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Hairy cell leukemia variant (HCL-v) is a rare malignancy of clonal mature B-cells that follows a chronic disease course. HCL-v patients are often resistant to purine nucleoside analogs, which are the first-line therapy. To address the shortcomings of current therapy for HCL-v, we investigated the activity of a BAFF ligand-based CAR-T cell which binds to all three BAFF receptors, BAFF-receptor, TACI, and BCMA. Here, we demonstrate that HCLv patient-derived cells highly express all three BAFF receptors and that BAFF CAR-T cells induce significant cytotoxicity in vitro against both cell lines and HCL-v patient cells. This cytotoxicity corresponds with significant CAR-T cell activation, degranulation, and release of pro-inflammatory cytokines after co-incubation with HCLv cells. Furthermore, we successfully generated BAFF CAR-T cells directly from an HCLv patient and observed direct autologous killing against patient tumor cells in vitro. These HCLv patient-derived CAR-T cells were also effective in killing the Hair-M cell line and tumor cells derived from a different HCLv patient. Lastly, we also developed two mouse xenograft models for HCL, a subcutaneous Bonna-12 model and intravenous Hair-M xenograft model. We observed decreases in tumor burden and prolonged overall survival without significant toxicity. In conclusion, here we show that BAFF CAR-T cells exert anti-tumor effects in vitro and in vivo against multiple cell lines and patient-derived HCL-v samples and may be a successful therapeutic strategy for HCLv patients.

Adoptive T cell therapy, using T cell receptor-engineered T (TCR-T) cells and chimeric antigen receptor T (CAR-T) cells, is a potent immunotherapy option. Bladder cancer is a prevalent urological malignancy, particularly in cases of muscle invasion and metastasis, for which systemic therapy is crucial. Immunotherapy utilizing immune checkpoint blockade has been approved for bladder cancer treatment. The antitumor effect of an immune checkpoint blockade based on cytotoxic T cells (CTLs) and the patient's immune status is essential. The chemotherapeutic drug cisplatin (CDDP) is a key drug in bladder cancer treatment. However, it has been shown to suppress T cells, making combination therapy with CDDP and immunotherapy difficult. To address this, we developed TCR-T cells specific for bladder cancer cells. In previous studies, we found that the tumor-associated antigen CLSPN is overexpressed in CDDP-resistant bladder cancer cells and that the antigenic peptide HLA-A*02:01/CLSPN 1254-1262 , encoded by CLSPN, could be targeted by a CTL clone. The TCR was cloned from the HLA-A*02:01/CLSPN 1254-1262 specific CTL clone yc3. We also designed a codon-optimized TCR sequence using GeneArt® GeneOptimizer® (Opt TCR) and compared the TCR-T cells using the original TCR sequence (Ori TCR-T cells) and the codon-optimized TCR sequence (Opt TCR-T cells). Opt TCR-T cells exhibited higher TCR transduction efficiency, higher TCR expression levels, higher avidity, and greater cytotoxicity than did Ori TCR-T cells. These results suggest that HLA-A*02:01/CLSPN 1254-1262 specific Opt TCR-T cells are promising candidates for CDDP combination therapy.

Chimeric antigen receptor-natural killer (CAR-NK) cells represent a breakthrough in cancer immunotherapy, making this a highly popular research area. However, comprehensive analyses of this field using bibliometric methods are rare. To our knowledge, this study has collected highest number of publications (1,259) on CAR-NK therapy from January 1, 2004, to December 31, 2023, and utilized CiteSpace and VOSviewer to analyze regions, institutions, journals, authors, and keywords to forecast the latest trends in CAR-NK therapy research. The United States and China, contributing over 60% of publications, are the primary drivers in this field. The Helmholtz Association and Harvard University are the most active institutions, with most publications appearing in Frontiers in Immunology. Winfried S. Wels is the most prolific author, while EL Liu is the most frequently co-cited author. "Immunotherapy," "T-cells," and "Cancer" are the most extensively covered topics in CAR-NK therapy research. Our study reveals current CAR-NK research trends, identifies potential research hotspots, and visualizes references through bibliometric methods, providing valuable guidance for future research in this field.

Despite success in treating some hematological malignancies, CAR-T cells have not yet produced similar outcomes in solid tumors due, in part, to the tumor microenvironment, poor persistence, and a paucity of suitable target antigens. Importantly, the impact of the CAR components on these challenges remains focused on the intracellular signaling and antigen-binding domains. In contrast, the flexible hinge and transmembrane domains have been commoditized and are the least studied components of the CAR. Here, we compared the hinge and transmembrane domains derived from either the CD8ɑ or CD28 molecule in identical GUCY2C-targeted third-generation designs for colorectal cancer. While these structural domains do not contribute to differences in antigen-independent contexts, such as CAR expression and differentiation and exhaustion phenotypes, the CD8ɑ structural domain CAR has a greater affinity for GUCY2C. This results in increased production of inflammatory cytokines and granzyme B, improved cytolytic effector function with low antigen-expressing tumor cells, and robust anti-tumor efficacy in vivo compared with the CD28 structural domain CAR. This suggests that CD8α structural domains should be considered in the design of all CARs for the generation of high-affinity CARs and optimally effective CAR-T cells in solid tumor immunotherapy.

CAR-T cell therapy has established itself as a highly effective treatment for hematological malignancies. There are currently six commercial CAR-T products that have been FDA approved for diseases such as B-ALL, LBCL, MCL, FL, MM, and CLL/SLL. "Real-world" studies allow us to evaluate outcomes from the general population to determine their efficacy and safety compared to those who were included in the original trials. Based on several well conducted "Real-world" studies that represent diverse populations, we report that outcomes from the original trials that led to the approval of these therapies are comparable to those in practice.

Recently, CAR-T cell therapy in hematological malignancies has received extensive attention. The objective of this study is to gain a comprehensive understanding of the current research status, development trends, research hotspots, and emerging topics pertaining to CAR-T cells in the treatment of hematological malignancies. Articles pertaining to CAR-T cell therapy for hematological malignancies from the years 2012 to 2023 were obtained and assessed from the Web of Science Core Collection (WoSCC). A bibliometric approach was employed to conduct a scientific, comprehensive, and objective quantitative analysis, as well as a visual analysis, of this particular research domain. A comprehensive analysis was conducted on a corpus of 3643 articles, which were collaboratively authored by 72 countries and various research institutions. CAR-T cell research in treating hematological malignancies shows an increasing trend each year. Notably, the study identified the countries and institutions displaying the highest level of activity, the journals with the most citations and output, as well as the authors who garnered the highest frequency of citations and co-citations. Furthermore, the analysis successfully identified the research hotspots and highlighted six emerging topics within this domain. This study conducted a comprehensive exploration and analysis of the research status, development trends, research hotspots, and emerging topics about CAR-T cells in the treatment of hematological malignancies from 2012 to 2023. The findings of this study will serve as a valuable reference and guide for researchers seeking to delve deeper into this field and determine the future direction of their research.

CAR-T cell therapy has emerged as a significant approach for the management of hematological malignancies. Over the past few years, the utilization of CAR-T cells in the investigation and treatment of solid tumors has gained momentum, thereby establishing itself as a prominent area of research. This descriptive study involved the retrieval of articles about CAR-T cell therapy for solid tumors from the Web of Science Core Collection (WoSCC) database. Subsequently, bibliometric analysis and knowledge map analysis were conducted on these articles. The field under consideration is currently experiencing a period of swift advancement, as evidenced by the escalating number of publications in this domain each year. The United States holds an indisputable position as the foremost leader in this particular field, with the University of Pennsylvania emerging as the most active institution. The authors with the highest citation frequency and co-citation frequency are Carl H. June and Shannon L. Maude, respectively. The research hotspots in this field mainly focus on five aspects. Additionally, 10 emerging themes were identified. This study undertakes a comprehensive, systematic, and objective analysis and exploration of the field of CAR-T cell treatment for solid tumors, utilizing bibliometric methods. The findings of this study are expected to serve as a valuable reference and enlightenment for future research endeavors in this particular domain.

