Allogeneic CD19-targeted CAR-T therapy in patients with severe myositis and systemic sclerosis.

Allogeneic chimeric antigen receptor (CAR)-T cells hold great promise for expanding the accessibility of CAR-T therapy, whereas the risks of allograft rejection have hampered its application. Here, we genetically engineered healthy-donor-derived, CD19-targeting CAR-T cells using CRISPR-Cas9 to address the issue of immune rejection and treated one patient with refractory immune-mediated necrotizing myopathy and two patients with diffuse cutaneous systemic sclerosis with these cells. This study was registered at ClinicalTrials.gov (NCT05859997). The infused cells persisted for over 3 months, achieving complete B cell depletion within 2 weeks of treatment. During the 6-month follow-up, we observed deep remission without cytokine release syndrome or other serious adverse events in all three patients, primarily shown by the significant improvement in the clinical response index scores for the two diseases, respectively, and supported by the observations of reversal of inflammation and fibrosis. Our results demonstrate the high safety and promising immune modulatory effect of the off-the-shelf CAR-T cells in treating severe refractory autoimmune diseases.

Recent updates on allogeneic CAR-T cells in hematological malignancies.

CAR-T cell therapy is known as an effective therapy in patients with hematological malignancies. Since 2017, several autologous CAR-T cell (auto-CAR-T) drugs have been approved by the US Food and Drug Administration (FDA) for the treatment of some kinds of relapsed/refractory hematological malignancies. However, some patients fail to respond to these drugs due to high manufacturing time, batch-to-batch variation, poor quality and insufficient quantity of primary T cells, and their insufficient expansion and function. CAR-T cells prepared from allogeneic sources (allo-CAR-Ts) can be an alternative option to overcome these obstacles. Recently, several allo-CAR-Ts have entered into the early clinical trials. Despite their promising preclinical and clinical results, there are two main barriers, including graft-versus-host disease (GvHD) and allo-rejection that may decline the safety and efficacy of allo-CAR-Ts in the clinic. The successful development of these products depends on the starter cell source, the gene editing method, and the ability to escape immune rejection and prevent GvHD. Here, we summarize the gene editing technologies and the potential of various cell sources for developing allo-CAR-Ts and highlight their advantages for the treatment of hematological malignancies. We also describe preclinical and clinical data focusing on allo-CAR-T therapy in blood malignancies and discuss challenges and future perspectives of allo-CAR-Ts for therapeutic applications.

Release Assays and Potency Assays for CAR T-Cell Interventions.

Chimeric antigen receptor (CAR) T-cells are considered "living drugs" and offer a compelling alternative to conventional anticancer therapies. Briefly, T-cells are redirected, using gene engineering technology, toward a specific cancer cell surface target antigen via a synthetic chimeric antigen receptor (CAR) protein. CARs have a modular design comprising four main structures: an antigen-binding domain, a hinge region, a transmembrane domain, and one or more intracellular signaling domains for T-cell activation. A major challenge in the CAR T-cell manufacturing field is balancing product quality with scalability and cost-effectiveness, especially when transitioning from an academic clinical trial into a marketed product, to be implemented across many collection, manufacturing, and treatment sites. Achieving product consistency while circumnavigating the intrinsic variability associated with autologous products is an additional barrier. To overcome these limitations, a robust understanding of the product and its biological actions is crucial to establish a target product profile with a defined list of critical quality attributes to be assessed for each batch prior to product certification. Additional challenges arise as the field progresses, such as new safety considerations associated with the use of allogenic T-cells and genome editing tools. In this chapter, we will discuss the release and potency assays required for CAR T-cell manufacturing, covering their relevance, current challenges, and future perspectives.

ReCARving the future: bridging CAR T-cell therapy gaps with synthetic biology, engineering, and economic insights.

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematologic malignancies, offering remarkable remission rates in otherwise refractory conditions. However, its expansion into broader oncological applications faces significant hurdles, including limited efficacy in solid tumors, safety concerns related to toxicity, and logistical challenges in manufacturing and scalability. This review critically examines the latest advancements aimed at overcoming these obstacles, highlighting innovations in CAR T-cell engineering, novel antigen targeting strategies, and improvements in delivery and persistence within the tumor microenvironment. We also discuss the development of allogeneic CAR T cells as off-the-shelf therapies, strategies to mitigate adverse effects, and the integration of CAR T cells with other therapeutic modalities. This comprehensive analysis underscores the synergistic potential of these strategies to enhance the safety, efficacy, and accessibility of CAR T-cell therapies, providing a forward-looking perspective on their evolutionary trajectory in cancer treatment.

Generating universal anti-CD19 CAR T cells with a defined memory phenotype by CRISPR/Cas9 editing and safety evaluation of the transcriptome.

