy was supported by his clinical course, imaging findings, and further confirmation with blood thiamine levels. This case presents unique ocular manifestations of Wernicke's encephalopathy and highlights the importance of early diagnosis in this potentially reversible condition., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: Children with Down syndrome (constitutive trisomy 21) that develop acute lymphoblastic leukemia (DS-ALL) have a 3-fold increased likelihood of treatment-related mortality coupled with a higher cumulative incidence of relapse, compared with other children with B-cell acute lymphoblastic leukemia (B-ALL). This highlights the lack of suitable treatment for Down syndrome children with B-ALL., Experimental Design: To facilitate the translation of new therapeutic agents into clinical trials, we built the first preclinical cohort of patient-derived xenograft (PDX) models of DS-ALL, comprehensively characterized at the genetic and transcriptomic levels, and have proven its suitability for preclinical studies by assessing the efficacy of drug combination between the MEK inhibitor trametinib and conventional chemotherapy agents., Results: Whole-exome and RNA-sequencing experiments revealed a high incidence of somatic alterations leading to RAS/MAPK pathway activation in our cohort of DS-ALL, as well as in other pediatric B-ALL presenting somatic gain of the chromosome 21 (B-ALL+21). In murine and human B-cell precursors, activated KRAS G12D functionally cooperates with trisomy 21 to deregulate transcriptional networks that promote increased proliferation and self renewal, as well as B-cell differentiation blockade. Moreover, we revealed that inhibition of RAS/MAPK pathway activation using the MEK1/2 inhibitor trametinib decreased leukemia burden in several PDX models of B-ALL+21, and enhanced survival of DS-ALL PDX in combination with conventional chemotherapy agents such as vincristine., Conclusions: Altogether, using novel and suitable PDX models, this study indicates that RAS/MAPK pathway inhibition represents a promising strategy to improve the outcome of Down syndrome children with B-cell precursor leukemia., (©2020 American Association for Cancer Research.)

Recently approved by the FDA and European Medicines Agency, CAR-T cell therapy is a new treatment option for B-cell malignancies. Currently, CAR-T cells are manufactured in centralized facilities and face bottlenecks like complex scaling up, high costs, and logistic operations. These difficulties are mainly related to the use of viral vectors and the requirement to expand CAR-T cells to reach the therapeutic dose. In this paper, by using Sleeping Beauty-mediated genetic modification delivered by electroporation, we show that CAR-T cells can be generated and used without the need for ex vivo activation and expansion, consistent with a point-of-care (POC) approach. Our results show that minimally manipulated CAR-T cells are effective in vivo against RS4;11 leukemia cells engrafted in NSG mice even when inoculated after only 4 h of gene transfer. In an effort to better characterize the infused CAR-T cells, we show that 19BBz T lymphocytes infused after 24 h of electroporation (where CAR expression is already detectable) can improve the overall survival and reduce tumor burden in organs of mice engrafted with RS4;11 or Nalm-6 B cell leukemia. A side-by-side comparison of POC approach with a conventional 8-day expansion protocol using Transact beads demonstrated that both approaches have equivalent antitumor activity in vivo . Our data suggest that POC approach is a viable alternative for the generation and use of CAR-T cells, overcoming the limitations of current manufacturing protocols. Its use has the potential to expand CAR immunotherapy to a higher number of patients, especially in the context of low-income countries., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The recent FDA approval of the first CAR immunotherapy marks a watershed moment in the advancement toward a cure for cancer. CD19 CAR treatment for B cell acute lymphocytic leukemia has achieved unprecedented remission rates. However, despite success in treating previously relapsed and refractory patients, CD19 CAR faces similar challenges as traditional chemotherapy, in that malignancy can adapt and overcome treatment. The emergence of both antigen positive and negative blasts after CAR treatment represents a need to bolster current CAR approaches. Here, we report on the anti-tumor activity of a CAR T cell possessing 2 discrete scFv domains against the leukemic antigens CD19 and CD123. We determined that the resulting compound CAR (cCAR) T cell possesses consistent, potent, and directed cytotoxicity against each target antigen population both in vitro and in vivo. Our findings indicate that targeting CD19 and CD123 on B-ALL cells may be an effective strategy for augmenting the response against leukemic blasts and reducing rates of relapse.