Chimeric antigen receptor (CAR) T cells have great potential in cancer therapy, particularly in treating hematologic malignancies. However, their efficacy in solid tumors remains limited, with a significant proportion of patients failing to achieve long-term complete remission. One major challenge is the premature exhaustion of CAR-T cells, often due to insufficient metabolic energy. The survival, function and metabolic adaptation of CAR-T cells are key determinants of their therapeutic efficacy. We explore how targeting metabolic pathways in the tumor microenvironment can enhance CAR-T cell therapy by addressing metabolic competition and immunosuppression that impair CAR-T cell function. Tumors undergo metabolically reprogrammed to meet their rapid proliferation, thereby modulating metabolic pathways in immune cells to promote immunosuppression. The distinct metabolic requirements of tumors and T cells create a competitive environment, affecting the efficacy of CAR-T cell therapy. Recent research on glucose, lipid and amino acid metabolism, along with the interactions between tumor and immune cell metabolism, has revealed that targeting these metabolic processes can enhance antitumor immune responses. Combining metabolic interventions with existing antitumor therapies can fulfill the metabolic demands of immune cells, providing new ideas for tumor immunometabolic therapies. This review discusses the latest advances in the immunometabolic mechanisms underlying tumor immunosuppression, their implications for immunotherapy, and summarizes potential metabolic targets to improve the efficacy of CAR-T therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Microplastics (MPs) are increasingly recognized as contaminants present in various environments and are widely acknowledged as potential hazards to the mammalian immune system. In our study of chimeric antigen receptor T cell (CAR-T) therapy, we observed the presence of MP in CAR-T cell products for the first time. It is worth exploring whether MP could enter CAR-T cells and how they might affect CAR-T cells' functionality. Therefore, we analyzed how MP affected CD19 and BCMA-CAR-T cells. Based on flow cytometry, ELISA, and cytotoxicity analysis of in vitro and in vivo experiments, MP suppressed the activity of CAR-T cells. Subsequent investigation revealed that the exposure of CAR-T cells to varying concentrations of MP resulted in a notable increase in apoptosis, ferroptosis, and exhaustion levels. Furthermore, the hyperactivation of the mTOR signaling pathway in MP-treated CAR-T cells was verified. The partial restoration of CAR-T cell function in MP was achieved by inhibiting the mTOR pathway. MP present a threat to CAR-T cell function due to their role in inducing CAR-T cell apoptosis, ferroptosis, and T-cell exhaustion through the hyperactivation of mTOR signaling pathways., Competing Interests: Declaration of Competing Interest Our authors claim no known personal relation or competing financial interests affecting this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In recent years, chimeric antigen receptor (CAR)-T cell therapy has been highly successful in treating hematological malignancies, leading to significant advancements in the cancer immunotherapy field. However, the typical CAR-T therapy necessitates the enrichment of patients' own leukocytes for ex vivo production of CAR-T cells, this customized pattern requires a complicated and time-consuming manufacturing procedure, making it costly and less accessible. The off-the-shelf universal CAR-T strategy could reduce manufacturing costs and realize timely drug administration, presenting as an ideal substitute for typical CAR-T therapy. Utilizing nanocarriers for targeted gene delivery is one of the approaches for the realization of universal CAR-T therapy, as biocompatible and versatile nanoparticles could deliver CAR genes to generate CAR-T cells in vivo. Nanoparticle-mediated in situ generation of CAR-T cells possesses multiple advantages, including lowered cost, simplified manufacturing procedure, and shortened administration time, this strategy is anticipated to provide a potentially cost-effective alternative to current autologous CAR-T cell manufacturing, thus facilitating the prevalence and improvement of CAR-T therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Single-targeted CAR-T has exhibited notable success in treating B-cell tumors, effectively improving patient outcomes. However, the recurrence rate among patients remains above fifty percent, primarily attributed to antigen escape and the diminished immune persistence of CAR-T cells. Over recent years, there has been a surge of interest in bispecific CAR-T cell therapies, marked by an increasing number of research articles and clinical applications annually. This paper undertakes a comprehensive review of influential studies on the design of bispecific CAR-T in recent years, examining their impact on bispecific CAR-T efficacy concerning disease classification, targeted antigens, and CAR design. Notable distinctions in antigen targeting within B-ALL, NHL, and MM are explored, along with an analysis of how CAR scFv, transmembrane region, hinge region, and co-stimulatory region design influence Bi-CAR-T efficacy across different tumors. The summary provided aims to serve as a reference for designing novel and improved CAR-Ts, facilitating more efficient treatment for B-cell malignant tumors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Systemic Lupus Erythematosus (SLE) is a rare, heterogeneous, potentially life-threatening autoimmune disease. Presence of kidney or other major organ (brain, heart or lung) involvement are predictors of poor outcome and in a subset of patients resistant to 1st or 2nd line conventional treatment. The 10-year mortality remains around 10-15 %. Chimeric Antigen Receptors (CAR) are molecules that allow to redirect the engineered immune cells towards specific target antigens and to simultaneously boost their activation. Following breakthrough results observed in the treatment of hematological malignancies, conventional CAR T-cell therapy has recently been applied to refractory SLE patients. Compared to the use of monoclonal antibodies, anti-CD19 CAR T-cells allow to achieve deeper depletion of autoreactive B cells, notably at site of inflamed tissues and lymphoid organs (i.e. lymph node), to suppress interferon signature and to restore the immune tolerance with the reemergence of naïve B-cells with a new repertoire. All clinical data reported in SLE patients so far showed that autologous anti-CD19 CAR T-cell treatment allowed impressive short- and longer-term resolution of lupus nephritis and other severe disease-related manifestations, without major toxicities and only mild cytokine-release syndrome. These clinical effects persisted after B-cell reconstitution and were associated with normalization of double-stranded DNA antibodies and complement levels in drug-free patients until three years after the procedure. Overall, these pioneering experiences show unique clinical and immunological response to CAR T-cell therapy in SLE, and the need for extended follow-up to determine its long-term efficacy., Competing Interests: Declaration of competing interest We have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose of Review: To review the landscape of chimeric antigen receptor T-cell (CAR T) therapy for gliomas as seen in recently published trials and discuss on-going challenges with new cancer immunotherapy treatments., Recent Findings: Given how CAR T therapy has revolutionized the treatment of several hematologic malignancies, there has been increasing interest in using immunotherapy, and particularly CAR T therapy for gliomas. Within the past decade, several first in human trials have published early patient experiences showing treatment is generally well tolerated but with limited efficacy, which may be improving with newer evolutions in CAR T design to overcome known resistance mechanisms in glioma treatment., Summary: CAR T therapy is a promising avenue of treatment for high-grade gliomas, which have a universally poor prognosis as well as limited therapeutics. There are a growing number of CAR T clinical trials for CNS tumors and thus, an understanding of their treatment strategies, toxicity management, and overcoming resistance mechanisms will be important for both clinical practice and to identify areas for future research., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Cytokine release syndrome (CRS) can occur following cancer immunotherapies, but is most often mild and of limited duration. International Classification of Diseases (ICD)-10 codes allowing identification of CRS were introduced in 2020 but may be underutilized. We evaluated the performance of a published claims-based algorithm to detect CRS (any grade) and high-grade CRS (HG, grades 2-5), as well as identified indicators of HG CRS in retrospective data. Adults with low-grade and HG CRS during an encounter coinciding with administrations of blinatumomab or chimeric antigen receptor-T therapy were identified in three types of retrospective databases (hospital chargemaster data, electronic health records, and administrative claims). The algorithm's sensitivity in detecting any CRS and HG CRS was reported. A least absolute shrinkage and selection operator (LASSO) regression model was developed to identify indicators of HG CRS. Performance of the model was evaluated using area under the curve (AUC). The sensitivity of the algorithm to detect any grade CRS ranged between 77%-100% and between 8%-80% for HG CRS, depending on the type of database. The LASSO model identified hypotension, positive pressure (including mechanical ventilation), tocilizumab, and vasopressors as indicators of HG CRS. AUC varied between 60% and 75%. The algorithm accurately detected any grade CRS for over three-quarters of instances, but was not as reliable for HG CRS. Results varied based on database attributes. Hypotension, vasopressors, positive pressure, and tocilizumab were associated with HG CRS and may be methodologically helpful signals of CRS severity in retrospective data., (© 2024 Janssen Scientific Affairs, LLC. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Although major progress has been made in the use of chimeric antigen receptor (CAR)-T-cell therapy for hematological malignancies, this method is ineffective against solid tumors largely because of the limited infiltration, activation and proliferation of CAR-T cells. To overcome this issue, we engineered CAR-T cells with synthetic Notch (synNotch) receptors, which induce local tumor-specific secretion of extracellular matrix (ECM)-degrading enzymes at the tumor site. SynNotch CAR-T cells achieve precise ECM recognition and robustly kill targeted tumors, with synNotch-induced enzyme production enabling the degradation of components of the tumor ECM. In addition, this regulation strongly increased the infiltration of CAR-T cells and the clearance of solid tumors, resulting in tumor regression without toxicity in vivo. Notably, synNotch CAR-T cells also promoted the persistent activation of CAR-T cells in patient-derived tumor organoids. Thus, we constructed a synthetic T-cell system that increases the infiltration and antitumor function of CAR-T cells, providing a strategy for targeting ECM-rich solid tumors., Competing Interests: Competing interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest., (© 2024. The Author(s), under exclusive licence to CSI and USTC.)

