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Background: Chimeric antigen receptor T-cell (CAR-T) therapy, a rapidly emerging treatment for cancer that has gained momentum since its approval by the FDA in 2017, involves the genetic engineering of patients' T cells to target tumors. Although significant therapeutic benefits have been observed, lifethreatening adverse pulmonary events have been reported. Methods: Using SAS 9.4 with MedDRA 26.1, we retrospectively analyzed data from the Food and Drug Administration's Adverse Event Reporting System (FAERS) database, covering the period from 2017 to 2023. The analysis included the Reporting Odds Ratio Proportional Reporting Ratio Information Component and Empirical Bayes Geometric Mean to assess the association between CAR-T cell therapy and adverse pulmonary events (PAEs). Results: The FAERS database recorded 9,400 adverse events (AEs) pertaining to CAR-T therapies, of which 940 (10%) were PAEs. Among these CAR-T cell-related AEs, hypoxia was the most frequently reported (344 cases), followed by respiratory failure (127 cases). Notably, different CAR-T cell treatments demonstrated varying degrees of association with PAEs. Specifically, Tisa-cel was associated with severe events including respiratory failure and hypoxia, whereas Axi-cel was strongly correlated with both hypoxia and tachypnea. Additionally, other CAR-T therapies, namely, Brexu-cel, Liso-cel, Ide-cel, and Cilta-cel, have also been linked to distinct PAEs. Notably, the majority of these PAEs occurred within the first 30 days post-treatment. The fatality rates varied among the different CAR-T therapies, with Tisa-cel exhibiting the highest fatality rate (43.6%), followed by Ide-cel (18.8%). Conclusion: This study comprehensively analyzed the PAEs reported in the FAERS database among recipients of CAR-T cell therapy, revealing conditions such as hypoxia, respiratory failure, pleural effusion, and atelectasis. These CAR-T cell therapy-associated events are clinically significant and merit the attention of clinicians and researchers. [ABSTRACT FROM AUTHOR]

Simple Summary: This review explores the emerging area of the therapeutic use of chimeric antigen receptor (CAR)-T cells in nervous tissue tumors. Through a detailed analysis of the existing literature, this paper highlights the most recent applications of CAR-T cells to the therapy of pediatric and adult nervous system neoplasia. Furthermore, it discusses the potential mechanisms underlying sensitivity or resistance to CAR-T cell-based therapies. Overall, this review underscores the importance of CAR-T cell therapies to the treatment of some nervous system tumors. Chimeric antigen receptor T cells (CAR-Ts) have shown a remarkable efficacy in hematological malignancies but limited responses in solid tumors. Among solid tumors, CAR-T cell therapy has been particularly explored in brain tumors. CAR-T cells have shown a limited clinical efficacy in various types of brain tumors due to several factors that have hampered their activity, including tumor antigen heterogeneity, the limited access of CAR-T cells to brain tumor cells, limited CAR-T cell trafficking and in vivo persistence and the presence of a highly immunosuppressive tumor microenvironment. Despite these considerations, some recent studies have shown promising antitumor activity of GD2-CAR-T cells on diffuse midline gliomas and neuroblastomas and of CARv3-TEAM-E cells in glioblastomas. However, strategies are required to improve the effect of CAR-T cells in brain tumors, including advanced CAR-T cell design with multiple antigenic targeting and incorporation of combination therapies. [ABSTRACT FROM AUTHOR]

Objective: To construct chimeric antigen receptor (CAR)-T cells targeting epithelial cell adhesion molecule (EpCAM) antigen (anti-EpCAM-CAR-T). Methods: A third-generation CAR-T cell construct used a single-chain variable fragment derived from monoclonal antibody against human EpCAM. Peripheral blood mononuclear cells were extracted from volunteers. The proportion of cluster of differentiation 8 positive (CD8+) and CD4 + T cells was measured using flow cytometry. Western blot was used to detect the expression of EpCAM-CAR. The killing efficiency was detected using the MTT assay and transwell assay, and the secretion of killer cytokines tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was detected using the ELISA. The inhibitory effect of EpCAM-CAR-T on colorectal cancer in vivo was detected using xenografts. Results: It was found that T cells expanded greatly, and the proportion of CD3+, CD8 + and CD4 + T cells was more than 60%. Furthermore, EpCAM-CAR-T cells had a higher tumour inhibition rate in the EpCAM expression positive group than in the negative group (P < 0.05). The secretion of killer cytokines TNF-α and IFN-γ in the EpCAM expression positive cell group was higher than that in the negative group (P < 0.05). In the experimental group treated with EpCAM-CAR-T cells, the survival rate of nude mice was higher (P < 0.05), and the tumour was smaller than that in the blank and control groups (P < 0.05). The secretion of serum killer cytokines TNF-α and IFN-γ in tumour-bearing nude mice in the experimental group treated with EpCAM-CAR-T cells was higher than that in the blank and control groups (P < 0.05). Conclusion: This study successfully constructed EpCAM-CAR cells and found that they can target and recognise EpCAM-positive tumour cells, secrete killer cytokines TNF-α and IFN-γ and better inhibit the growth and metastasis of colorectal cancer in vitro and in vivo than unmodified T cells. [ABSTRACT FROM AUTHOR]

Background: Tocilizumab is commonly used for the management of chimeric antigen receptor (CAR) T‐cell therapy–associated cytokine release syndrome (CRS). However, it remains unknown whether tocilizumab or its dosage affects the efficacy and safety of CAR T‐cell therapy. The objective of this multicenter retrospective study was to explore the impact of tocilizumab on CAR T‐cell therapy. Methods: In total, 93 patients with B‐cell acute lymphoblastic leukemia (B‐ALL) receiving humanized anti‐CD19 CAR T cells were recruited from May 2016 to November 2022. Forty‐five patients received tocilizumab (tocilizumab group), whereas 48 patients did not (nontocilizumab group). Thirteen patients received >1 dose of tocilizumab. The primary end point was the effect of tocilizumab on the efficacy and safety of CAR T cells. Additionally, proliferation, killing, and cytokine assays of CAR T cells were performed in vitro in the presence of tocilizumab. Results: The median age of the patients was 33 years, with 47 males and 46 females. Patients in the tocilizumab group showed similar complete response (CR) rate, overall survival (OS), and event‐free survival (EFS) compared with the nontocilizumab group. Compared with patients who received ≤1 dose of tocilizumab, receiving >1 dose of tocilizumab did not affect their CR rate, OS, or EFS. In the tocilizumab group, all patients experienced CRS and 26.7% experienced immune effector cell–associated neurotoxicity syndrome (ICANS). In the nontocilizumab group, 64.6% of patients experienced CRS and 8.3% experienced ICANS. Up to 75% of ICANS and 87.5% of grade ≥3 ICANS occurred in the tocilizumab group. In vitro, tocilizumab did not impair the proliferation and killing effects of CAR T cells. Conclusions: Tocilizumab does not affect the efficacy of CAR T cells but may increase the likelihood of ICANS. Tocilizumab does not affect the efficacy of anti‐CD19 chimeric antigen receptor (CAR) T cells and the survival outcome of patients with B‐cell acute lymphoblastic leukemia. Tocilizumab may increase the likelihood of immune effector cell–associated neurotoxicity syndrome after CAR T‐cell therapy. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor T (CAR-T) cells therapy is a milestone achievement in the immunotherapy of relapsed and refractory (R/R) B cell acute lymphoblastic leukemia (B-ALL). However, some patients treated with CAR-T cells do not achieve complete remission, the mechanisms of which have not been elucidated. In the present study, we report a 9-year-old pediatric patient with refractory B-ALL received a triple infusion of autologous CD19 CAR-T cells therapy after the second relapse. CAR-T cells expanded in the peripheral blood and bone marrow. However, the patient did not achieve complete remission, indicating a lack of response to CAR-T cells therapy. Analysis of etiological factors revealed that the number of CD4 and CD8 double-negative T (DNT) cells was significantly upregulated in the peripheral blood, bone marrow, and autologous CAR-T cells products. In conclusiont, these findings indicate that DNT cells mediated resistance to CAR-T cells therapy in this pediatric patient with R/R B-ALL. [ABSTRACT FROM AUTHOR]