CAR-T-cell therapy has shown considerable advance in recent years, being approved by regulatory agencies in US, Europe, and Japan for the treatment of refractory patients with CD19+ B-cell leukemia or diffuse large B-cell lymphoma. Current methods for CAR-T-cell production use viral vectors for T-cell genetic modification and can take up to 15 days to generate the infusion product. The development of simple and less costly manufacturing protocols is needed in order to meet the increasing demand for this therapy. In this present work, we generated 19BBz CAR-T cells in 8 days using a protocol based on the non-viral transposon-based vector Sleeping Beauty. The expanded cells display mostly a central memory phenotype, expressing higher levels of inhibitory receptors when compared with mock cells. In addition, CAR-T cells were cytotoxic against CD19+ leukemia cells in vitro and improved overall survival rates of mice xenografted with human RS4;11 or Nalm-6 B-cell leukemias. Infused CAR-T cells persisted for up to 28 days, showing that they are capable of long-term persistence and antitumor response. Altogether, these results demonstrate the effectiveness of our protocol and pave the way for a broader application of CAR-T-cell therapy.

Cytokine release syndrome (CRS) counteracts the effectiveness of chimeric antigen receptor (CAR) T cell therapy in cancer patients, but the mechanism underlying CRS remains unclear. Here, we show that tumor cell pyroptosis triggers CRS during CAR T cell therapy. We find that CAR T cells rapidly activate caspase 3 in target cells through release of granzyme B. The latter cleaves gasdermin E (GSDME), a pore-forming protein highly expressed in B leukemic and other target cells, which results in extensive pyroptosis. Consequently, pyroptosis-released factors activate caspase 1 for GSDMD cleavage in macrophages, which results in the release of cytokines and subsequent CRS. Knocking out GSDME, depleting macrophages, or inhibiting caspase 1 eliminates CRS occurrence in mouse models. In patients, GSDME and lactate dehydrogenase levels are correlated with the severity of CRS. Notably, we find that the quantity of perforin/granzyme B used by CAR T cells rather than existing CD8 + T cells is critical for CAR T cells to induce target cell pyroptosis., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Immunotherapy using T cells modified with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemia and lymphomas resistant to chemotherapy. Recent clinical studies have shown excellent responses of CAR-T cells in a variety of B cell tumors. However, it is important to validate in vitro activity of these cells, though different sorts of assays, which are capable of measuring the cytotoxic potential of these cells. In this chapter, it will be pointed two methods to evaluate CAR-T cell killing potential against B cell malignancy cell lines.

At present, the only curative therapy for patients with T-cell malignancies is allogeneic stem cell transplant, which has associated risks and toxicities. Novel agents have been tried in relapsed T-cell acute lymphoblastic leukemia (T-ALL), but only one, with 20%-30% complete remission rates, has been approved by the US Food and Drug Administration. T-ALL is a heterogeneous disease, but it has universal overexpression of CD7 as well as several other T-cell markers, such as CD2 and CD5. T cells engineered to express a chimeric antigen receptor (CAR) are a promising cancer immunotherapy. Such targeted therapies have shown great potential for inducing both remissions and even long-term relapse-free survival in patients with B-cell leukemia and lymphoma. UCART7 for CD7 + T-cell malignancies is in development for treatment of relapsed T-ALL in children and adults. It may also have potential in other CD7 + hematologic malignancies that lack both effective therapies and targeted therapies. The challenges encountered and progress made in developing a novel fratricide-resistant "off-the-shelf" CAR-T (or UCART7) that targets CD7 + T-cell malignancies are discussed here., (Copyright © 2019. Published by Elsevier Ltd.)

Autologous T cells that have been genetically modified to express a chimeric antigen receptor (CAR) targeting the B cell antigen CD19 have yielded remarkable clinical responses in patients with B cell malignancies, and are now on the market as anticancer 'drugs'. Riding on this success, the field of immune cell engineering is rapidly growing, with creative solutions to major outstanding challenges, such as limitations in target antigen selection, the hostility of the tumor microenvironment and the logistical challenges of generating autologous therapies. Innovations in antigen receptor design, coupled with advances in gene transfer and gene-editing technologies, have enabled the engineering of T cells to have sophisticated sensing circuits, to have synthetic functionalities, and to be used as off-the-shelf, universal cellular products. As these technologies are applied to other immune cells, such as natural killer cells, hematopoietic cells or induced pluripotent stem cells, the potential to transform the treatment of many cancers, as well as other diseases, is palpably exciting. We discuss the pipeline of several influential innovations in the preclinical setting, the early translational results from clinical trials of these next-generation approaches, and the outlook for gene-modified or gene-edited cell therapies.

The approval of tisagenlecleucel and axicabtagene ciloleucel represents a breakthrough in the field of immune and cellular therapy for hematologic malignancies. These anti-CD19 chimeric antigen receptor-T cells (CAR) proved to be highly effective in the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) and specific histologic subtypes of B-cell non-Hodgkin lymphomas. This expert review aims to summarize the current available research evidence in this field, with a special focus on the different challenges faced by treating physicians, and we also provide future perspectives.

