Exploring miRNA therapies and gut microbiome-enhanced CAR-T cells: advancing frontiers in glioblastoma stem cell targeting.

Glioblastoma multiforme (GBM) presents a formidable challenge in oncology due to its aggressive nature and resistance to conventional treatments. Recent advancements propose a novel therapeutic strategy combining microRNA-based therapies, chimeric antigen receptor-T (CAR-T) cells, and gut microbiome modulation to target GBM stem cells and transform cancer treatment. MicroRNA therapies show promise in regulating key signalling pathways implicated in GBM progression, offering the potential to disrupt GBM stem cell renewal. CAR-T cell therapy, initially successful in blood cancers, is being adapted to target GBM by genetically engineering T cells to recognise and eliminate GBM stem cell-specific antigens. Despite early successes, challenges like the immunosuppressive tumour microenvironment persist. Additionally, recent research has uncovered a link between the gut microbiome and GBM, suggesting that gut dysbiosis can influence systemic inflammation and immune responses. Novel strategies to modulate the gut microbiome are emerging, enhancing the efficacy of microRNA therapies and CAR-T cell treatments. This combined approach highlights the synergistic potential of these innovative therapies in GBM treatment, aiming to eradicate primary tumours and prevent recurrence, thereby improving patient prognosis and quality of life. Ongoing research and clinical trials are crucial to fully exploit this promising frontier in GBM therapy, offering hope to patients grappling with this devastating disease.

The Therapeutic Potential of Targeting Tumor Microenvironment and Modulation of Immunotherapy in Gastrointestinal Cancer.

Immunotherapy, as a novel treatment approach for various disorders, including cancers, is designed to either stimulate or suppress the immune system with high speci-ficity. The recent achievements of this therapy in clinical trials are set to transform tradi-tional treatment methods. Furthermore, it holds promise for enhancing the survival rates of patients suffering from both metastatic cancers and primary stages. Gastrointestinal Cancers (GI) account for 26% of global incidence and 35% of worldwide deaths. Treat-ment can be carried out using targeted immunotherapy in these cancers. If the tiers are superior, improvement could require more enterprise. On account that the function of immunotherapy in GI has been so promising, solely in sufferers with severe metastatic levels, within the literature, the immune checkpoint inhibitors in cancer immunotherapy of GI cancers, chimeric antigen receptor T-cell (vehicle-T), modulators of the tumor mi-croenvironment, and drug resistance mechanisms in immunotherapy as an effective treatment approach to GI cancers along with colon, pancreas, gastric, and esophageal cancers have been addressed. This review provides an overview of FDA-approved im-munotherapy drugs and ongoing preclinical developments. Additionally, we offer in-sights into the future of immunotherapy for GI cancer patients, addressing the associated challenges.

Next-Generation Therapies for Multiple Myeloma.

Recent therapeutic advances have significantly improved the outcome for patients with multiple myeloma (MM). The backbone of successful standard therapy is the combination of Ikaros degraders, glucocorticoids, and proteasome inhibitors that interfere with the integrity of myeloma-specific superenhancers by directly or indirectly targeting enhancer-bound transcription factors and coactivators that control expression of MM dependency genes. T cell engagers and chimeric antigen receptor T cells redirect patients' own T cells onto defined tumor antigens to kill MM cells. They have induced complete remissions even in end-stage patients. Unfortunately, responses to both conventional therapy and immunotherapy are not durable, and tumor heterogeneity, antigen loss, and lack of T cell fitness lead to therapy resistance and relapse. Novel approaches are under development to target myeloma-specific vulnerabilities, as is the design of multimodality immunological approaches, including and beyond T cells, that simultaneously recognize multiple epitopes to prevent antigen escape and tumor relapse.

The efficacy and applicability of chimeric antigen receptor (CAR) T cell-based regimens for primary bone tumors: A comprehensive review of current evidence.