Objective To investigate the feasibility of granzyme B (GB) promoter-controlled ferritin heavy chain (FTH1) reporter gene expression for monitoring the activation status of chimeric antigen receptor T cells (CAR-T) by magnetic resonance imaging (MRI). Methods Cytotoxic T lymphocytes (CTLs) were screened by Ficoll density gradient centrifugation and flow sorting. The GB promoter and FTH1 gene were ligated together with disialoganglioside 2 (GD2) CAR, and lentiviral vectors were transfected into CTLs to construct GD2-CAR-T/pGB-FTH1 cells. GD2-CAR-T/pCMV-FTH1, GD2-CAR-T, and T cells served as control cells. CytoTox96@non-radioactive cytotoxicity was used to detect the killing effect of each group of cells after co-culture with human neuroblastoma cells (SK-N-SH). ELISA was employed to detect the coincubation factor as well as the amount of GB secretion. Western blotting, Prussian blue staining and cellular MRI were applied to detect the expression of the FTH1 gene after co-culture. Results CTLs were successfully obtained, and then GD2-CAR-T/pGB-FTH1, GD2-CAR-T/pCMV-FTH1 and GD2-CAR-T cells were constructed. The killing effect, co-incubation factor and GB secretion of the above 3 groups of cells were significantly higher than those of the T cells, and the level of GB expression was highest at day 1, and then decreased in order at day 3 and day 7 after co-culturing with SK-N-SH cells. The relative expression of FTH1 and iron content of the GD2-CAR-T/pGB-FTH1 cells showed the same trend as GB expression, and the MRI signals were gradually increased. There were no significant differences in the relative expression of FTH1, iron content and MRI signals in the GD2-CAR-T/pCMV-FTH1 cells at all time points. No FTH1 expression or iron aggregation was observed in the GD2-CAR-T and T cells groups. Conclusion MRI based on the FTH1 reporter gene driven by the granzyme B promoter can reflect the GB expression level and tumor killing effect of CAR-T cells, which provides a potential real-time visual means to monitor the cell activation status for CAR-T therapy.

Abstract Objective To construct chimeric antigen receptor (CAR)-T cells targeting epithelial cell adhesion molecule (EpCAM) antigen (anti-EpCAM-CAR-T). Methods A third-generation CAR-T cell construct used a single-chain variable fragment derived from monoclonal antibody against human EpCAM. Peripheral blood mononuclear cells were extracted from volunteers. The proportion of cluster of differentiation 8 positive (CD8+) and CD4 + T cells was measured using flow cytometry. Western blot was used to detect the expression of EpCAM-CAR. The killing efficiency was detected using the MTT assay and transwell assay, and the secretion of killer cytokines tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was detected using the ELISA. The inhibitory effect of EpCAM-CAR-T on colorectal cancer in vivo was detected using xenografts. Results It was found that T cells expanded greatly, and the proportion of CD3+, CD8 + and CD4 + T cells was more than 60%. Furthermore, EpCAM-CAR-T cells had a higher tumour inhibition rate in the EpCAM expression positive group than in the negative group (P

Chordoma is a rare bone tumor that frequently recurs after surgery, and the prognosis is poor with current treatments. This study aimed to identify potential novel immunotherapeutic targets for chordomas by identifying target proteins in clinical samples as well as tumor microenvironmental factors to enhance efficacy. Fourteen chordoma samples were analyzed by single-cell RNA sequencing, and B7–H3 and IL-7 were identified as potential targets and potentiators, respectively. B7–H3-targeted chimeric antigen receptor T (CAR-T) cells and B7–H3 CAR-T cells expressing IL-7 were synthesized and their anti-tumor activity evaluated in vitro, including in primary chordoma organoid models. The B7–H3 CAR-T/IL-7 therapy showed enhanced cytotoxicity and prolonged duration of action against tumor cells. Additionally, IL-7 modulated favorable subpopulations of cultured CAR-T cells, diminished immune checkpoint expression on T-cell surfaces, and enhanced T-cell functionality. The incorporation of IL-7 molecules into the B7–H3 CAR structure augmented CAR-T-cell function and improved CAR-T-cell efficacy, thus providing a novel dual therapeutic strategy for chordoma treatment. [ABSTRACT FROM AUTHOR]

Acute myeloid leukaemia (AML) is a fatal and refractory haematologic cancer that primarily affects adults. It interferes with bone marrow cell proliferation. Patients have a 5 years survival rate of less than 30% despite the availability of several treatments, including chemotherapy, allogeneic haematopoietic stem cell transplantation (Allo‐HSCT), and receptor antagonist drugs. Allo‐HSCT is the mainstay of acute myeloid leukaemia treatment. Although it does work, there are severe side effects, such as graft‐versus‐host disease (GVHD). In recent years, chimeric antigen receptor (CAR)‐T cell therapies have made significant progress in the treatment of cancer. These engineered T cells can locate and recognize tumour cells in vivo and release a large number of effectors through immune action to effectively kill tumour cells. CAR‐T cells are among the most effective cancer treatments because of this property. CAR‐T cells have demonstrated positive therapeutic results in the treatment of acute myeloid leukaemia, according to numerous clinical investigations. This review highlights recent progress in new targets for AML immunotherapy, and the limitations, and difficulties of CAR‐T therapy for AML. [ABSTRACT FROM AUTHOR]

Simple Summary: Light-chain (AL) amyloidosis is a rare disease similar to the more common disease, multiple myeloma (MM). Both are caused by proliferation of malignant plasma cells. In AL amyloidosis, disease is a result of the deposition of aggregates of proteins, namely immunoglobulin light chains, secreted by the malignant plasma cells, in target organs such as the heart or kidneys. Historically, treatment of AL amyloidosis has followed that of MM. A wide range of novel immunotherapies, i.e., therapies which utilize or activate immune mechanisms to eliminate the disease, are already established in MM and are gradually being adopted in AL amyloidosis as well. Although promising, the increased frailty of typical AL amyloidosis compared to MM patients is a concern in the administration of these therapies, which may be associated with severe side effects. We review both the promise and the challenges with the expansion of MM immunotherapies to AL amyloidosis. Light-chain (AL) amyloidosis is a rare plasma cell disorder characterized by the deposition of misfolded immunoglobulin light chains in target organs, leading to multi-organ dysfunction. Treatment approaches have historically mirrored but lagged behind those of multiple myeloma (MM). Recent advancements in MM immunotherapy are gradually being evaluated and adopted in AL amyloidosis. This review explores the current state of immunotherapeutic strategies in AL amyloidosis, including monoclonal antibodies, antibody–drug conjugates, bispecific antibodies, and chimeric antigen receptor T-cell therapy. We discuss the unique challenges and prospects of these therapies in AL amyloidosis, including the exposure of frail AL amyloidosis patients to immune-mediated toxicities such as cytokine release syndrome (CRS) and immune effector-cell-associated neurotoxicity syndrome (ICANS), as well as their efficacy in promoting rapid and deep hematologic responses. Furthermore, we highlight the need for international initiatives and compassionate programs to provide access to these promising therapies and address critical unmet needs in AL amyloidosis management. Finally, we discuss future directions, including optimizing treatment sequencing and mitigating toxicities, to improve outcomes for AL amyloidosis patients. [ABSTRACT FROM AUTHOR]