The long-term effects of CD19-targeted chimeric antigen receptor-modified T-cell immunotherapy (CD19-CARTx) for B-cell malignancies on humoral immunity are unclear. We examined antiviral humoral immunity in 39 adults with B-cell malignancies who achieved durable complete remission without additional therapy for >6 months after CD19-CARTx. Despite CD19+ B-cell aplasia in all patients, the incidence of viral infections occurring >90 days post-CD19-CARTx was low (0.91 infections per person-year). Because long-lived plasma cells are CD19- and should not be direct targets of CD19-targeted chimeric antigen receptor T cells, we tested the hypothesis that humoral immunity was preserved after CD19-CARTx based on linear mixed-effects models of changes in serum total immunoglobulin G (IgG) concentration, measles IgG concentration, and the number of viruses or viral epitopes to which serum IgG was directed (the "antivirome") using the novel VirScan assay. Samples were tested pre-CD19-CARTx and ∼1, 6, and 12 months post-CD19-CARTx. Although total IgG concentration was lower post-CD19-CARTx (mean change, -17.5%), measles IgG concentration was similar (mean change, 1.2%). Only 1 participant lost measles seroprotection post-CD19-CARTx but had undergone allogeneic hematopoietic cell transplantation before CD19-CARTx. The antivirome was also preserved, with mean absolute losses of 0.3 viruses and 6 viral epitopes detected between pre- and post-CD19-CARTx samples. Most participants gained IgG to ≥2 epitopes for ≥2 viruses, suggesting that humoral immunity to some viruses may be maintained or recover after successful CD19-CARTx. These findings may differ in children. Studies of immunoglobulin replacement and vaccination after CARTx are warranted., (© 2019 by The American Society of Hematology.)

Objective: To study the safety and effectiveness of humanized CD19-targeted CAR-T cells (hCART19s) for treatment of patients with refractory/relapsed (R/R) B-ALL., Methods: The analyzed patients were 15 children and adults with relapsed/refractory B-ALL who not received treatment with murine CD19 CAR-T cells. The patients received a single dose (1×10 6 /kg) of autologous hCART19 infusion after lymphodepletion chemotherapy based on cyclophosphamide and fludarabine., Results: Among the 15 patients, 13/14 (92.9%) evaluable patients achieved complete remission (CR) or CR with incomplete recovery of blood cells (CRi) on day 30 after hCART19s infusion. At day 180 after the infusion, the overall survival rate was 73.3%, and the leukemia-free survival rate was 69.2%. The cumulative incidence of relapse was 24.5% and non-relapse mortality rate was 7.7%. During treatment,12/15 patients (80%) developed cytokine release syndrome (CRS) of grade 1-2, and 3 patients (20.0%) developed CRS of grade 3-5. Only one patient (6.7%) suffered from the reversible neurotoxicity., Conclusion: hCART19s can effectively treat refractory/relapsed (R/R) adult and children with B-ALL, and the incidence of treatment-related CRS and neurotoxicity is low.

In 2017, the US Food and Drug Administration approved the first chimeric antigen receptor (CAR) T-cell therapies for patients with relapsed or refractory B-cell leukemia and selected B-cell lymphomas. This novel form of cellular immunotherapy creates a "living drug" that effectively reprograms a patient's T cells to target specific antigens on the surface of a tumor. The therapy has high response rates in patients with refractory disease, although a single infusion of CAR T cells costs hundreds of thousands of dollars. A value analysis is required to determine whether and how to offer patients these expensive, customized drugs., (© 2019 American Medical Association. All Rights Reserved.)

CAR T cells targeting CD19 provide promising options for treatment of B cell malignancies. However, tumor relapse from antigen loss can limit efficacy. We developed humanized, second-generation CAR T cells against another B cell-specific marker, B cell activating factor receptor (BAFF-R), which demonstrated cytotoxicity against human lymphoma and acute lymphoblastic leukemia (ALL) lines. Adoptively transferred BAFF-R-CAR T cells eradicated 10-day preestablished tumor xenografts after a single treatment and retained efficacy against xenografts deficient in CD19 expression, including CD19-negative variants within a background of CD19-positive lymphoma cells. Four relapsed, primary ALLs with CD19 antigen loss obtained after CD19-directed therapy retained BAFF-R expression and activated BAFF-R-CAR, but not CD19-CAR, T cells. BAFF-R-CAR, but not CD19-CAR, T cells also demonstrated antitumor effects against an additional CD19 antigen loss primary patient-derived xenograft (PDX) in vivo. BAFF-R is amenable to CAR T cell therapy, and its targeting may prevent emergence of CD19 antigen loss variants., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)