Primary bone tumors (PBT), although rare, could pose significant mortality and morbidity risks due to their high incidence of lung metastasis. Survival rates of patients with PBTs may vary based on the tumor type, therapeutic interventions, and the time of diagnosis. Despite advances in the management of patients with these tumors over the past four decades, the survival rates seem not to have improved significantly, implicating the need for novel therapeutic interventions. Surgical resection with wide margins, radiotherapy, and systemic chemotherapy are the main lines of treatment for PBTs. Neoadjuvant and adjuvant chemotherapy, along with emerging immunotherapeutic approaches such as chimeric antigen receptor (CAR)-T cell therapy, have the potential to improve the treatment outcomes for patients with PBTs. CAR-T cell therapy has been introduced as an option in hematologic malignancies, with FDA approval for several CD19-targeting CAR-T cell products. This review aims to highlight the potential of immunotherapeutic strategies, specifically CAR T cell therapy, in managing PBTs.

Homology-independent targeted insertion-mediated derivation of M1-biased macrophages harbouring Megf10 and CD3ζ from human pluripotent stem cells.

BACKGROUND: Macrophages engineered with chimeric antigen receptors (CAR) are suitable for immunotherapy based on their immunomodulatory activity and ability to infiltrate solid tumours. However, the production and application of genetically edited, highly effective, and mass-produced CAR-modified macrophages (CAR-Ms) are challenging. METHODS: Here, we used homology-independent targeted insertion (HITI) for site-directed CAR integration into the safe-harbour region of human pluripotent stem cells (hPSCs). This approach, together with a simple differentiation protocol, produced stable and highly effective CAR-Ms without heterogeneity. FINDINGS: These engineered cells phagocytosed cancer cells, leading to significant inhibition of cancer-cell proliferation in vitro and in vivo. Furthermore, the engineered CARs, which incorporated a combination of CD3ζ and Megf10 (referred to as FRP5Mζ), markedly enhanced the antitumour effect of CAR-Ms by promoting M1, but not M2, polarisation. FRP5Mζ promoted M1 polarisation via nuclear factor kappa B (NF-κB), ERK, and STAT1 signalling, and concurrently inhibited STAT3 signalling even under M2 conditions. These features of CAR-Ms modulated the tumour microenvironment by activating inflammatory signalling, inducing M1 polarisation of bystander non-CAR macrophages, and enhancing the infiltration of T cells in cancer spheroids. INTERPRETATION: Our findings suggest that CAR-Ms have promise as immunotherapeutics. In conclusion, the guided insertion of CAR containing CD3ζ and Megf10 domains is an effective strategy for the immunotherapy of solid tumours. FUNDING: This work was supported by KRIBB Research Initiative Program Grant (KGM4562431, KGM5282423) and a Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korean government (Ministry of Science and ICT,Ministry of Health and Welfare) (22A0304L1-01).

Therapeutic targets of armored chimeric antigen receptor T cells navigating the tumor microenvironment.

Chimeric antigen receptor (CAR) T cell therapy, which targets tumors with high specificity through the recognition of particular antigens, has emerged as one of the most rapidly advancing modalities in immunotherapy, demonstrating substantial success against hematological malignancies. However, previous generations of CAR-T cell therapy encountered numerous challenges in treating solid tumors, such as the lack of suitable targets, high immunosuppression, suboptimal persistence, and insufficient infiltration owing to the complexities of the tumor microenvironment, all of which limited their efficacy. In this review, we focus on the current therapeutic targets of fourth-generation CAR-T cells, also known as armored CAR-T cells, and explore the mechanisms by which these engineered cells navigate the tumor microenvironment by targeting its various components. Enhancing CAR-T cells with these therapeutic targets holds promise for improving their effectiveness against solid tumors, thus achieving substantial clinical value and advancing the field of CAR-T cell therapy. Additionally, we discuss potential strategies to overcome existing challenges and highlight novel targets that could further enhance the efficacy of CAR-T cell therapy in treating solid tumors.

Regulatory CD4+ T cells redirected against pathogenic CD8+ T cells protect NOD mice from development of autoimmune diabetes.