Several anti-mesothelin (MSLN) chimeric antigen receptor (CAR) T cells are in phase 1/2 clinical trials to treat solid-organ malignancies. The effect of MSLN antigen density on MSLN CAR cytotoxicity against tumor cells has not been examined previously, nor are there data regarding the effect of agents that increase MSLN antigen density on anti-MSLN CAR T cell efficacy. MSLN antigen density was measured on a panel of pancreatic cancer and mesothelioma cell lines by flow cytometry. In parallel, the cytotoxicity and specificity of two anti-MSLN CAR T cells (m912 and SS1) were compared against these cell lines using a real-time impedance-based assay. The effect of two MSLN 'sheddase' inhibitors (lanabecestat and TMI-1) that increase MSLN surface expression was also tested in combination with CAR T cells. SS1 CAR T cells were more cytotoxic compared with m912 CAR T cells against cell lines that expressed fewer than ∼170 000 MSLN molecules/cell. A comparison of the m912 and amatuximab (humanized SS1) antibodies identified that amatuximab could detect and bind to lower levels of MSLN on pancreatic cancer and mesothelioma cell lines, suggesting that superior antibody/scFv affinity was the reason for the SS1 CAR's superior cytotoxicity. The cytotoxicity of m912 CAR T cells was improved in the presence of sheddase inhibitors, which increased MSLN antigen density. These data highlight the value of assessing CAR constructs against a panel of cells expressing varying degrees of target tumor antigen as occurs in human tumors. Furthermore, the problem of low antigen density may be overcome by concomitant administration of drugs that inhibit enzymatic shedding of MSLN. [Display omitted] [ABSTRACT FROM AUTHOR]

Gastric cancer is a malignant tumor that seriously endangers human health. Claudin18.2 (CLDN18.2) is a transmembrane protein and a main component of tight junctions that plays an important role in maintaining barrier function. Under normal physiological conditions, CLDN18.2 is not expressed in healthy tissues, but it is expressed in differentiated epithelial cells of the gastric mucosa. With the progress of research, it has been found that CLDN18.2 is highly expressed or ectopically activated in primary malignant tumors such as gastric cancer, esophageal cancer, ovarian cancer, pancreatic cancer, lung cancer, and breast cancer. Due to its potential for specific expression, CLDN18.2 has become a new emerging target in anti- tumor drug development. This article reviews the expression characteristics, regulation and clinical pathological features, of CLDN18.2 in gastric cancer, and the progress of clinical drug research targeting CLDN18.2.

Objective To preliminarily explore the efficacy of chimeric antigen receptor T cells (CAR-T) targeting CD123 in the treatment of acute myeloid leukemia (AML) and the role of dasatinib in the treatment of CD123 targeting CAR-T induced side effects. Methods Clinical data of 1 patient with relapsed AML admitted to No.920 Hospital of PLA Joint Logistic Support Force in September, 2019 were collected.The patient relapsed after previous multi-line chemotherapy and was treated with CD123 targeting CAR-T therapy.The routine blood changes of the patient after treatment were observed.Dasatinib was used when agranulocytosis occurred, 40 mg orally 3 times per day, and was stopped when agranulocytosis was relieved.Changes in blood cells, CAR-T amplification, and disease control were observed.The patient was followed up for over 1 year. Results Flow cytometry for bone marrow showed that minimal residual disease negative result was observed in 30 d after infusion.The patient remained disease-free for over 1 year.After CD123 CAR-T cells infusion, significant expansion of CAR-T cells was observed, accompanied by granulocyte deficiency and cytokine release syndrome (CRS).After using dasatinib, inhibition of CAR-T cell expansion was observed, accompanied by blood cell recovery, and CRS symptoms were alleviated.After stop of dasatinib, CAR-T cells expanded again and blood cells decreased again. Conclusion CAR-T cells targeting CD123 have certain efficacy in the treatment for relapsed AML.Dashatinib has a blocking effect on the amplification and function of CAR-T, which can alleviate bone marrow suppression caused by CD123 targeting CAR-T and avoid severe CRS.

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In solid tumors, there are multiple barriers for a chimeric antigen receptor (CAR) T cell to surmount in order to reach the tumor site. For better understanding whether CAR T cells effectively infiltrate into tumor site, and simultaneously, whether there are off-target effects, real-time monitoring technologies need to be established. Cell-based positron emission tomography reporter genes have been developed to monitor engineered cells in living subjects. In this study, we reported the construction of a novel reporter gene truncated prostate-specific membrane antigen (ΔPSMA) pending for monitoring CAR T cells using 68Ga-PSMA-617 and a method for tracking the distribution of CAR T cells in vivo was developed. Data were provided to demonstrate that ΔPSMA was predominantly localized on the plasma membrane and could take up 68Ga-PSMA-617 in vitro in a time-dependent manner. And the expression of ΔPSMA did not affect CAR expression and cytolytic capacity of CAR T cells. CAR-ΔPSMA T cell xenografts in nude mice were clearly imaged by positron emission tomography 60 min after injection of 68Ga-PSMA-617. PSMA paired with 68Ga-PSMA-617 was capable of identifying approximately 1 × 104 engineered CAR T cells. The ability to image small numbers of CAR T cells in vivo would be helpful to accelerate the translation of cell-based therapies into the clinic, and it may reinforce our understanding of treatment success, failure, and toxicity. [ABSTRACT FROM AUTHOR]

Primary central nervous system lymphomas (PCNSL) are rare highly aggressive diffuse large B cell non-Hodgkin lymphomas confined to the brain, meninges, the spinal cord and the eyes. Although the implementation of high-dose methotrexate-based chemotherapy has significantly improved the prognosis of PCNSL during the last decades, about one third of patients show refractory disease and about half of the patients eventually relapse after having achieved complete response. This highlights the need for novel treatment strategies. The most promising progress has been made in the field of molecular targeted therapy that interferes with the oncogenic signaling pathways of PCNSL. These include inhibitors of Bruton tyrosine kinase and inhibitors of the PI3K/mTOR signaling pathway. In addition, the thalidomide analogues lenalidomide and pomalidomide, which belong to the class of immunomodulators, show efficacy in the treatment of PCNSL. As immune evasion appears to play a relevant pathogenetic role in PCNSL, immunotherapies in the treatment of PCNSL are the subject of intensive research. Promising initial clinical data are available for both immune checkpoint inhibitors and cellular immunotherapy with chimeric antigen receptor (CAR) T cells. Before the widespread clinical application of these novel therapies, the efficacy needs to be confirmed in larger prospective studies. Despite high response rates, targeted therapies and immunotherapy often fail to achieve lasting tumor control. Therefore, novel approaches are currently being investigated in combination protocols. [ABSTRACT FROM AUTHOR]

Objectives: We aim to analyze the predictive value of thromboelastography on bleeding severity of patients with chimeric antigen receptor (CAR)‐T cell therapy. Methods: A total of 80 patients with refractory/relapsed hematological malignancy were enrolled and divided into two groups: the severe bleeding group and the non‐severe bleeding group. The thromboelastography data was collected on the day of CAR‐T infusion and the 3rd, 7th, 10th, 13th, 17th, and 20th day after CAR‐T cell infusion. Results: The patients of the severe bleeding group had lower platelet (p <.007), maximum amplitude (p =.002), coagulation index (p =.005), and longer coagulation time (p =.019). Increasing trend in reaction time and coagulation time and decreasing trend in Alpha, maximum amplitude, and coagulation index on Days 0–10, opposite on Days 10–20. Univariate logistic regression analysis and multivariable logistic regression analysis showed maximum amplitude on the 3rd day after CAR‐T cell infusion (MA3) (OR = 0.9; 95% CI = 0.84–0.95; p <.001) and cytokine release syndrome grade (OR = 2.57; 95% CI = 1.35–5.32; p =.006) were significantly associated with high bleeding severity. Conclusions: Thromboelastography was considered to be a good predictor of bleeding severity. [ABSTRACT FROM AUTHOR]