Like their natural counterparts, chimeric antigen receptor-engineered cells are prone to suppression by inhibitory signals, such as PD-L1, expressed by tumors or suppressor cells in the tumor microenvironment. Consequently, they become impaired, resulting in immune cell exhaustion, tumor progression, and resistance to other therapies. In this study, we developed an anti-PD-L1-CAR NK cell with efficient activity and a notable PD-L1-specific response toward tumor cell lines. The degranulation assay demonstrated that CD107a frequencies between the PD-L1 med and PD-L1 high groups and between Herceptin-treated and non-treated groups were not statistically different. Further investigation into NK cell characterization, considering different markers such as CD57, KIR2D, and CD25, revealed that the majority of the population are activated expanding NK cells. At the same time, immune checkpoint inhibitors, including PD-1, PD-L1, and LAG-3, showed increased levels following activation and expansion. Regarding the efficient functional activity of PD-L1-CAR NK cells and the instinctive receptor balance-based response of NK cells, this observation could point to the inhibition of NK cell overactivation or even higher cytotoxicity and cytokine production rather than exhaustion, especially in the case of healthy NK cells. These findings can contribute to a better understanding of the potential and challenges of using primary NK cells for CAR-NK cell therapy., (© 2024 Scandinavian Societies for Pathology, Medical Microbiology and Immunology.)

Chimeric Antigen Receptor (CAR)-T-cell therapy has revolutionized cancer immune therapy. However, challenges remain including increasing efficacy, reducing adverse events and increasing accessibility. Use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology can effectively perform various functions such as precise integration, multi-gene editing, and genome-wide functional regulation. Additionally, CRISPR screening using large-scale guide RNA (gRNA) genetic perturbation provides an unbiased approach to understanding mechanisms underlying anti-cancer efficacy of CAR T-cells. Several emerging CRISPR tools with high specificity, controllability and efficiency are useful to modify CAR T-cells and identify new targets. In this review we summarize potential uses of the CRISPR system to improve results of CAR T-cells therapy including optimizing efficacy and safety and, developing universal CAR T-cells. We discuss challenges facing CRISPR gene editing and propose solutions highlighting future research directions in CAR T-cell therapy., Competing Interests: Competing interests: RPG is a consultant to Antengene Biotech LLC, Ascentage Pharma Group and NexImmune Inc.; Medical Director, FFF Enterprises Inc.; A speaker for Janssen Pharma and Hengrui Pharma; Board of Directors: Russian Foundation for Cancer Research Support; and Scientific Advisory Boards, Nanexa AB and StemRad Ltd. AH receives research support from Novartis, BMS, Incyte and Pfizer., (© 2024. The Author(s).)

Lymphomatous infiltration of the peripheral nervous system (PNS), termed neurolymphomatosis, represents a distinct extranodal non-Hodgkin lymphoma variant with dismal outcome. CD19-directed chimeric antigen receptor (CD19-CAR) T-cell therapy has emerged as a safe and effective treatment for B-cell lymphomas. We aimed to assess toxicity and efficacy of CD19-CAR T-cells in neurolymphomatosis. Neurolymphomatosis patients treated with CD19 CAR T-cells were retrospectively identified at Massachusetts General Hospital over a six-year period. Toxicities were graded according to the ASTCT classification, management, and response rates were recorded. Eleven neurolymphomatosis patients were identified with a median of 2 lines of PNS-directed treatments (range: 1-3) prior to receiving CD19-CAR T-cells. Neurolymphomatosis localized to the nerve roots (8/11, 73%), plexus (5/11, 45%), peripheral (4/11, 36%) and cranial nerves (5/11, 45%). Low grade cytokine release syndrome (CRS) was detected in 8/11 (73%; grade 1: N = 7; grade 2: N = 1) cases. Low- and high-grade immune cell-associated neurotoxicity syndrome (ICANS) were recorded in 5/11 (45%; grade 1: N = 4; grade 2: N = 1) and 1/11 (9%; grade 4) patients, respectively. CRP levels at infusion were predictive of ICANS (area under the curve: 0.96, p = 0.01). Seven of eleven neurolymphomatosis patients (64%) responded to CD19-CAR T-cells. Complete remissions (CR) were achieved in three cases (27%), with 2 patients in sustained CR nine and 46 months after CD19-CAR infusion. Median progression-free survival (PFS) was 4 months. Collectively, CD19-CAR T-cell treatment was well tolerated and showed promising efficacy in recurrent neurolymphomatosis, a difficult to treat condition with unmet medical need. Findings suggest that CD19-CAR may sufficiently penetrate the blood-nerve barrier. Toxicity and outcomes were overall similar to CAR-T cell therapy in CNS lymphoma., (© 2024 Wiley Periodicals LLC.)