Introduction: In this study, we report a novel therapeutic approach redirecting antigen-specific CD4+ T cells recognizing a hybrid insulin peptide (BDC2.5 T cell receptor (TCR) transgenic CD4+ T cells) to attract and suppress islet-specific CD8+ T cells T cells in the non-obese diabetic (NOD) mouse model, and prevent the development of autoimmune diabetes. Methods: Purified BDC2.5 CD4+ T cells were induced to differentiate into regulatory T cells (Tregs). The Tregs were then electroporated with mRNA encoding chimeric human ß2 microglobulin (hß2m) covalently linked to insulin B chain amino acids 15-23 (designated INS-eTreg) or islet-specific glucose-6-phosphatase related protein (IGRP) peptide 206-214 (designated IGRP-eTreg). Immunoregulatory functions of these engineered regulatory T cells (eTregs) were tested by in vitro assays and in vivo co-transfer experiments with ß-cell-antigen-specific CD8+ T cells in NOD.Scid mice or by adoptive transfer into young, pre-diabetic NOD mice. Results: These eTregs were phenotyped by flow cytometry, and shown to have high expression of FoxP3, as well as other markers of Treg function, including IL-10. They suppressed polyclonal CD4+ T cells and antigen-specific CD8+ T cells (recognizing insulin or IGRP), decreasing proliferation and increasing exhaustion and regulatory markers in vitro. In vivo, eTregs reduced diabetes development in co-transfer experiments with pathogenic antigen-specific CD8+ T cells (INS-CD8+ or IGRP-CD8+ cells) into NOD.Scid mice. Finally, when the eTreg were injected into young NOD mice, they reduced insulitis and prevented spontaneous diabetes in the recipient mice. Conclusion: Our results suggest a novel therapeutic strategy to protect NOD mice by targeting antigen-specific cytotoxic CD8+ T cells, using redirected antigen-specific CD4+ Treg cells, to suppress autoimmune diabetes. This may suggest an innovative therapy for protection of people at risk of development of type 1 diabetes.

Case report: Successful combination of CLL1 CAR-T therapy and hematopoietic stem cell transplantation in a 73-year-old patient diagnosed with refractory acute myeloid leukemia.

The incidence of Acute myeloid leukemia (AML) increases with advancing age, and the prognosis for elderly patients is significantly poorer compared to younger patients. Although the combination therapy of venetoclax and hypomethylating agents has demonstrated improved prognosis in patients unable to tolerate intensive chemotherapy, there remains a therapeutic blank for those who fail to achieve remission with current treatment regimens. Here, we report the successful clinical utilization of autogenous CLL1 CAR-T therapy combined with hematopoietic stem cell transplantation in a 73-year-old patient diagnosed with refractory AML. The patient achieved morphological complete remission (CR) with incomplete marrow recovery and a slight presence of minimal residual disease (MRD) after receiving CLL1 CAR-T therapy. To further enhance the treatment and promote the recovery of hemopoiesis, we performed bridged allogenic hematopoietic stem cell transplantation (allo-HSCT) 20 days after the infusion of CLL1 CAR-T cells. The patient achieved MRD-negative CR following HSCT treatment. His primary disease maintained a complete remission status during the 11-month follow-up period. The patient encountered grade 2 cytokine release syndrome and grade 4 granulocytopenia subsequent to the infusion of CAR-T cells, while several rounds of infection and graft-versus-host disease were observed following allo-HSCT. Nevertheless, all these concerns were successfully addressed through comprehensive provision of supportive treatments. We have successfully demonstrated a highly effective and safe combination strategy involving CLL1 CAR-T therapy and allo-HSCT, which has exhibited remarkable tolerability and holds great promise even for elderly patients with AML.

Case report: sub-clinical extramedullary B-ALL in the setting of relapse following targeted therapy.