Background: Chimeric antigen receptor (CAR) T cells for refractory or relapsed (r/r) B cell no-Hodgkin lymphoma (NHL) patients have shown promising clinical effectiveness. However, the factors impacting the clinical response of CAR-T therapy have not been fully elucidated. We here investigate the independent influencing factors of the efficacy of CD19 CAR-T cell infusion in the treatment of r/r B-NHL and to establish an early prediction model. Methods: A total of 43 r/r B-NHL patients were enrolled in this retrospective study. The patients' general data were recorded, and the primary endpoint is the patients' treatment response. The independent factors of complete remission (CR) and partial remission (PR) were investigated by univariate and binary logistic regression analysis, and the prediction model of the probability of CR was constructed according to the determined independent factors. Receiver operating characteristic (ROC) and calibration plot were used to assess the discrimination and calibration of the established model. Furthermore, we collected 15 participators to validate the model. Results: Univariate analysis and binary logistic regression analysis of 43 patients showed that the ratio of central memory T cell (Tcm) and naïve T cell (Tn) in cytotoxic T cells (Tc) was an independent risk factor for response to CD19 CAR-T cell therapy in r/r B-NHL. On this basis, the area under the curve (AUC) of Tcm in the Tc and Tn in the Tc nomogram model was 0.914 (95%CI 0.832–0.996), the sensitivity was 83%, and the specificity was 74.2%, which had excellent predictive value. We did not found the difference of the progression-free survival (PFS). Conclusions: The ratio of Tcm and Tn in Tc was found to be able to predict the treatment response of CD19 CAR-T cells in r/r B-NHL. We have established a nomogram model for the assessment of the CD19 CAR-T therapy response presented high specificity and sensitivity. [ABSTRACT FROM AUTHOR]

Efficient engineering of T cells to express exogenous tumor-targeting receptors such as chimeric antigen receptors (CARs) or T-cell receptors (TCRs) is a key requirement of effective adoptive cell therapy for cancer. Genome editing technologies, such as CRISPR/Cas9, can further alter the functional characteristics of therapeutic T cells through the knockout of genes of interest while knocking in synthetic receptors that can recognize cancer cells. Performing multiple rounds of gene transfer with precise genome editing, termed multiplexing, remains a key challenge, especially for non-viral delivery platforms. Here, we demonstrate the efficient production of primary human T cells incorporating the knockout of three clinically relevant genes (B2M, TRAC, and PD1) along with the non-viral transfection of a CAR targeting disialoganglioside GD2. Multiplexed knockout results in high on-target deletion for all three genes, with low off-target editing and chromosome alterations. Incorporating non-viral delivery to knock in a GD2-CAR resulted in a TRAC-B2M-PD1-deficient GD2 CAR T-cell product with a central memory cell phenotype and high cytotoxicity against GD2-expressing neuroblastoma target cells. Multiplexed gene-editing with non-viral delivery by CRISPR/Cas9 is feasible and safe, with a high potential for rapid and efficient manufacturing of highly potent allogeneic CAR T-cell products.

Adoptive cell therapy (ACT) is a rapidly expanding area within the realm of transfusion medicine, focusing on the delivery of lymphocytes to trigger responses against tumors, viruses, or inflammation. This area has quickly evolved from its initial promise in immuno-oncology during preclinical trials to commercial approval of chimeric antigen receptor (CAR) T-cell therapies for leukemia and lymphoma (Jun and et al., 2018) [1]. CAR T-cell therapy has demonstrated success in treating hematological malignancies, particularly relapsed/refractory B-cell acute lymphoblastic leukemia and non-Hodgkin's lymphoma (Qi and et al., 2022) [2]. However, its success in treating solid tumors faces challenges due to the short-lived presence of CAR-T cells in the body and diminished T cell functionality (Majzner and Mackall, 2019) [3]. CAR T-cell therapy functions by activating immune effector cells, yet significant side effects and short response durations remain considerable obstacles to its advancement. A prior study demonstrated that the therapeutic regimen can induce systemic inflammatory reactions, such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), tumor lysis syndrome (TLS), off-target effects, and other severe complications. This study aims to explore current research frontiers in this area.

Chimeric antigen receptor T cells (CAR-Ts) have shown a remarkable efficacy in hematological malignancies but limited responses in solid tumors. Among solid tumors, CAR-T cell therapy has been particularly explored in brain tumors. CAR-T cells have shown a limited clinical efficacy in various types of brain tumors due to several factors that have hampered their activity, including tumor antigen heterogeneity, the limited access of CAR-T cells to brain tumor cells, limited CAR-T cell trafficking and in vivo persistence and the presence of a highly immunosuppressive tumor microenvironment. Despite these considerations, some recent studies have shown promising antitumor activity of GD2-CAR-T cells on diffuse midline gliomas and neuroblastomas and of CARv3-TEAM-E cells in glioblastomas. However, strategies are required to improve the effect of CAR-T cells in brain tumors, including advanced CAR-T cell design with multiple antigenic targeting and incorporation of combination therapies.

Unlimited generation of chimeric antigen receptor (CAR) T cells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR T cell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αβ T cell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L+ naive and memory T cells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αβ-positive CAR T cell characteristics. Expanded iPSC CD19-CAR T cells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR T cells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR T cells also mediated potent antitumor activity in vivo, prolonging survival of mice with CD19+ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR T cells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.

Background: Anaplastic Large Cell Lymphoma (ALCL) is one of the most common subtypes of T-cell lymphoma. Among these, refractory and relapsed (r/r) ALK positive ALCL lacks effective therapies. The chimeric antigen receptormodified T (CAR-T) cell therapy holds great promise as a therapeutic strategy for this disease. However, it is not known yet whether anti-CD5 CAR-T cells are sufficient for the definitive treatment of relapsed ALK+ ALCL, nor the role of accurate laboratory-based diagnoses during CAR-T treatment. Case presentation: The adolescent patient received autologous T cells containing sequences encoding VH domains specific to CD5. Following the infusion, there was an increase in both the copy number and proportion of CAR-T cells in peripheral blood. IL-6 and ferritin levels in the patient exhibited significant fluctuations, with increases of 13 and 70 folds respectively, compared to baseline after the treatment. Additionally, adverse effects were observed, including grade 4 rash, grade 1 headache, nausea, and neck-pain. Surprisingly, a relapsed disease phenotype was identified based on the results of PET/CT and histopathological analysis of the inguinal lymph node biopsy. After conducting a thorough diagnostic assessment, which included flow cytometry, nextgeneration sequencing (NGS), examination of immune-related gene rearrangements, and analysis of the immune repertoire of T-cell receptors (TCR), we conclusively determined that the hyperplastic T cells identified in the lymph node were the result of an expansion of CAR-T cells. Ultimately, the patient has attained complete remission (CR) and has sustained a disease-free survival state for 815 days as of the cutoff date on August 30, 2023. Conclusion: Taken together, the results demonstrate that anti-CD5 CAR-T cells can induce a clinical response in r/r ALK+ ALCL patient. Furthermore, this case underscores the importance of utilizing advanced technologies with high sensitivity and accuracy for biological detection in clinical laboratory diagnosis and prognosis in CAR-T cell treatment. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor T (CAR-T) cell therapy exhibits remarkable efficacy against refractory or relapsed multiple myeloma (RRMM); however, the immune deficiency following CAR-Ts infusion has not been well studied. In this study, 126 patients who achieved remission post-CAR-Ts infusion were evaluated for cellular immunity. Following lymphodepletion (LD) chemotherapy, the absolute lymphocyte count (ALC) and absolute counts of lymphocyte subsets were significantly lower than baseline at D0. Grade ≥ 3 lymphopenia occurred in 99% of patients within the first 30 days, with most being resolved by 180 days. The median CD4+ T-cell count was consistently below baseline and the lower limit of normal (LLN) levels at follow-up. Conversely, the median CD8+ T-cell count returned to the baseline and LLN levels by D30. The median B-cell count remained lower than baseline level at D60 and returned to baseline and LLN levels at D180. In the first 30 days, 27 (21.4%) patients had 29 infections, with the majority being mild to moderate in severity (21/29; 72.4%). After day 30, 44 (34.9%) patients had 56 infections, including 20 severe infections. One patient died from bacteremia at 3.8 months post-CAR-Ts infusion. In conclusion, most patients with RRMM experienced cellular immune deficiency caused by LD chemotherapy and CAR-Ts infusion. The ALC and most lymphocyte subsets gradually recovered after day 30 of CAR-Ts infusion, except for CD4+ T cells. Some patients experience prolonged CD4+ T-cell immunosuppression without severe infection. [ABSTRACT FROM AUTHOR]