Background: Chimeric antigen receptor T cell (CAR-T) therapy is an emerging form of immunotherapy that has recently gained recognition for treating hematological malignancies. This successful utilization of CAR-T therapy has attracted interest in its application in refractory rheumatological diseases. Here, we will review the use of CAR-T therapy in rheumatological diseases., Methods: Per PRISMA guidelines, a comprehensive literature search was performed on PubMed, Cochrane, and ClinicalTrials.gov using keywords for 'CAR-T cell therapy' and 'Rheumatological diseases' from inception to December 9, 2023. After screening 2977 articles, six studies reporting outcomes of CAR-T cell therapies in patients with underlying autoimmune /rheumatological diseases. Descriptive analysis was performed to represent demographics and clinical outcomes., Results: A total of 101 adult patients from six studies were included in this systematic review. The median age of the participants was 50.8 years (IQR: 14.875), with ages ranging from 18 to 83 years. The included studies comprised 2 case reports, 1 case series, one observational study, and two clinical trials. The studies were conducted globally, including USA, Germany, and China. The underlying rheumatologic conditions were systemic lupus erythematosus (17.8 %), rheumatoid arthritis (23.8 %), myasthenia gravis (13.8 %), neuromyelitis optica (11.9 %), and others (32.7 %). The target of CAR-T therapy included CD-19 in four studies and B cell maturation antigen (BCMA) in two studies. All the patients were on prior therapy, including glucocorticoids and disease-modifying antirheumatic drugs. Follow-up ranged from a month to 1.5 years. Most of the studies reported improvement in the symptoms and decline in serological biomarkers of the underlying disease. The notable outcomes in the included studies were a 100 % response rate in five out of six studies. Grade 1 and 2 cytokine release syndrome (CRS) was observed in five studies. Only one study reported Grade 3 or higher CRS. 2 patients (1.98 %) developed neurotoxicity among the adverse effects., Conclusion: CAR-T cell therapy is a paradigm shift in managing rheumatologic diseases, with symptomatic improvement and biochemical control of these diseases. Although preliminary evidence indicates promising results, long-term follow-up and prospective clinical trials are needed to establish optimal timing and assess the safety and efficacy of CAR-T immunotherapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Ovarian cancer is the most malignant gynecological tumor. Previous studies have demonstrated that chimeric antigen receptor (CAR)-engineered NK-92 cells targeting folate receptor α (αFR) (NK-92-αFR-CAR) can specifically kill αFR-positive ovarian cancer cells. However, the migration barrier restricts antitumor effects of CAR-engineered cells., Objectives: To elucidate the mechanism by which NK-92-αFR-CAR cells induce the secretion of chemokine CXCL10 during killing ovarian cancer cells. It is speculated that NK-92-αFR-CAR-CXCR3A can target αFR and have chemotaxis of CXCL10, and they may have stronger killing effect of ovarian cancer., Methods: Study the mechanism of CXCL10 expression strongly induced by TNF-α and IFN-γ combined stimulation in ovarian cancer cells. Construct the fourth generation of NK-92-αFR-CAR-CXCR3A cells, which were co-expressed CXCR3A and αFR-CAR. Evaluate the killing and migration effects of NK-92-αFR-CAR-CXCR3A in vitro and in vivo., Results: RNA sequencing (RNA-seq) first revealed that the expression level of the chemokine CXCL10 was most significantly increased in ovarian cancer cells co-cultured with NK-92-αFR-CAR. Secondly, cytokine stimulation experiments confirmed that IFN-γ and TNF-α secreted by NK-92-αFR-CAR synergistically induced high CXCL10 expression in ovarian cancer cells. Further signaling pathway experiments showed that IFN-γ and TNF-α enhanced the activation level of the IFN-γ-IFNGR-JAK1/2-STAT1-CXCL10 signaling axis. Cytotoxicity experiments showed that NK-92-αFR-CAR-CXCR3A cells could not only efficiently kill αFR-positive ovarian cancer cells in vitro but also secrete IFN-γ and TNF-α. Higher migration than that of NK-92-αFR-CAR was detected in NK-92-αFR-CAR-CXCR3A using transwell assay. NK-92-αFR-CAR-CXCR3A effectively killed tumor cells in different mouse xenograft models of ovarian cancer and increased infiltration into tumor tissue., Conclusion: This study confirmed that IFN-γ and TNF-α secreted by αFR-CAR-engineered NK cells can synergistically induce high expression of CXCL10 in ovarian cancer cells and constructed self-driving αFR-CAR-engineered NK cells that can break through migration barriers based on CXCL10, which may provide a new therapeutic weapon for ovarian cancer., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest, (Copyright © 2024. Published by Elsevier Inc.)

Purpose of Review: Infections are the leading cause of non-relapse mortality following chimeric antigen receptor (CAR)-T-cell therapy, with viral infections being frequent both in the early and late phases post-infusion. We review the epidemiology of viral infections and discuss critical approaches to prevention and management strategies in this setting., Recent Findings: Herpesviruses dominate the early period. herpes simplex virus and varicella zoster virus infections are rare due to widespread antiviral prophylaxis, but cytomegalovirus (CMV) reactivation is increasingly observed, particularly in high-risk groups including B cell maturation antigen (BCMA)-CAR-T-cell therapy recipients and patients receiving corticosteroids. While CMV end-organ disease is rare, CMV is associated with increased mortality, emphasizing the need to evaluate the broader impact of CMV on long-term hematological, infection, and survival outcomes. Human herpesvirus-6 (HHV-6) has also emerged as a concern, with its diagnosis complicated by overlapping symptoms with neurotoxicity, underscoring the importance of considering viral encephalitis in differential diagnoses. Respiratory viruses are the most common late infections with a higher incidence after BCMA CAR-T-cell therapy. Vaccination remains a critical preventive measure against respiratory viruses but may be less immunogenic following CAR-T-cell therapy. The optimal timing, type of vaccine, and dosing schedule require further investigation., Summary: A better understanding of viral epidemiology and preventive trials are needed to improve infection prevention practices and outcomes following CAR-T-cell therapies., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The remarkable success of mRNA-based coronavirus 2019 (COVID-19) vaccines has propelled the advancement of nanomedicine, specifically in the realm of RNA technology and nanomaterial delivery systems. Notably, significant strides have been made in the development of RNA-based in vivo chimeric antigen receptor (CAR) therapy. In comparison to the conventional ex vivo CAR therapy, in vivo CAR therapy offers several benefits including simplified preparation, reduced costs, broad applicability and decreased potential for carcinogenic effects. This review summarises the RNA-based CAR constructs in in vivo CAR therapy, discusses the current applications of in vivo delivery vectors and outlines the immune cells edited with CAR molecules. We aim for the conveyed messages to contribute towards the advancement of in vivo CAR application., (© 2024 John Wiley & Sons Ltd.)

Despite the great success that chimeric antigen receptor (CAR) T-cells have had in patients with B-cell malignancies and multiple myeloma, they continue to have limited efficacy against most solid tumors. Especially in the pediatric population, pre- and post-treatment biopsies are rarely performed due to ethical reasons, and thus, our understanding is still very limited regarding the mechanisms in the tumor microenvironment by which tumor cells exclude effectors and attract immune-suppressive cells. Nevertheless, based on the principles that are known, current T-cell engineering has leveraged some of these processes and created more potent CAR T-cells. The recent discovery of new oncofetal antigens and progress made in CAR design have expanded the potential pool of candidate antigens for therapeutic development. The most promising approaches to enhance CAR T-cells are novel CAR gating strategies, creative ways of cytokine delivery to the TME without enhancing systemic toxicity, and hijacking the chemokine axis of tumors for migratory purposes. With these new modifications, the next step in the era of CAR T-cell development will be the clinical validation of these promising preclinical findings., Competing Interests: Declarations Ethical approval N/A. Informed consent N/A Competing interests R.N. filed a patent (WO2023158986A1) for GPC2-CARs., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

CAR T cell therapy, hailed as a breakthrough in cancer treatment due to its remarkable outcomes in hematological malignancies, encounters significant hurdles when applied to solid tumors. While notable responses to CAR T cells remain sporadic in these patients, challenges persist due to issues such as on-target off-tumor toxicity, difficulties in their trafficking and infiltration into the tumor, and the presence of a hostile and immunosuppressive microenvironment. This review aims to explore recent endeavors aimed at overcoming these obstacles in CAR T cell therapy for solid tumors. Specifically, we will delve into promising strategies for enhancing tumor specificity through antigen targeting, addressing tumor heterogeneity, overcoming physical barriers, and counteracting the immune-suppressive microenvironment., Competing Interests: Declarations Conflict of interest M.C. has received research support from Kite/Gilead, is a consultant of Alia Therapeutics and is an inventor on different patents on cancer immunotherapy and genetic engineering., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