Standard testing for disease evaluation in B-cell acute lymphoblastic leukemia (B-ALL) includes examination of the bone marrow and cerebrospinal fluid. Radiographic or functional imaging are indicated when clinical signs of non-CNS extramedullary disease are present but are not standard in the relapsed/refractory setting. We describe two cases of patients with relapsed/refractory B-ALL with prior exposure to blinatumomab and/or inotuzumab ozogamicin presenting for CAR-T cell treatment. Both patients were thought to only have minimal residual disease (MRD) at the pre-CAR disease assessment, with MRD of 6,648 (0.66%) and 100 (0.01%) cells per million cells, respectively, as measured by next-generation sequencing (NGS) in their bone marrows. Both patients for distinct reasons unrelated to non-CNS extra-medullary (EM) symptoms had PET-MRIs prior to lymphodepletion and CAR T cell infusion. In both cases patients were found to have significant bulky subclinical EM disease that required changes in clinical management. In the newly-emergent era of antigen-targeted immunotherapy, it is foundational that incidence and relapse patterns following targeted therapy are well-understood. Herein we contribute to a growing body of literature addressing this fundamental clinical gap and highlight a future role for formal prospective imaging studies to better establish response, toxicity and relapse patterns following CAR-T cell therapy in EM B-ALL.

[Cost-effectiveness analysis in the treatment of hematologic malignancies].

Recent advancements in treatment have improved the prognosis of hematologic malignancies. However, the increasing cost of therapeutic drugs has become an urgent issue. Cost-effectiveness analysis is performed using the incremental cost-effective ratio (ICER), a value calculated by dividing the incremental cost by the incremental quality-adjusted life year (QALY). The ICER must be compared with the willingness-to-pay (WTP) threshold, which differs between countries. Since the analysis should be made over a long time horizon, it is necessary to model and extrapolate the long-term outcomes of clinical trials to calculate cumulative costs and QALYs. This article discusses several approaches to cost-effectiveness analysis for chronic myelogenous leukemia, multiple myeloma, and CAR-T therapy. As even expensive treatments could be cost-effective if they provide long treatment-free survival, it is essential to judge cost-effectiveness by an appropriate method, rather than price alone.

Long-Term Survival and Immune Reconstitution of Donor-Derived Chimeric Antigen Receptor T-Cell Therapy for Childhood Molecular Relapse of B-Cell Acute Lymphoblastic Leukemia After Allogeneic Hematopoietic Stem Cell Transplantation.

Measurable residual disease (MRD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an independent risk factor for relapse in patients with acute lymphoblastic leukemia (ALL). This study aimed to assess the efficacy, safety, and immune reconstitution of chimeric antigen receptor T-cell (CAR-T) therapy in patients with molecular relapse after allo-HSCT. Eleven patients with molecular relapse of B-cell-ALL who underwent CAR-T therapy after allo-HSCT were enrolled. The rate of MRD negativity after a month of CAR-T infusion was 81.8%. Patients who bridged to second-HSCT after CAR-T therapy (n = 3) showed a trend of higher 3-year leukemia-free survival and 3-year overall survival than those who did not (n = 8; 100% vs. 75.0%; 95% CI, 45.0-104.9%; p = 0.370). No treatment-related mortalities were observed. Among patients who did not bridge to second-HSCT and remained in complete remission until the last follow-up (n = 6), five of them had not recovered normal immunoglobulin concentrations with a median follow-up of 43 months. CAR-T therapy may be a safe and effective treatment strategy to improve survival after allo-HSCT; however, the problem of prolonged hypogammaglobulinemia in patients who do not bridge to second-HSCT is worth noting.

S100 as marker for immune effector cell-associated neurotoxicity syndrome.

Chimeric antigen receptor (CAR)-T cell therapy is a new and successful treatment for otherwise refractory malignancies but despite the growing number of applications, this form of treatment is still associated with significant toxicity. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) in particular are common and dangerous side effects. This report is about two patients who received CAR­T cell therapy and subsequently developed ICANS. This was successfully treated. During CAR­T cell therapy, a blood marker, S100, was monitored daily. It correlated with the occurrence and progression of ICANS.

Therapeutic advantage of combinatorial CAR T cell and chemo-therapies.