Radiation therapy is the standard treatment for patients with nasopharyngeal carcinoma (NPC), but relapse occurs in 10% to 20% of patients. The treatment of recurrent nasopharyngeal carcinoma (rNPC) remains challenging. Chimeric antigen receptors (CAR)-T-cell therapy has achieved good outcomes in the treatment of leukemia and seems to be a promising therapeutic strategy for solid tumors. c-Met has been found to be highly expressed in multiple cancer types, and the activation of c-Met leads to the proliferation and metastasis of cancer cells. However, the expression of c-Met in rNPC tissues and whether it can be used as a target for CAR-T therapy in rNPC remain to be investigated. We detected the expression of c-Met in 24 primary human rNPC tissues and three NPC cell lines and constructed two different antibody-derived anti-c-Met CARs, namely, Ab928z and Ab1028z. To estimate the function of these two different c-Met-targeted CAR-T cells, CD69 expression, cytotoxicity and cytokine secretion of CAR-T cells were assessed after coculture with target cells. A cell line-derived xenograft mouse model also was used to evaluate these two anti-c-Met CAR-T cells. Furthermore, we determined whether combination with an anti-EGFR antibody could promote the antitumor effect of CAR-T cells in a patient-derived xenograft mouse model. High c-Met expression was detected in 23 of 24 primary human rNPC tissues by immunohistochemistry staining and in three NPC cell lines by flow cytometry. Ab928z-T cells and Ab1028z-T cells showed significantly upregulated expression of CD69 after coculture with targeted cells. However, Ab1028z-T cells showed superior cytokine secretion and antitumor activity. Furthermore, Ab1028z-T cells effectively suppressed tumor growth compared with control CAR-T cells, and the combination with nimotuzumab further enhanced the tumor-clearing ability of Ab1028z-T cells. We found that c-Met is highly expressed in rNPC tissues and confirmed its potential as a CAR-T target for rNPC. Our study provides a new idea for the clinical treatment of rNPC. [ABSTRACT FROM AUTHOR]

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Since the FDA’s approval of chimeric antigen receptor (CAR) T cells in 2017, significant improvements have been made in the design of chimeric antigen receptor constructs and in the manufacturing of CAR T cell therapies resulting in increased in vivo CAR T cell persistence and improved clinical outcome in certain hematological malignancies. Despite the remarkable clinical response seen in some patients, challenges remain in achieving durable long-term tumor-free survival, reducing therapy associated malignancies and toxicities, and expanding on the types of cancers that can be treated with this therapeutic modality. Careful analysis of the biological factors demarcating efficacious from suboptimal CAR T cell responses will be of paramount importance to address these shortcomings. With the ever-expanding toolbox of experimental approaches, single-cell technologies, and computational resources, there is renowned interest in discovering new ways to streamline the development and validation of new CAR T cell products. Better and more accurate prognostic and predictive models can be developed to help guide and inform clinical decision making by incorporating these approaches into translational and clinical workflows. In this review, we provide a brief overview of recent advancements in CAR T cell manufacturing and describe the strategies used to selectively expand specific phenotypic subsets. Additionally, we review experimental approaches to assess CAR T cell functionality and summarize current in silico methods which have the potential to improve CAR T cell manufacturing and predict clinical outcomes.

Objective To investigate the killing efficacy of chimeric antigen receptor T (CAR-T) cells targeting epithelial cellular adhesion molecule (EpCAM) in several hepatoma cell lines. Methods Flow cytometry was used to detect the expression of EpCAM in hepatoma cells (SK-hep1, Hep-G2, HuH7 and BEL-7402). After EpCAM -targeting CAR-T cells were constructed using lentiviral vectors with CAR genes, flow cytometry was used to detect the expression of EpCAM-CAR in the obtained cells, and to analyze the CD3/4/8 phenotype of CAR-T cells without transfection of lentiviral vectors. Then the effector cells and target cells were co-cultured, and LDH release assay and ELISA were used to detect the release of LDH and major cytokines in the supernatant. Results EpCAM was highly expressed in Hep-G2, HuH7 and BEL-7402 cells, but not in SK-hep1 cells. Flow cytometry showed that the expression rate of EpCAM-CAR was 43.21% in CAR-T cells after lentivirus infection. Flow cytometry also indicated that up to 95.1% of peripheral blood mononuclear cells (PBMCs) were CD3 positive, of which 61.40% were CD8+ T cells and 32.13% were CD4+ T cells. LDH release assay revealed that the obtained CAR-T cells had stronger direct killing ability on EpCAM-positive hepatoma cell lines Hep-G2, HuH7, and BEL-7402. ELISA showed that EpCAM-positive target cells Hep-G2, HuH7, and BEL-7402 induced CAR-T cells to release more IFN-γ and TNF-α. For SK-hep1 cells with negative EpCAM expression, IFN-γ and TNF-α releases were not significantly different between the CAR-T group and the control group, and the levels of these 2 cytokines werw increased with the increase of E ∶T ratio. Results Compared with ordinary CAR-T cells, EpCAM-targeting CAR-T cells have a stronger killing effect on EpCAM-positive hepatocellular carcinoma cells.

The success of chimeric antigen receptor (CAR) T cell therapies in refractory hematologic malignancies has prompted investigation of their efficacy in solid tumors. AUTO6NG is a dual-transduced GD2-targeting CAR that encodes distinct modules designed to enhance T cell activity in relapsed/refractory neuroblastoma. The ability to detect and precisely quantify vector copy number (VCN) for each integrated vector is essential for assessing the effect of each module on T cell tumor infiltration, persistence, and clinical activity. Droplet digital PCR (ddPCR) enables accurate, sensitive, and absolute quantification of specific nucleic acid sequences. Compared to standard detection of two targets, multiplex ddPCR assays allow simultaneous detection of up to four targets by selective modulation of signal amplitude while retaining the ability to quantify the target. We have developed a multiplex assay based on the two-channel system for simultaneous detection and quantification of three targets in AUTO6NG CAR T cells. The assay was highly specific, sensitive, accurate, and reproducible across time and samples. No differences were observed in measuring VCN between standard duplex and multiplex assays. Our results demonstrate that ddPCR is an accurate and cost-effective method for simultaneous detection of multiple targets in genomic DNA derived from engineered CAR T cells.