Introduction: Chimeric antigen receptor-expressing T cells (CAR T cells) have revolutionized cancer treatment, particularly in B cell malignancies. However, the use of autologous T cells for CAR T therapy presents several limitations, including high costs, variable efficacy, and adverse effects linked to cell phenotype. Methods: To overcome these challenges, we developed a strategy to generate universal and safe anti-CD19 CAR T cells with a defined memory phenotype. Our approach utilizes CRISPR/Cas9 technology to target and eliminate the B2M and TRAC genes, reducing graft-versus-host and host-versus-graft responses. Additionally, we selected less differentiated T cells to improve the stability and persistence of the universal CAR T cells. The safety of this method was assessed using our CRISPRroots transcriptome analysis pipeline, which ensures successful gene knockout and the absence of unintended off-target effects on gene expression or transcriptome sequence. Results: In vitro experiments demonstrated the successful generation of functional universal CAR T cells. These cells exhibited potent lytic activity against tumor cells and a reduced cytokine secretion profile. The CRISPRroots analysis confirmed effective gene knockout and no unintended off-target effects, validating it as a pioneering tool for on/off-target and transcriptome analysis in genome editing experiments. Discussion: Our findings establish a robust pipeline for manufacturing safe, universal CAR T cells with a favorable memory phenotype. This approach has the potential to address the current limitations of autologous CAR T cell therapy, offering a more stable and persistent treatment option with reduced adverse effects. The use of CRISPRroots enhances the reliability and safety of gene editing in the development of CAR T cell therapies. Conclusion: We have developed a potent and reliable method for producing universal CAR T cells with a defined memory phenotype, demonstrating both efficacy and safety in vitro. This innovative approach could significantly improve the therapeutic landscape for patients with B cell malignancies.

Chimeric Antigen Receptor-T Cell Therapy for Lymphoma: New Settings and Future Directions.

In the last decade, anti-CD19 CAR-T cell therapy has led to a treatment paradigm shift for B-cell non-Hodgkin lymphomas, first with the approval for relapsed/refractory (R/R) large B-cell lymphomas and subsequently for R/R mantle cell and follicular lymphoma. Many efforts are continuously being made to extend the therapeutic setting in the lymphoma field. Several reports are supporting the safety and efficacy of CAR-T cells in patients with central nervous system disease involvement. Anti-CD30 CAR-T cells for the treatment of Hodgkin lymphoma are in development and early studies looking for the optimal target for T-cell malignancies are ongoing. Anti-CD19/CD20 and CD19/CD22 dual targeting CAR-T cells are under investigation in order to increase anti-lymphoma activity and overcome tumor immune escape. Allogeneic CAR product engineering is on the way, representing a rapidly accessible 'off-the-shelf' and potentially more fit product. In the present manuscript, we will focus on recent advances in CAR-T cell therapy for lymphomas, including new settings and future perspectives in the field, reviewing data reported in literature in the last decade up to October 2023.

Custom CARs: Leveraging the Adaptability of Allogeneic CAR Therapies to Address Current Challenges in Relapsed/Refractory DLBCL.

Cellular therapies have transformed the treatment of relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL), which typically does not respond well to salvage chemotherapy. Recently, approximately 40% of r/r DLBCL patients across three different trials achieved a complete remission at 1 year after receiving treatment with autologous chimeric antigen receptor (CAR) T cells (auto-CARs). These successes have prompted studies of auto-CARs in second-line settings, in which axicabtagene ciloleucel and lisocabtagene maraleucel both showed improved event-free survival over autologous hematopoietic cell transplantation (AHCT). While encouraging, this data also highlights that 60% of patients relapse or progress following treatment with auto-CARs. Individual disease characteristics and logistical challenges of cell engineering also limit patients' eligibility for auto-CARs. Allogeneic CAR T cells (allo-CARs) may address some of these limitations as they may mitigate delays associated with auto-CARs, thereby reducing the need for bridging chemotherapies and increasing availability of cellular products for patients with aggressive lymphomas. By being sourced from healthy donors who have never been exposed to cytotoxic chemotherapy, allo-CARs can be created from T cells with better fitness. Allo-CARs made from specific cellular subsets (e.g., stem cell memory or naïve/early memory T cells) may also have increased efficacy and long-term persistence. Additionally, allo-CARs have been successfully created from other cell types, including natural killer cells, gamma-delta T-cells and induced pluripotent stem cells. These cell types can be engineered to target viral antigens, enabling precision targeting of virally driven DLBCL. As allogeneic donor cells can be banked and cryopreserved in batches, they can be made more readily available, potentially reducing logistical hurdles and costs compared to engineering auto-CARs. This may ultimately create a more sustainable platform for cell therapies. Challenges with allo-CARs that will need to be addressed include graft versus host disease, alloimmunization, potentially decreased persistence relative to auto-CARs, and antigen escape. In short, the adaptability of allo-CARs makes them ideal for treating patients with r/r DLBCL who have progressed through standard chemotherapy, AHCT, or auto-CARs. Here, we review the published literature on patients with r/r DLBCL treated with allogeneic CAR products manufactured from various cell types as well as forthcoming allogeneic CAR technologies.

Use of chimeric antigen receptor T cells in allogeneic hematopoietic stem cell transplantation.

The chimeric antigen receptor T (CAR-T) cells play an antileukemia role, and can be used to treat or prevent relapse by targeting minimal residual disease for patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the infusion of allogeneic CAR-T cells may also cause graft-versus-host disease, which limited their applications during and after allo-HSCT. In this review, we discuss the clinical trials that applying CAR-T cells before allo-HSCT and the use of donor-derived CAR-T cells as conditioning regimen during allo-HSCT. At last, we analyzed the effect of donor-derived CAR-T cells on preventive infusion after allo-HSCT.