CAR T-cell products targeting lineage-specific cell-of-origin antigens, thereby eliminating both tumor and healthy counterpart cells, are currently clinically approved therapeutics in B- and plasma-cell malignancies. While they represent a major clinical improvement, they are still limited in terms of efficacy by e.g. single, sometimes low-expressed antigen targeting, and in terms of safety by e.g., lack of on-off activity. Successful cell-of-origin non-discriminative targeting of heterogeneous hematopoietic stem and progenitor cell malignancies, such as acute myeloid leukemia (AML), will require antigen-versatile targeting and off-switching of effectors in order to then allow rescue by hematopoietic stem cell transplantation (HSCT), preventing permanent myeloablation. To address this, we developed adaptor-CAR (AdFITC-CAR) T-cells targeting fluoresceinated AML antigen-binding diabody adaptors. This platform enables the use of adaptors matching the AML-antigen-expression profile and conditional activity modulation. Combining adaptors significantly improved lysis of AML cells in vitro. In therapeutic xenogeneic mouse models, AdFITC-CAR T-cells co-administered with single diabody adaptors were as efficient as direct CAR T-cells, and combinatorial use of adaptors further enhanced therapeutic efficacy against both, cell lines and primary AML. Collectively, this study provides proof-of-concept that AdFITC-CAR T-cells and combinations of adaptors can efficiently enhance immune-targeting of AML., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Chimeric antigen receptor (CAR)-T therapy has demonstrated sustained clinical remission in numerous hematologic malignancies and has expanded to encompass solid tumors and autoimmune diseases. While progress is being made in establishing optimal culture conditions for CAR-T cells, the identification of the most effective cytokine for promoting their persistence in vitro remains elusive., Methods: Here, we employed scRNA-seq (single-cell RNA sequencing) analysis to investigate the potential alterations in biological processes within CAR-T cells following exposure to cytokines (IL-2, IL-12, and IL-21) and antigens. Transcriptomic changes in diverse CAR-T groups were compared following various treatments, with a focus on epigenetic modifications, metabolic shifts, cellular senescence, and exhaustion., Results: Our study reveals that CAR-T cells treated with antigen, IL-2, and IL-12 exhibit signs of exhaustion and senescence, whereas those treated with IL-21 do not display these characteristics. The activities of glycolysis and epigenetic changes were significantly increased by treatments with antigens, IL-2, and IL-12, while IL-21 treatment maintained the oxidative phosphorylation (OXPHOS) of CAR-T cells., Conclusions: Our findings suggest that IL-21 may play a role in preventing senescence and could be utilized in combination with other strategies, such as IL-2 and IL-12, for CAR-T culture., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children. Typical therapy for CNS tumors in children involves a combination of surgery, radiation, and chemotherapy. While upfront therapy is effective for many high-grade tumors, therapy at the time of relapse remains limited. Furthermore, for diffuse intrinsic pontine glioma (DIPG) and diffuse midline glioma (DMG), there are currently no curative therapies. Chimeric antigen receptor T (CAR T) cell therapy is a promising novel treatment avenue for these tumors. Here, we review the preclinical evidence for CAR T cell use in pediatric brain tumors, the preliminary clinical experience of CNS CAR T cell trials, toxicity associated with systemic and locoregional CAR T cell therapy for CNS tumors, challenges in disease response evaluation with CAR T cell therapy, and the knowledge gained from correlative biologic studies from these trials in the pediatric and young adult population., Competing Interests: Declarations Competing interests Dr. Vitanza is chair of the Scientific Board for Brain Child Bio. Otherwise, the authors have no financial or nonfinancial interests that are directly or indirectly related to the work submitted for publication., (© 2024. The Author(s).)

This year marks the tenth anniversary of the world's first transplantation of tissue generated from induced pluripotent stem cells (iPSCs). There is now a growing number of clinical trials worldwide examining the efficacy and safety of autologous and allogeneic iPSC-derived products for treating various pathologic conditions. As we patiently wait for the results from these and future clinical trials, it is imperative to strategize for the next generation of iPSC-based therapies. This review examines the lessons learned from the development of another advanced cell therapy, chimeric antigen receptor (CAR) T cells, and the possibility of incorporating various new bioengineering technologies in development, from RNA engineering to tissue fabrication, to apply iPSCs not only as a means to achieve personalized medicine but also as designer medical applications., (© 2024 Wiley Periodicals LLC.)

The rise of immunotherapy provided new approaches to cancer treatment. We aimed to describe the contribution of chimeric antigen receptor T cell immunotherapy to future prospects. We analyzed 8035 articles from the Web of Science Core Collection with CiteSpace that covered with various aspects with countries, institutions, authors, co-cited authors, journals, keywords, and references. The USA was the most prolific country, with the University of Pennsylvania being the most published institution. Among individual authors, June Carl H published the most articles, while Maude SL was the most frequently co-cited author. "Blood" emerged as the most cited journal. Keyword clustering revealed six core themes: "Expression," "Chimeric Antigen Receptor," "Tumor Microenvironment," "Blinatumomab," "Multiple Myeloma," and "Cytokine Release Syndrome." In the process of researching the timeline chart of keywords and references, "Large B-cell lymphoma" was located on the right side of the timeline. In the keyword prominence analysis, we found that the keywords "biomarkers," "pd-1," "antibody drug conjugate," "BCMA," and "chimeric antigen" had high explosive intensity in the recent past. We found that in terms of related diseases, "large B-cell lymphoma" and "cytokine release syndrome" are still difficult problems in the future. In the study of therapeutic methods, "BCMA," "PD-1," "chimeric antigen," and "antibody drug conjugate" deserve more attention from researchers in the future., Competing Interests: Declarations. Ethical approval: No ethical approval was required for all analyses. Consent to participate: Informed consent was obtained from all individual participants included in the study. Consent for publication: The authors affirm that human research participants provided informed consent for publication. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The promising results seen in the treatment of refractory hematologic malignancies with tisagenlecleucel (Kymriah), the pioneering Chimeric Antigen Receptor (CAR) T-cell immunotherapy, has stimulated a fast growth in research and clinical testing of novel CAR-T constructs, targets, and various generations of CAR T-cells. Pharmacists may receive inquiries about active clinical trials or may be contributing to the care of patients participating in these studies. This briefing discusses the on-going clinical trials that explore novel CAR T-cell immunotherapies in pediatric refractory malignancies of hematologic and solid organ origins. It can be valuable in assisting practitioners in navigating the rapidly evolving field of CAR T-cell immunotherapies., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Clinical trials evaluating chimeric antigen receptor (CAR) T-cell therapy in relapsed/refractory multiple myeloma (RRMM) have typically excluded patients with AL amyloidosis. As a result, there are limited data on the safety and efficacy of CAR T-cell therapy in this patient population. We retrospectively reviewed eight consecutive patients with RRMM and AL amyloidosis who were treated with standard of care CAR T-cell therapy. Cytokine release syndrome was seen in 75% of patients (grade ≥3: 0%) and immune effector cell-associated neurotoxicity syndrome (grade 1) in only one patient. Low-grade cytopenias were common (any grade/grade ≥3: neutropenia 62.5%/37.5%, anemia 37.5%/0%, thrombocytopenia 25%/0%). CAR T-cell therapy led to rapid and deep responses with a median time to best response of 43 days and a hematologic very good partial response or better rate of 62.5%. Overall, we found that commercial CAR T-cell therapy was feasible, and effective in patients with RRMM and concurrent AL amyloidosis., (© 2024 The Author(s). European Journal of Haematology published by John Wiley & Sons Ltd.)