Chimeric antigen receptor (CAR) T cell therapies have transformed outcomes for many patients with hematological malignancies. However, some patients do not respond to CAR T cell treatment, and adapting CAR T cells for solid and brain tumors has been met with many challenges including a hostile tumor microenvironment and poor CAR T cell persistence. Thus, it is unlikely that CAR T cell therapy alone will be sufficient for consistent, complete tumor clearance across cancer patients. Combinatorial therapies of CAR T cells and chemotherapeutics are a promising approach for overcoming this as chemotherapeutics could augment CAR T cells for improved anti-tumor activity or work in tandem with CAR T cells to clear tumors. Herein, we review efforts towards achieving successful CAR T cell and chemical drug combination therapies. We focus on combination therapies with approved chemotherapeutics as these will be more easily translated to the clinic, but also review non-approved chemotherapeutics and drug screens designed to reveal promising new CAR T cell and chemical drug combinations. Together, this review highlights the promise of CAR T cell and chemotherapy combinations with specific focus on how combinatorial therapy overcomes challenges faced by either monotherapy and supports the potential of this therapeutic strategy to improve outcomes for cancer patients. Significance Statement Improving currently available CAR T cell products via combinatorial therapy with chemotherapeutics has the potential to drastically expand the types of cancers and number of patients that could benefit from these therapies when neither alone has been sufficient to achieve tumor clearance. Herein, we provide a thorough review of the current efforts towards studying CAR T and chemotherapy combinatorial therapies and provide perspectives on optimal ways to identify new and effective combinations moving forward.

Generating advanced CAR-based therapy for hematological malignancies in clinical practice: targets to cell sources to combinational strategies.

Chimeric antigen receptor T (CAR-T) cell therapy has been a milestone breakthrough in the treatment of hematological malignancies, offering an effective therapeutic option for multi-line therapy-refractory patients. So far, abundant CAR-T products have been approved by the United States Food and Drug Administration or China National Medical Products Administration to treat relapsed or refractory hematological malignancies and exhibited unprecedented clinical efficiency. However, there were still several significant unmet needs to be progressed, such as the life-threatening toxicities, the high cost, the labor-intensive manufacturing process and the poor long-term therapeutic efficacy. According to the demands, many researches, relating to notable technical progress and the replenishment of alternative targets or cells, have been performed with promising results. In this review, we will summarize the current research progress in CAR-T eras from the "targets" to "alternative cells", to "combinational drugs" in preclinical studies and clinical trials.

Immunoediting Dynamics in Glioblastoma: Implications for Immunotherapy Approaches.

Glioblastoma is an aggressive primary brain tumor that poses many therapeutic difficulties because of the high rate of proliferation, genetic variability, and its immunosuppressive microenvironment. The theory of cancer immunoediting, which includes the phases of elimination, equilibrium, and escape, offers a paradigm for comprehending interactions between the immune system and glioblastoma. Immunoediting indicates the process by which immune cells initially suppress tumor development, but thereafter select for immune-resistant versions leading to tumor escape and progression. The tumor microenvironment (TME) in glioblastoma is particularly immunosuppressive, with regulatory T cells and myeloid-derived suppressor cells being involved in immune escape. To achieve an efficient immunotherapy for glioblastoma, it is crucial to understand these mechanisms within the TME. Existing immunotherapeutic modalities such as chimeric antigen receptor T cells and immune checkpoint inhibitors have been met with some level of resistance because of the heterogeneous nature of the immune response to glioblastoma. Solving these issues is critical to develop novel strategies capable of modulating the TME and re-establishing normal immune monitoring. Further studies should be conducted to identify the molecular and cellular events that underlie the immunosuppressive tumor microenvironment in glioblastoma. Comprehending and modifying the stages of immunoediting in glioblastoma could facilitate the development of more potent and long-lasting therapies.

Antigen-independent activation is critical for the durable antitumor effect of GUCY2C-targeted CAR-T cells.