Light-chain (AL) amyloidosis is a rare plasma cell disorder characterized by the deposition of misfolded immunoglobulin light chains in target organs, leading to multi-organ dysfunction. Treatment approaches have historically mirrored but lagged behind those of multiple myeloma (MM). Recent advancements in MM immunotherapy are gradually being evaluated and adopted in AL amyloidosis. This review explores the current state of immunotherapeutic strategies in AL amyloidosis, including monoclonal antibodies, antibody–drug conjugates, bispecific antibodies, and chimeric antigen receptor T-cell therapy. We discuss the unique challenges and prospects of these therapies in AL amyloidosis, including the exposure of frail AL amyloidosis patients to immune-mediated toxicities such as cytokine release syndrome (CRS) and immune effector-cell-associated neurotoxicity syndrome (ICANS), as well as their efficacy in promoting rapid and deep hematologic responses. Furthermore, we highlight the need for international initiatives and compassionate programs to provide access to these promising therapies and address critical unmet needs in AL amyloidosis management. Finally, we discuss future directions, including optimizing treatment sequencing and mitigating toxicities, to improve outcomes for AL amyloidosis patients.

Synthetic biology has made it possible to rewire natural cellular responses to treat disease, notably demonstrated by chimeric antigen receptor (CAR) T cells as cancer immunotherapy. Building on the success of T‐cell activation using synthetic receptors, the field is now investigating how induction of noncanonical signalling pathways and sophisticated synthetic gene circuitry can enhance the antitumour phenotype of engineered T cells. This commentary explores two recently published studies that provide proof of concept for how new technologies achieve this. The first demonstrated that non‐naturally occurring combinations of signalling motifs derived from various immune receptors and arranged as a CAR drove unique signal transduction pathways in T cells and improved their tumour killing ability. Here, machine learning complemented the screening process and successfully predicted CAR T‐cell phenotype dependent on signalling motif choice. The second explored how synthetic zinc fingers can be engineered into controllable transcriptional regulators, where their activity was dependent on the presence or absence of FDA‐approved small‐molecule drugs. These studies are pivotal in expanding the design choices available for gene circuits of the future and highlight how a single cellular therapy could respond to multiple environmental cues including target cell antigen expression, the tumour microenvironment composition and small molecule drugs.

Abstract Over the last decade, the emergence of several novel therapeutic approaches has changed the therapeutic perspective of human malignancies. Adoptive immunotherapy through chimeric antigen receptor T cell (CAR‐T), which includes the engineering of T cells to recognize tumor‐specific membrane antigens and, as a result, death of cancer cells, has created various clinical benefits for the treatment of several human malignancies. In particular, CAR‐T‐cell‐based immunotherapy is known as a critical approach for the treatment of patients with hematological malignancies such as acute lymphoblastic leukemia (ALL), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), Hodgkin lymphoma (HL), and non‐Hodgkin's lymphoma (NHL). However, CAR‐T‐cell therapy of hematological malignancies is associated with various side effects. There are still extensive challenges in association with further progress of this therapeutic approach, from manufacturing and engineering issues to limitations of applications and serious toxicities. Therefore, further studies are required to enhance efficacy and minimize adverse events. In the current review, we summarize the development of CAR‐T‐cell‐based immunotherapy and current clinical antitumor applications to treat hematological malignancies. Furthermore, we will mention the current advantages, disadvantages, challenges, and therapeutic limitations of CAR‐T‐cell therapy.

Objective To evaluate the efficacy and safety of allogeneic hematopoietic stem cell transplantation (allo-HSCT) before leukocyte differentiation antigen (CD19) chimeric antigen receptor T cell (CD19 CAR-T) immunotherapy in patients with relapse/refractory B lymphoblastic leukemia (r/r B-ALL) . Methods The data of r/r B-ALL patients treated with homologous CD19 CAR-T in this hospital from June 2015 to June 2019 were collected. According to whether allo-HSCT was performed before treatment, the patients were divided into the transplantation group and the non-transplantation group. The overall response rate (ORR),relapse-free survival time (RFS),overall survival time (OS) and general adverse events, special adverse reactions were compared between the two groups. Results A total of 47 r/r B-ALL patients treated with homologous CD19 CAR-T were screened, of which nine were treated with allo-HSCT before the treatment. The ORR of the transplant group and the non-transplant group was 88.9% and 78. 9%,respectively, and the difference was not significant (P=0. 667) . After excluded patients treated with bridging allo-HSCT after CD19 CAR-T immuno therapy, the median RFS were 386 (434,237) d and 95 (168,37) d (Wilcoxon test, P=0.028; Log-Rank test, P=0. 231) ; the first year RFS rates were 62. 5% and 20.0%,respectively (P=0. 068) ; the median OS were 852 (1 168,590) d and 222 (809,141) d (Wilcoxon test, P=0. 049; Log-Rank test, P=0.186) ; the first year OS rates were 77. 8% and 35. 7% (P=0.052) in the transplant group and the non-transplant group. Within four weeks after CD19 CAR-T infusion, there was no significant difference in the incidence of general adverse events or specific adverse reactions between the transplant group and the non-transplant group (P＞0.05) . Conclusion B-ALL patients who received allo-HSCT prior to CD19 CAR-T immuno therapy may achieve better survival without increased adverse reactions. [ABSTRACT FROM AUTHOR]

Aims: Mesothelin (MSLN) is a cancer‐associated antigen that is overexpressed in malignancies such as mesothelioma, pancreatic and ovarian cancer. It is also a target for novel personalised therapies, including antibodies, antibody–drug conjugates and chimeric antigen receptor T cells. Immunohistochemistry may predict those who would best respond to anti‐mesothelin therapies and guide decisions in therapeutic strategy. This study aimed to assess the intensity and distribution of MSLN immunostaining in mesothelioma, and to determine the prognostic value of MSLN expression by histochemical‐score (H‐score). Methods and results: The MN1 anti‐MSLN antibody was used to stain a formalin‐fixed paraffin‐embedded tissue microarray of histologically confirmed mesothelioma from 75 consecutive patients who had undergone pleurectomy with or without decortication. MSLN positivity, the staining intensity, distribution of staining and H‐score were evaluated. The correlation of H‐score with prognosis was investigated. Sixty‐six per cent of epithelioid tumours were MSLN‐positive (with expression in > 5% tumour cells). Of MSLN‐expressing epithelioid tumours, 70.4% had moderate (2+) or strong (3+) intensity MSLN immunostaining, although only 37% of samples had staining in ≥ 50% of tumour cells. In multivariate analysis, MSLN H‐score as a continuous variable and an H‐score ≥ 33 were independent predictors of improved survival (P = 0.04 and P < 0.001, respectively). Conclusions: MSLN expression was more heterogenous in epithelioid mesothelioma than reported previously. Therefore, it would be appropriate to perform an immunohistochemical assessment of MSLN expression to stratify and assess patient suitability for mesothelin‐targeted personalised therapies, such as chimeric antigen receptor T cells. [ABSTRACT FROM AUTHOR]

Purpose: Autologous chimeric antigen receptor (CAR) T cell therapy is one of the most significant breakthroughs in hematological malignancies. However, a three-week manufacturing cycle and ineffective T cell dysfunction in some patients hinder the widespread application of auto-CAR T cell therapy. Studies suggest that cord blood (CB), with its unique biological properties, could be an optimal source for CAR T cells, providing a product with 'off-the-shelf' availability. Therefore, exploring the potential of CB as an immunotherapeutic agent is essential for understanding and promoting the further use of CAR T cell therapy. Experimental design: We used CB to generate CB-derived CD19-targeting CAR T (CB CD19-CAR T) cells. We assessed the anti-tumor capacity of CB CD19-CAR T cells to kill diffuse large B cell lymphoma (DLBCL) in vitro and in vivo. Results: CB CD19-CAR T cells showed the target-specific killing of CD19+ T cell lymphoma cell line BV173 and CD19+ DLBCL cell line SUDHL-4, activated various effector functions, and inhibited tumor progression in a mouse (BALB/c nude) model. However, some exhaustion-associated genes were involved in off-tumor cytotoxicity towards activated lymphocytes. Gene expression profiles confirmed increased chemokines/chemokine receptors and exhaustion genes in CB CD19-CAR T cells upon tumor stimulation compared to CB T cells. They indicated inherent changes in the associated signaling pathways in the constructed CB CAR T cells and targeted tumor processes. Conclusion: CB CD19-CAR T cells represent a promising therapeutic strategy for treating DLBCL. The unique biological properties and high availability of CB CD19-CAR T cells make this approach feasible. [ABSTRACT FROM AUTHOR]