Background: Chimeric antigen receptor T-cell (CAR-T) therapy has shown promise in treating hematologic malignancies, yet its potential cardiotoxic effects require thorough investigation., Objectives: We aim to conduct a systematic review and meta-analysis to examine the cardiotoxic effects of CAR-T therapy in adults with hematologic malignancies., Methods: We searched PubMed, Embase, and the Cochrane Central Register of Controlled Trials for studies reporting cardiovascular outcomes, such as arrhythmias, heart failure, and reduced left ventricle ejection fraction (LVEF)., Results: Our analysis of 20 studies involving 4789 patients revealed a 19.68% incidence rate of cardiovascular events, with arrhythmias (7.70%), heart failure (5.73%), and reduced LVEF (3.86%) being the most prevalent. Troponin elevation was observed in 23.61% of patients, while NT-Pro-BNP elevation was observed in 9.4. Subgroup analysis showed higher risks in patients with pre-existing conditions, such as atrial arrhythmia (OR 3.12; p < .001), hypertension (OR 1.85; p = .002), previous heart failure (OR 3.38; p = .003), and coronary artery disease (OR 2.80; p = .003)., Conclusion: Vigilant cardiovascular monitoring is crucial for patients undergoing CAR-T therapy to enhance safety and treatment efficacy.Novelty Statements., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Chimeric antigen receptor (CAR) T-cell therapy is highly effective for treating blood cancers such as B-cell malignancies, however, its effectiveness as an approach to treat solid tumors remains to be further explored. Here, we focused on the development of CAR-T cell therapies targeting tropomyosin-related kinase receptor B (TRKB), a highly expressed protein that is significantly associated with tumor progression, malignancy, and drug resistance in multiple forms of aggressive solid tumors. To achieve this, we screened brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NTF4) ligand-based CAR-T cells for their efficiency in targeting the TRKB receptor in the context of solid tumors, particularly hepatocellular carcinoma and pancreatic cancer. We demonstrated that TRKB is overexpressed not only in hepatocellular carcinoma and pancreatic carcinoma cell lines but also in cancer stem-like cells (CSCs). Notably, BDNF-CAR T and NTF4-CAR T cells could not only effectively target and kill TRKB-expressing pan-cancer cell lines in a dose-dependent manner but also effectively kill CSCs. We also performed in vivo studies to show that NTF4-CAR T cells have a better potential to inhibit the tumor growth of hepatocellular carcinoma xenografts in mice, compared with BDNF-CAR T cells. Taken together, our findings suggest that CAR-T targeting TRKB may be a promising approach for developing novel therapies to treat solid cancers., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Relapse/refractory B-cell acute lymphoblastic leukaemia (r/r B-ALL) represents paediatric cancer with a challenging prognosis. CAR T-cell treatment, considered an advanced treatment, remains controversial due to high relapse rates and adverse events. This study assessed the efficacy and safety of CAR T-cell therapy for r/r B-ALL., Methods: The literature search was performed on four databases. Efficacy parameters included minimal residual disease negative complete remission (MRD-CR) and relapse rate (RR). Safety parameters constituted cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS)., Results: Anti-CD22 showed superior efficacy with the highest MRD-CR event rate and lowest RR, compared to anti-CD19. Combining CAR T-cell therapy with haploidentical stem cell transplantation improved RR. Safety-wise, bispecific anti-CD19/22 had the lowest CRS rate, and anti-CD22 showed the fewest ICANS. Analysis of the costimulatory receptors showed that adding CD28ζ to anti-CD19 CAR T-cell demonstrated superior efficacy in reducing relapses with favorable safety profiles., Conclusion: Choosing a more efficacious and safer CAR T-cell treatment is crucial for improving overall survival in acute leukaemia. Beyond the promising anti-CD22 CAR T-cell, exploring costimulatory domains and new CD targets could enhance treatment effectiveness for r/r B-ALL.

Over the past decade, chimeric antigen receptor (CAR)-T cell therapy has emerged as a revolutionary immunotherapeutic approach to combat cancer. This therapy constructs a CAR on the surface of T cells through genetic engineering techniques. The CAR is formed from a combination of antibody-derived or ligand-derived domains and T-cell receptor (TCR) domains. This enables T cells to specifically bind to and activate against tumor cells. However, the efficacy of CAR-T cells in solid tumors remains inconclusive due to several challenges such as poor tumor trafficking, infiltration, and the immunosuppressive tumor microenvironment (TME). In response, CAR natural killer (CAR-NK) and CAR macrophages (CAR-M) have been developed as complementary strategies for solid tumors. CAR-NK cells do not require HLA compatibility, demonstrate reduced toxicity, and are thus seen as potential substitutes for CAR-T cells. Furthermore, CAR-M immunotherapy is also being researched and has shown phagocytic capabilities and tumor-antigen presentation. This study discusses the features, advantages, and limitations of CAR-T, CAR-NK, and CAR-M cells in the treatment of solid tumors and suggests prospective solutions for enhancing the efficacy of CAR host-cell-based immunotherapy., Competing Interests: Declarations. Conflict of interest: 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., (© 2024. The Author(s).)

Background: Pancreatic cancer is known for its poor prognosis and resistance to conventional therapies, largely due to the presence of cancer stem cells (CSCs) and aggressive angiogenesis. Effectively targeting these CSCs and associated angiogenic pathways is crucial for effective treatment. This study leverages single-cell multi-omics to explore a novel therapeutic approach involving Chimeric Antigen Receptor (CAR) macrophages engineered to target the c-Met protein on pancreatic CSCs., Methods: We employed single-cell RNA sequencing to analyze pancreatic cancer tissue, identifying c-Met as a key marker of CSCs. CAR macrophages were engineered using a lentiviral system to express a c-Met-specific receptor. The phagocytic efficiency of these CAR macrophages against pancreatic CSCs was assessed in vitro, along with their ability to inhibit angiogenesis. The in vivo efficacy of CAR macrophages was evaluated in a mouse model of pancreatic cancer., Results: CAR macrophages demonstrated high specificity for c-Met + CSCs, significantly enhancing phagocytosis and reducing the secretion of angiogenic factors such as VEGFA, FGF2, and ANGPT. In vivo, these macrophages significantly suppressed tumor growth and angiogenesis, prolonging survival in pancreatic cancer-bearing mice., Conclusion: CAR macrophages targeting c-Met represent a promising therapeutic strategy for pancreatic cancer, offering targeted elimination of CSCs and disruption of tumor angiogenesis. This study highlights the potential of single-cell multi-omics in guiding the development of precision immunotherapies., Competing Interests: Declarations. Ethical statement: All animal experiments reported in this study were approved by the Animal Ethics Committee of Jiaxing University Medical Center (Approval No. JUMC2024-087). Consent for publication: Not Applicable. Consent for publication: The authors declare that this manuscript is original, has not been published before, and is not currently being considered for publication elsewhere. All authors have approved the manuscript and agree with its submission to Molecular Medicine. Competing interests: The authors declare no competing interests. Conflict of interest: The author declares no conflict of interest., (© 2024. The Author(s).)

The treatment of multiple myeloma has changed dramatically in recent years, with huge strides forward made in the field. Chimeric antigen receptor T-cell therapy targeting the B cell maturation antigen (BCMA) is now widely approved in relapsed refractory patients and is moving into earlier treatment lines. In this review, we discuss the evidence underpinning current regulatory approvals and consider mechanisms through which CAR-T cell efficacy could be improved. These include tackling BCMA-loss, harnessing the immunosuppressive tumour microenvironment, manufacturing concerns including the potential role of other cellular sources, safety issues such as cytokine release syndrome and neurotoxicity, and optimal patient selection., Competing Interests: Competing interests: DS: none; DM: Employment: Johnson and Johnson; JH: none., (© 2024. The Author(s).)