BACKGROUND: Chimeric antigen receptor (CAR)-T cells face many obstacles in solid tumor therapy, including heterogeneous antigen expression and inefficient T cell persistence. Guanylyl cyclase C (GUCY2C) has been identified as a suitable tumor antigen for targeted therapy due to its intestinal-restricted expression pattern in normal tissues and steady overexpression in gastrointestinal tumors, especially colorectal cancer. An antigen-sensitive and long-lasting CAR-T cell targeting GUCY2C was investigated in this study. METHODS: Using constructed tumor cell lines with various GUCY2C expression densities, we screened out an antigen-sensitive single chain variable fragment (scFv) that enabled CAR-T cells to efficiently eradicate the GUCY2C lowly expressed tumor cells. CAR-T cells with different compositions of the hinge, transmembrane and costimulatory domains were also constructed for selection of the long-lasting CAR-T format with durable antitumor efficacy in vitro and in tumor-bearing mice. The underlying mechanism was further investigated based on mutation of the hinge and transmembrane domains. RESULTS: We found that the composition of the antigen-sensitive scFv, CD8α hinge, CD8α transmembrane, and CD28 costimulatory domains boosted CAR-T cells to rapidly kill tumors, maintain high expansion capacity, and long-term efficacy in various colorectal cancer models. The durable antitumor function was attributed to the optimal CAR tonic signaling that conferred CAR-T cells with autonomous activation, proliferation, survival and cytokine release in the absence of antigen stimulation. The tonic signaling was associated with the length and the cysteine residues in the CD8α hinge and transmembrane domains. CONCLUSIONS: This study demonstrated a potent GUCY2C-targeted CAR-T cell for gastrointestinal tumor therapy and highlights the importance of adequate tonic signaling for effective CAR-T cell therapy against solid tumors.

EASIX-guided risk stratification for complications and outcome after CAR T-cell therapy with ide-cel in relapsed/refractory multiple myeloma.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy has demonstrated significant benefits in the treatment of relapsed/refractory multiple myeloma (RRMM). However, these outcomes can be compromised by severe complications, including cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS) and immune effector cell-associated hematotoxicity (ICAHT), predisposing for life-threatening infections. METHODS: This retrospective observational study examined a total of 129 patients with RRMM who had received idecabtagene vicleucel (ide-cel) at two major myeloma centers in Germany and one center in the USA to assess the Endothelial Activation and Stress Index (EASIX) as a risk marker for an unfavorable clinical course and outcome after CAR T-cell therapy. EASIX is calculated by lactate dehydrogenase (U/L) × creatinine (mg/dL) / platelets (109 cells/L) and was determined before lymphodepletion (baseline) and at the day of CAR T-cell infusion (day 0). The analysis was extended to EASIX derivatives and the CAR-HEMATOTOX score. RESULTS: An elevated baseline EASIX (>median) was identified as a risk marker for severe late ICAHT, manifesting with an impaired hematopoietic reconstitution and pronounced cytopenias during the late post-CAR-T period. Patients with high EASIX levels (>upper quartile) were particularly at risk, as evidenced by an increased rate of an aplastic phenotype of neutrophil recovery, severe late-onset infections and ICANS. Finally, we found associations between baseline EASIX and an inferior progression-free and overall survival. Moreover, the EASIX at day 0 also demonstrated potential to serve as a risk marker for post-CAR-T complications and adverse outcomes. CONCLUSIONS: In conclusion, EASIX aids in risk stratification at clinically relevant time points prior to CAR T-cell therapy with ide-cel. Increased EASIX levels might help clinicians to identify vulnerable patients to adapt peri-CAR-T management at an early stage.

CAR T-cell therapy in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive leukemic malignancy that affects myeloid lineage progenitors. Relapsed or refractory AML patients continue to have poor prognoses, necessitating the development of novel therapy alternatives. Adoptive T-cell therapy with chimeric antigen receptors (CARs) is an intriguing possibility in the field of leukemia treatment. Chimeric antigen receptor T-cell therapy is now being tested in clinical trials (mostly in phase I and phase II) using AML targets including CD33, CD123, and CLL-1. Preliminary data showed promising results. However, due to the cellular and molecular heterogeneity of AML and the co-expression of some AML targets on hematopoietic stem cells, these clinical investigations have shown substantial "on-target off-tumor" toxicities, indicating that more research is required. In this review, the latest significant breakthroughs in AML CAR T cell therapy are presented. Furthermore, the limitations of CAR T-cell technology and future directions to overcome these challenges are discussed.