Tumor cells and the immunosuppressive tumor microenvironment suppress the antitumor activity of T cells through immune checkpoints, including the PD-L1/PD-1 axis. Cytokine-inducible SH2-containing protein (CISH), a member of the suppressor of cytokine signaling (SOCS) family, inhibits JAK-STAT and T cell receptor (TCR) signaling in T and natural killer (NK) cells. However, its role in the regulation of immune checkpoints in T cells remains unclear. In this study, we ablated CISH in T cells with CRISPR-Cas9 and found that the sensitivity of T cells to TCR and cytokine stimulation was increased. In addition, chimeric antigen receptor T cells with CISH deficiency exhibited longer survival and higher cytokine secretion and antitumor activity. Notably, PD-1 expression was decreased in activated CISH-deficient T cells in vitro and in vivo. The level of FBXO38, a ubiquitination-regulating protein that reduces PD-1 expression, was elevated in activated T cells after CISH ablation. Hence, this study reveals a mechanism by which CISH promotes PD-1 expression by suppressing the expression of FBXO38 and proposes a new strategy for augmenting the therapeutic effect of CAR-T cells by inhibiting CISH.

Gene therapy is the treatment of abnormal or mutated genes present in cells through the addition of healthy genes or replacement/deletion/site-specific modification of faulty genes. Deoxyribonucleic acid, messenger ribonucleic acid (RNA), small interference RNA, microsomal RNA and antisense oligonucleotides are the genetic materials implicated in gene therapy. They are inserted into the diseased cells using viral or non-viral vectors through an in vivo or ex vivo transduction. Gamma retrovirus, lentivirus, herpesvirus, adenovirus and adeno-associated virus are common viral vectors, while transposons, cationic polymers, dendrimers and cell-penetrating peptides or liposomes are common non-viral vectors. Allologous or autologous T cells, haematopoietic stem cells and chimeric antigen receptor T cells are used for ex vivo gene transduction. Conventional gene therapy of inserting new genetic material shows toxicity such as off-target effects, altered immune responses, inflammatory reactions and possible oncogenic transformation in the recipient. Newer gene editing techniques such as zinc-finger nuclease, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeats allow the site-specific correction or control of expression of mutated genes present in cells. Until August 2020, 23 gene-based medicines received approval from drug regulatory agencies in various countries and 362 were in development. Single-gene disorders have shown encouraging results, but evidence of using gene therapy in polygenic and common age-related diseases is still required. Recently, the horizon of gene therapy widened to include COVID vaccines and as an adjunct to chemotherapy. If we could overcome its limitations such as immunogenicity, mutagenicity and high costs, gene therapy can be the medicine of the next generation.

Although exponential progress in treating advanced malignancy has been made in the modern era with immune checkpoint blockade, survival outcomes remain suboptimal. Cellular immunotherapy, such as chimeric antigen receptor T cells, has the potential to improve this. CAR T cells combine the antigen specificity of a monoclonal antibody with the cytotoxic 'power' of T-lymphocytes through expression of a transgene encoding the scFv domain, CD3 activation molecule, and co-stimulatory domains. Although, very rarely, fatal cytokine-release syndrome may occur, CAR T-cell therapy gives patients with refractory CD19-positive B-lymphoid malignancies an important further therapeutic option. However, low-level expression of epithelial tumour-associated-antigens on non-malignant cells makes the application of CAR T-cell technology to common solid cancers challenging, as does the potentially limited ability of CAR T cells to traffic outside the blood/lymphoid microenvironment into metastatic lesions. Despite this, in advanced neuroblastoma refractory to standard therapy, 60% long-term overall survival and an objective response in 63% was achieved with anti GD2-specific CAR T cells. [ABSTRACT FROM AUTHOR]

Introduction: Chimeric antigen receptor T (CAR-T) cell therapy presents a promising treatment option for various cancers, including solid tumors. Carcinoembryonic antigen (CEA) is an attractive target due to its high expression in many tumors, particularly gastrointestinal cancers, while limited expression in normal adult tissues. In our previous clinical study, we reported a 70% disease control rate with no severe side effects using a humanized CEAtargeting CAR-T cell. However, the selection of the appropriate single-chain variable fragment (scFv) significantly affects the therapeutic efficacy of CAR-T cells by defining their specific behavior towards the target antigen. Therefore, this study aimed to identify the optimal scFv and investigate its biological functions to further optimize the therapeutic potential of CAR-T cells targeting CEA-positive carcinoma. Methods: We screened four reported humanized or fully human anti-CEA antibodies (M5A, hMN-14, BW431/26, and C2-45), and inserted them into a 3rd-generation CAR structure. We purified the scFvs and measured the affinity. We monitored CAR-T cell phenotype and scFv binding stability to CEA antigen through flow cytometry. We performed repeated CEA antigen stimulation assays to compare the proliferation potential and response of the four CAR-T cells, then further evaluated the anti-tumor efficacy of CAR-T cells ex vivo and in vivo. Results: M5A and hMN-14 CARs displayed higher affinity and more stable CEA binding ability than BW431/26 and C2-45 CARs. During CAR-T cell production culture, hMN-14 CAR-T cells exhibit a larger proportion of memory-like T cells, while M5A CAR-T cells showed a more differentiated phenotype, suggesting a greater tonic signal of M5A scFv. M5A, hMN-14, and BW431/26 CAR-T cells exhibited effective tumor cell lysis and IFN-g release when cocultured with CEApositive tumor cells in vitro, correlating with the abundance of CEA expression in target cells. While C2-45 resulted in almost no tumor lysis or IFN-g release. In a repeat CEA antigen stimulation assay, M5A showed the best cell proliferation and cytokine secretion levels. In a mouse xenograft model, M5A CAR-T cells displayed better antitumor efficacy without preconditioning. Discussion: Our findings suggest that scFvs derived from different antibodies have distinctive characteristics, and stable expression and appropriate affinity are critical for robust antitumor efficacy. This study highlights the importance of selecting an optimal scFv in CAR-T cell design for effective CEA-targeted therapy. The identified optimal scFv, M5A, could be potentially applied in future clinical trials of CAR-T cell therapy targeting CEA-positive carcinoma. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor T cell therapy (CAR-T) is a novel treatment that has produced unprecedented clinical effects in patients with hematological malignancies. Acute adverse events often occur following adoptive immunotherapy. Therefore, a suicide gene is helpful, which is a genetically encoded mechanism that allows selective destruction of adoptively transferred T cells in the face of unacceptable toxicity. RQR8 is a gene that integrates CD34 and CD20 epitopes. In our study, we incorporated the suicide gene RQR8 into CAR-T cells, so it enabled rituximab to eliminate vector/transgene-expressing T cells via antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity. In this work, we explored the functionality of RQR8 CAR-T cells in vitro and in vivo. We believe that RQR8 as a safety switch will make CAR-T cell therapy safer and less costly. [ABSTRACT FROM AUTHOR]