Regenerative medicine is a rapidly expanding field harnessing human pluripotent stem cell (hPSC)-derived cells and tissues to treat many diseases, including type 1 diabetes. However, graft immune protection remains a key challenge. Chimeric antigen receptor (CAR) technology confers new specificities to effector T cells and immunosuppressive regulatory T cells (Tregs). One challenge in CAR design is identifying target molecules unique to the cells of interest. Here, we employ combinatorial genetic engineering to confer CAR-Treg-mediated localized immune protection to stem cell-derived cells. We engineered hPSCs to express truncated epidermal growth factor receptor (EGFRt), a biologically inert and generalizable target for CAR-Treg homing and activation, and generated CAR-Tregs recognizing EGFRt. Strikingly, CAR-Tregs suppressed innate and adaptive immune responses in vitro and prevented EGFRt-hPSC-derived pancreatic beta-like cell (sBC [stem cell-derived beta cell]) graft immune destruction in vivo. Collectively, we provide proof of concept that hPSCs and Tregs can be co-engineered to protect hPSC-derived cells from immune rejection upon transplantation., Competing Interests: Declaration of interests A provisional patent on the strategy described in this article has been submitted with L.M.R.F. and H.A.R. as inventors. L.M.R.F. is an inventor in and has received royalties from patents on genetically modified human stem cells and T cells and consults for Guidepoint Global and McKesson. H.A.R. holds patents in the regenerative medicine space and served as a science advisory board (SAB) member of Sigilon Therapeutics and Prellis Biologics and consults or has consulted for Sigilon Therapeutics, Eli Lilly, Minutia, Guidepoint Global, Axon Advisors, and Tolerance Bio. H.A.R. is a scientific cofounder of Tolerance Bio., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Targeting tumor-specific neoantigens holds promise for cancer immunotherapy, but their ultra-low expression on tumor cells poses challenges for T cell therapies. Here, we found that chimeric antigen receptors (CARs) exhibit 10-100 times lower sensitivity than T cell receptors (TCRs) when targeting human leukocyte antigen (HLA) class I-presented p53 R175H neoantigen. To enhance CAR functionality, we introduced T cell receptor fusion constructs (TRuCs) and synthetic TCRs and antigen receptors (STARs). Our data show that STARs optimally reproduce TCR antigen sensitivity, outperforming CARs and TRuCs in redirecting CD8 + and CD4 + T cells to recognize HLA class I neoantigens. STAR-T cells demonstrate superior killing of cancer cell lines with low neoantigen density in vitro and improved tumor control in mouse models compared to CAR-T and TRuC-T cells. These findings highlight CAR sensitivity limitations and present STARs as more effective synthetic receptors for T cell-based immunotherapy against tumors with low neoantigen density., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Patients who develop chimeric antigen receptor (CAR) T-cell-related immune effector cell-associated neurotoxicity syndrome (ICANS) frequently undergo evaluation with electroencephalography (EEG). We hypothesize that EEG features and Synek scale score, a measure of degree of EEG abnormality, are associated with ICANS severity. Here, we performed a retrospective review of 125 adult patients at Memorial Sloan Kettering Cancer Center (MSKCC) who received CAR-T cell therapy from 2010 to 2019, including 53 patients with B-acute lymphoblastic leukemia treated with 1928z CAR T cells (NCT01044069) and 72 patients with large B-cell lymphoma (LBCL) treated with the commercial CAR T products axicabtagene ciloleucel or tisagenlecleucel. We collected video EEG monitoring (27 with B-ALL and 20 with LBCL) and recorded daily EEG features, Synek scores, and ICANS grade for 47 eligible patients. Synek scale and ICANS grade were positively correlated (correlation coefficient 0.47, 95% CI: 0.31-0.60). This was further corroborated in the univariable model associating high Synek scale (3 or 4) with high ICANS grade (OR = 15.2; 95%CI:7.8-29.7, p < 0.0001). EEG features such as discontinuity, absence of posterior dominant rhythm, and presence of generalized sharp waves were statistically significantly associated with higher ICANS grade in univariable models. In the multivariable model, discontinuity (OR = 4.2 (95%CI:1.3-13.8, p = 0.02) and absence of posterior dominant rhythm (OR = 10.5 (95%CI:4.6-23.9, p < 0.0001) were statistically associated with higher ICANS grade. Overall, EEG discontinuity and absence of posterior dominant rhythm were independently associated with higher severity of neurotoxicity. Further, our data suggest that Synek Scale, may be a severity marker for neurotoxicity., Competing Interests: Declarations. Competing interests: Dr. Perales reports honoraria from Adicet, Allovir, Caribou Biosciences, Celgene, Bristol-Myers Squibb, Equilium, Exevir, Incyte, Karyopharm, Kite/Gilead, Merck, Miltenyi Biotec, MorphoSys, Nektar Therapeutics, Novartis, Omeros, OrcaBio, Syncopation, VectivBio AG, and Vor Biopharma. He serves on DSMBs for Cidara Therapeutics, Medigene, and Sellas Life Sciences, and the scientific advisory board of NexImmune. He has ownership interests in NexImmune, Omeros and OrcaBio. He has received institutional research support for clinical trials from Incyte, Kite/Gilead, Miltenyi Biotec, Nektar Therapeutics, and Novartis., (© 2024. The Author(s).)

The application of chimeric antigen receptor (CAR) T cell therapy in solid tumors is hindered by life-threatening toxicities resulting from on-target, off-tumor killing of nonmalignant cells that express low levels of the target antigen. Mesothelin (MSLN) has been identified as a target antigen for CAR T cell treatment of mesothelioma, lung, ovarian, and other cancers because of its high expression on tumor cells and limited expression on mesothelial cells. However, fatal off-tumor toxicity of high-affinity MSLN-targeting CAR T cells has been reported in multiple clinical trials. In this study, we constructed CARs using mutant variants of a single-domain nanobody that bind both human and mouse MSLN with a wide range of affinities and examined tumor responses and their toxicities from on-target, off-tumor interactions in mouse models. CAR T cells with low nanomolar affinity (equilibrium dissociation constant, KD) exhibited profound systemic expansion with no apparent infiltration into the tumor. With a gradual reduction of CAR affinity toward the micromolar KD, the expansion of CAR T cells became more restricted to tumors. Our preclinical studies demonstrated that high-affinity MSLN CARs were associated with fatal on-target, off-tumor toxicity and that affinity-tuned CARs rendered T cells more selective for MSLN-high tumors.

Background: Oncofetal splice variants of extracellular matrix (ECM) proteins present a unique group of target antigens for the immunotherapy of pediatric cancers. However, limited data is available if these splice variants can be targeted with T cells expressing chimeric antigen receptors (CARs)., Methods: To determine the expression of the oncofetal version of tenascin C (TNC) encoding the C domain (C.TNC) in pediatric brain and solid tumors, we used quantitative reverse transcription PCR and immunohistochemistry. Genetically modified T cells were generated from human peripheral blood mononuclear cells and evaluated in vitro and in vivo., Results: We demonstrate that C.TNC is expressed on a protein level in pediatric tumors, including diffuse intrinsic pontine glioma, osteosarcoma, rhabdomyosarcoma, and Ewing sarcoma. We generate C.TNC-CAR T cells and establish that these recognize and kill C.TNC-positive tumor cells. However, their antitumor activity in vivo is limited. To improve the effector function of C.TNC-CAR T cells, we design a leucine zipper-based chimeric cytokine receptor that activates interleukin-18 signaling pathways (Zip18R). Expression of Zip18R in C.TNC-CAR T cells improves their ability to secrete cytokines and expand in repeat stimulation assays. C.TNC-CAR.Zip18R T cells also have significantly greater antitumor activity in vivo compared with unmodified C.TNC-CAR T cells., Conclusions: Our study identifies the C domain of the ECM protein TNC as a promising CAR T-cell therapy for pediatric solid tumors and brain tumors. While we focus here on pediatric cancer, our work has relevance to a broad range of adult cancers that express C.TNC., Competing Interests: Competing interests: EW, SL, JW, TIS, GK, JZ, and SG have patent applications in the fields of cell or gene therapy for cancer. MB, GK, and SG are coinventors on a patent application for the developed Zip receptor technology. SG is a member of the Scientific Advisory Board of Be Biopharma and CARGO, and the Data and Safety Monitoring Board (DSMB) of Immatics and has received honoraria from TESSA Therapeutics within the last year. The other authors declare no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The development of chimeric antigen receptor (CAR)-T cell therapies for solid tumors has attracted considerable attention, yet their clinical efficacy remains limited. Therefore, various efforts have been made to improve the efficacy of CAR-T cell therapy. As one promising strategy, incorporating the T-cell receptor (TCR) machinery into CAR structures has been reported to improve the efficacy of CAR-T cells in studies using conventional CARs targeting such as EGFR. However, in the case of peptide/major histocompatibility complex (pMHC)-targeted CARs, the advantages of exploiting TCR machinery have not been fully elucidated. We recently developed MAGE-A4-derived pMHC (MAGE-A4 pMHC)-targeted CAR-T cells (MA-CAR-T cells) using a highly specific human scFv antibody against MAGE-A4 p230-239 /HLA-A*02:01. We aimed to determine whether MAGE-A4 pMHC-targeted CAR-T cells using the TCR machinery (Hybrid MA-TCR-T cells) exhibit superior functionality without compromising antigen specificity., Methods: We constructed a retroviral vector expressing Hybrid MA-TCR where MAGE-A4 pMHC-specific scFv are fused to human TCR constant chains., Results: Hybrid MA-TCR-T cells demonstrated superior in vitro functions compared with MA-CAR-T cells, while maintaining strict antigen specificity. In addition, functional superiority of Hybrid MA-TCR-T cells to MA-CAR-T cells became more pronounced on repetitive antigen stimulation. In particular, Hybrid MA-TCR-T cells significantly inhibited tumor growth in an immunodeficient mouse model more effectively than MA-CAR-T cells. Ex vivo analyses indicated that their enhanced therapeutic efficacy might result from higher infiltration of functionally active, less differentiated Hybrid MA-TCR-T cells in tumor tissues., Conclusions: These findings suggest that leveraging the TCR machinery is a promising strategy for enhancing pMHC-targeted CAR-T cell therapy for solid tumors, potentially leading to more effective treatments., Competing Interests: Competing interests: The Department of Personalized Cancer Immunotherapy, Mie University Graduate School of Medicine, is an endowment department supported by a grant from T Cell Nouveau Inc. NI and KN are employees of T Cell Nouveau Inc., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