Synthetic biology has made it possible to rewire natural cellular responses to treat disease, notably demonstrated by chimeric antigen receptor (CAR) T cells as cancer immunotherapy. Building on the success of T‐cell activation using synthetic receptors, the field is now investigating how induction of noncanonical signalling pathways and sophisticated synthetic gene circuitry can enhance the antitumour phenotype of engineered T cells. This commentary explores two recently published studies that provide proof of concept for how new technologies achieve this. The first demonstrated that non‐naturally occurring combinations of signalling motifs derived from various immune receptors and arranged as a CAR drove unique signal transduction pathways in T cells and improved their tumour killing ability. Here, machine learning complemented the screening process and successfully predicted CAR T‐cell phenotype dependent on signalling motif choice. The second explored how synthetic zinc fingers can be engineered into controllable transcriptional regulators, where their activity was dependent on the presence or absence of FDA‐approved small‐molecule drugs. These studies are pivotal in expanding the design choices available for gene circuits of the future and highlight how a single cellular therapy could respond to multiple environmental cues including target cell antigen expression, the tumour microenvironment composition and small molecule drugs. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cell is a novel therapy for relapse and refractory hematologic malignancy. Characteristics of CAR T cells are associated with clinical efficacy and toxicity. The type of serum supplements used during cultivation affects the immunophenotype and function of viral-based CAR T cells. This study explores the effect of serum supplements on nonviral piggyBac transposon CAR T-cell production. PiggyBac CD19 CAR T cells were expanded in cultured conditions containing fetal bovine serum, human AB serum or xeno-free serum replacement. We evaluated the effect of different serum supplements on cell expansion, transduction efficiency, immunophenotypes and antitumor activity. Xeno-free serum replacement exhibited comparable CAR surface expression, cell expansion and short-term antitumor activity compared with conventional serum supplements. However, CAR T cells cultivated with xeno-free serum replacement exhibited an increased naïve/stem cell memory population and better T-cell expansion after long-term co-culture as well as during the tumor rechallenge assay. Our study supports the usage of xeno-free serum replacement as an alternative source of serum supplements for piggyBac-based CAR T-cell expansion. [ABSTRACT FROM AUTHOR]

Over the last decade, the emergence of several novel therapeutic approaches has changed the therapeutic perspective of human malignancies. Adoptive immunotherapy through chimeric antigen receptor T cell (CAR‐T), which includes the engineering of T cells to recognize tumor‐specific membrane antigens and, as a result, death of cancer cells, has created various clinical benefits for the treatment of several human malignancies. In particular, CAR‐T‐cell‐based immunotherapy is known as a critical approach for the treatment of patients with hematological malignancies such as acute lymphoblastic leukemia (ALL), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), Hodgkin lymphoma (HL), and non‐Hodgkin's lymphoma (NHL). However, CAR‐T‐cell therapy of hematological malignancies is associated with various side effects. There are still extensive challenges in association with further progress of this therapeutic approach, from manufacturing and engineering issues to limitations of applications and serious toxicities. Therefore, further studies are required to enhance efficacy and minimize adverse events. In the current review, we summarize the development of CAR‐T‐cell‐based immunotherapy and current clinical antitumor applications to treat hematological malignancies. Furthermore, we will mention the current advantages, disadvantages, challenges, and therapeutic limitations of CAR‐T‐cell therapy. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor T (CAR-T) cell therapy has become a research hotspot in the field of hematological malignancies. However, CAR-T cell therapy can lead to immunotherapy-associated side effects including cytokine release syndrome and neurotoxicity. Gene depletion of GM-CSF in CAR-T cells was found preventive against adverse effects, but additional transfections were required to produce CAR-T cells. In this study, we interrupted GM-CSF expression in CAR-T cells by inserting the GM-CSF shRNA-expression cassette in the CAR vector. Reduction of GM-CSF in CAR-T cells could decrease the level of several proinflammatory cytokines without hampering the killing capacity. The manufacture of GM-CSF knockdown CAR-T cells does not require complicated transfections, which makes it more practical and feasible for clinical application. [ABSTRACT FROM AUTHOR]

Background: Immunological treatments (immune checkpoint inhibitors [ICIs], chimeric antigen receptor T [CAR-T] cells, bispecific T-cell engagers [BiTEs]) have deeply changed the treatment of several cancers. However, the impact of these treatments on the risk of developing infections has not been completely ascertained yet. Methods: We reviewed all the registration studies of currently approved ICIs, CAR-T cells, and BiTEs to collect all the reported infections. For each drug, we have generated a report with the infections occurring in at least 10% of the patients enrolled. Results: The most frequently reported infections involving patients treated with ICIs involved the respiratory tract, including nasopharyngitis, upper respiratory tract infections, and pneumonia and the urinary tract. Those treated with CAR-T cells frequently reported the incidence of unspecified infections and infestations, bacterial infections, and viral infections. In patients treated with BiTEs, nasopharyngitis, pneumonia, and device-related infections were the most frequently reported conditions. Conclusions: A wide range of infections are reported in registration studies and clinical trials of ICIs, CAR-T cells, and BiTEs.

Background: Chimeric antigen receptor T cell infusion (CAR T) therapy has revolutionized the treatment of hematologic malignancies, but treatmentrelated toxicities are of concern. Understanding the timing and reasons for which patients present to the emergency department (ED) after CAR T therapy can assist with the early recognition and management of toxicities. Methods: A retrospective observational cohort study was conducted for patients who had undergone CAR T therapy in the past 6 months and visited the ED of The University of Texas MD Anderson Cancer Center between 04/01/2018 and 08/ 01/2022. The timing of presentation after CAR T product infusion, patient characteristics, and outcomes of the ED visit were examined. Survival analyses were conducted using Cox proportional hazards regression and Kaplan-Meier estimates. Results: During the period studied, there were 276 ED visits by 168 unique patients. Most patients had diffuse large B-cell lymphoma (103/168; 61.3%), multiple myeloma (21/168; 12.5%), or mantle cell lymphoma (16/168; 9.5%). Almost all 276 visits required urgent (60.5%) or emergent (37.7%) care, and 73.5% of visits led to admission to the hospital or observation unit. Fever was the most frequent presenting complaint, reported in 19.6% of the visits. The 30- day and 90-day mortality rates after the index ED visits were 17.0% and 32.2%, respectively. Patients who had their first ED visit >14 days after CAR T product infusion had significantly worse overall survival (multivariable hazard ratio 3.27; 95% confidence interval 1.29–8.27; P=0.012) than patients who first visited the ED within 14 days of CAR T product infusion. Conclusion: Cancer patients who receive CAR T therapy commonly visit the ED, and most are admitted and/or require urgent or emergent care. During early ED visits patients mainly present with constitutional symptoms such as fever and fatigue, and these early visits are associated with better overall survival. [ABSTRACT FROM AUTHOR]

Opinion statement: Although chimeric antigen receptor T cell immunotherapy has been successfully applied in patients with hematological malignancies, several obstacles still need to be overcome, such as high relapse rates and side effects. Overcoming the limitations of CAR-T cell therapy and boosting the efficacy of CAR-T cell therapy are urgent issues that must be addressed. The exploration of small-molecule compounds in combination with CAR-T cell therapies has achieved promising success in pre-clinical and clinical studies in recent years. Protein kinase inhibitors, demethylating drugs, HDAC inhibitors, PI3K inhibitors, immunomodulatory drugs, Akt inhibitors, mTOR inhibitors, and Bcl-2 inhibitors exhibited potential synergy in combination with CAR-T cell therapy. In this review, we will discuss the recent application of these combination therapies for improved outcomes of CAR-T cell therapy. [ABSTRACT FROM AUTHOR]