NKG2D ligands (NKG2DLs) are broadly expressed in cancer. To target these, we describe an adaptor chimeric antigen receptor (CAR) termed NKG2D/Dap10-12. Herein, T cells are engineered to co-express NKG2D with a fusion protein that comprises Dap10 joined to a Dap12 endodomain. NKG2D/Dap10-12 T cells elicit compelling efficacy, eradicating or controlling NKG2DL-expressing tumors in several established xenograft models. Importantly, durable responses, long-term survival, and rejection of tumor re-challenge are reproducibly achieved. Efficacy is markedly superior to a clinical stage CAR analog, comprising an NKG2D-CD3ζ fusion. Structure-function analysis using an extended CAR panel demonstrates that potency is dependent on membrane proximity of signaling units, high NKG2D cell surface expression, adaptor structure, provision of exogenous Dap10, and inclusion of one rather than three immune tyrosine activation motifs per signaling unit. Potent therapeutic impact of NKG2D/Dap10-12 T cells is also underpinned by enhanced oxidative phosphorylation, reduced senescence, and transcriptomic re-programming for increased ribosomal biogenesis., Competing Interests: Declaration of interests J.M. is CSO, scientific founder, and shareholder of Leucid Bio. M.G., F.K., C.M.H., R.M., C.B., C.T., and D.M.D. are employees of Leucid Bio while P.D. is a former Leucid Bio employee. D.L.-Y. is undertaking a PhD studentship funded by Leucid Bio and has acted as a consultant for Leucid Bio. J.M., D.M.D., D.L.-Y., and F.K. are co-inventors on patent filings in relation to adaptor CAR technology., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Glioblastoma is the most common lethal primary brain tumor, urging evaluation of new treatment options. Chimeric antigen receptor (CAR)-T cells targeting B7 homolog 3 (B7-H3) are promising because of the overexpression of B7-H3 on glioblastoma cells but not on healthy brain tissue. Nanobody-based (nano)CARs are gaining increasing attention as promising alternatives to classical single-chain variable fragment-based (scFv)CARs, because of their single-domain nature and low immunogenicity. Still, B7-H3 nanoCAR-T cells have not been extensively studied in glioblastoma., Methods: B7-H3 nanoCAR- and scFvCAR-T cells were developed and evaluated in human glioblastoma models. NanoCAR-T cells targeting an irrelevant antigen served as control. T cell activation, cytokine secretion and killing capacity were evaluated in vitro using ELISA, live cell imaging and flow cytometry. Antigen-specific killing was assessed by generating B7-H3 knock-out cells using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-genome editing. The tumor tracing capacity of the B7-H3 nanobody was first evaluated in vivo using nuclear imaging. Then, the therapeutic potential of the nanoCAR-T cells was evaluated in a xenograft glioblastoma model., Results: We showed that B7-H3 nanoCAR-T cells were most efficient in lysing B7-H3 pos glioblastoma cells in vitro. Lack of glioblastoma killing by control nanoCAR-T cells and lack of B7-H3 neg glioblastoma killing by B7-H3 nanoCAR-T cells showed antigen-specificity. We showed in vivo tumor targeting capacity of the B7-H3 nanobody-used for the nanoCAR design-in nuclear imaging experiments. Evaluation of the nanoCAR-T cells in vivo showed tumor control in mice treated with B7-H3 nanoCAR-T cells in contrast to progressive disease in mice treated with control nanoCAR-T cells. However, we observed limiting toxicity in mice treated with B7-H3 nanoCAR-T cells and showed that the B7-H3 nanoCAR-T cells are activated even by low levels of mouse B7-H3 expression., Conclusions: B7-H3 nanoCAR-T cells showed promise for glioblastoma therapy following in vitro characterization, but limiting in vivo toxicity was observed. Off-tumor recognition of healthy mouse tissue by the cross-reactive B7-H3 nanoCAR-T cells was identified as a potential cause for this toxicity, warranting caution when using highly sensitive nanoCAR-T cells, recognizing the low-level expression of B7-H3 on healthy tissue., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The impact of phenotypic, clonal, and functional heterogeneity of chimeric antigen receptor (CAR)-T cells on clinical outcome remains understudied. Here, we integrate clonal kinetics with transcriptomic heterogeneity resolved by single-cell omics to interrogate cellular dynamics of non-transduced (CAR neg ) and transduced (CAR pos ) T cells, in the infusion product (IP) and at the CAR-T cell expansion peak in five B cell acute lymphoblastic leukemia (B-ALL) patients treated with CD19CAR-T cells (varni-cel). We identify significant differences in cellular dynamics in response to therapy. CAR pos T cells at IP of complete response patients exhibit a significantly higher CD4:CD8 ratio, validated in a larger cohort B-ALL patients (n = 47). Conversely, at the expansion peak, there is a clonal expansion of CD8 + effector memory and cytotoxic T cells. Cytotoxic CAR pos γδ-T cells expansion correlates with treatment efficacy validated in a cohort of B-ALL (n = 18) and diffuse large B cell lymphoma (DLBCL) patients (n = 58). Our data provide insights into the complexity of T cell responses following CAR-T cell therapy and suggest drivers of immunotherapy response., Competing Interests: Declaration of interests P.M. is a founder of the spin-off OneChain ImmunoTx, which has no connection with the present research. V.O.-M. reports honoraria and/or consulting fees from BMS/Celgene, Novartis, Gilead/Kite, Miltenyi Biomedicine, Pfizer, and Janssen. A.M.G.-S. reports honoraria and/or consulting fees from Roche, BMS/Celgene, Kyowa Kirin, Novartis, Gilead/Kite, Incyte, Lilly, ADC Therapeutics America, Miltenyi, Ideogen, AbbVie, and Sobi., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lambert-Eaton myasthenic syndrome (LEMS) is an autoantibody-mediated disease of the neuromuscular junction characterized by muscular weakness. Autoantibodies to presynaptic P/Q-type voltage-gated calcium channels (VGCCs) induce defective neuromuscular function. In severe cases, current immunosuppressive and immunomodulatory treatment strategies are often insufficient. First reports show beneficial effects of anti-CD19 chimeric antigen receptor (CAR)-T cell therapy in patients with autoantibody-mediated myasthenia gravis. We report a patient with isolated idiopathic LEMS treated with autologous anti-CD19-CAR-T cells. In this patient, CAR-T infusion leads to expansion of predominantly CD4 + CAR-T cells with a terminally differentiated effector memory cells re-expressing CD45RA (TEMRA)-like phenotype indicating cytotoxic capabilities and subsequent B cell depletion. VGCC antibody titers decrease, resulting in a clinical improvement of LEMS symptoms, e.g., 8-fold increase in walking distance. The patient does not show relevant side effects except for cytokine release syndrome grade 2 and intermittent neutropenia suggesting that anti-CD19 CAR-T cell therapy may be a treatment option in patients with LEMS., Competing Interests: Declaration of interests D.B. is an employee and shareholder of Kyverna Therapeutics with pending patent applications., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)