Off-the-shelf CAR-T cell therapies for relapsed or refractory B-cell malignancies: latest update from ASH 2023 annual meeting.

Currently, many off-the-shelf chimeric antigen receptor (CAR)-T cell products are under investigation for the treatment of relapsed or refractory (R/R) B-cell neoplasms. Compared with autologous CAR-T cell therapy, off-the-shelf universal CAR-T cell therapies have many potential benefits, such as immediate accessibility for patients, stable quality due to industrialized manufacturing and additional infusions of CAR-T cells with different targets. However, critical challenges, including graft-versus-host disease and CAR-T cell elimination by the host immune system, still require extensive research. The most common technological approaches involve modifying healthy donor T cells via gene editing technology and altering different types of T cells. This article summarizes some of the latest data from preclinical and clinical studies of off-the-shelf CAR-T cell therapies in the treatment of R/R B-cell malignancies from the 2023 ASH Annual Meeting (ASH 2023).

Prospects and challenges of CAR-T in the treatment of ovarian cancer.

Ovarian cancer ranks as the seventh most prevalent cancer among women and is considered the most lethal gynecological malignancy on a global scale. The absence of reliable screening techniques, coupled with the insidious onset of nonspecific symptoms, often results in a delayed diagnosis, typically at an advanced stage characterized by peritoneal involvement. Management of advanced tumors typically involves a combination of chemotherapy and cytoreductive surgery. However, the therapeutic arsenal for ovarian cancer patients remains limited, highlighting the unmet need for precise, targeted, and sustained-release pharmacological agents. Genetically engineered T cells expressing chimeric antigen receptors (CARs) represent a promising novel therapeutic modality that selectively targets specific antigens, demonstrating robust and enduring antitumor responses in numerous patients. CAR T cell therapy has exhibited notable efficacy in hematological malignancies and is currently under investigation for its potential in treating various solid tumors, including ovarian cancer. Currently, numerous researchers are engaged in the development of novel CAR-T cells designed to target ovarian cancer, with subsequent evaluation of these candidate cells in preclinical studies. Given the ability of chimeric antigen receptor (CAR) expressing T cells to elicit potent and long-lasting anti-tumor effects, this therapeutic approach holds significant promise for the treatment of ovarian cancer. This review article examines the utilization of CAR-T cells in the context of ovarian cancer therapy.

Secondary bone marrow graft loss after third-party virus-specific T cell infusion: Case report of a rare complication.

Virus-specific T cells (VST) from partially-HLA matched donors have been effective for treatment of refractory viral infections in immunocompromised patients in prior studies with a good safety profile, but rare adverse events have been described. Here we describe a unique and severe adverse event of VST therapy in an infant with severe combined immunodeficiency, who receives, as part of a clinical trial (NCT03475212), third party VSTs for treating cytomegalovirus viremia following bone marrow transplantation. At one-month post-VST infusion, rejection of graft and reversal of chimerism is observed, as is an expansion of T cells exclusively from the VST donor. Single-cell gene expression and T cell receptor profiling demonstrate a narrow repertoire of predominantly activated CD4+ T cells in the recipient at the time of rejection, with the repertoire overlapping more with that of peripheral blood from VST donor than the infused VST product. This case thus demonstrates a rare but serious side effect of VST therapy.

Naturally occurring T cell mutations enhance engineered T cell therapies.

Adoptive T cell therapies have produced exceptional responses in a subset of patients with cancer. However, therapeutic efficacy can be hindered by poor T cell persistence and function1. In human T cell cancers, evolution of the disease positively selects for mutations that improve fitness of T cells in challenging situations analogous to those faced by therapeutic T cells. Therefore, we reasoned that these mutations could be co-opted to improve T cell therapies. Here we systematically screened the effects of 71 mutations from T cell neoplasms on T cell signalling, cytokine production and in vivo persistence in tumours. We identify a gene fusion, CARD11-PIK3R3, found in a CD4+ cutaneous T cell lymphoma2, that augments CARD11-BCL10-MALT1 complex signalling and anti-tumour efficacy of therapeutic T cells in several immunotherapy-refractory models in an antigen-dependent manner. Underscoring its potential to be deployed safely, CARD11-PIK3R3-expressing cells were followed up to 418 days after T cell transfer in vivo without evidence of malignant transformation. Collectively, our results indicate that exploiting naturally occurring mutations represents a promising approach to explore the extremes of T cell biology and discover how solutions derived from evolution of malignant T cells can improve a broad range of T cell therapies.

Using Oncolytic Virus to Retask CD19-Chimeric Antigen Receptor T Cells for Treatment of Pancreatic Cancer: Toward a Universal Chimeric Antigen Receptor T-Cell Strategy for Solid Tumor.

BACKGROUND: Chimeric antigen receptor (CAR) T cells targeting the B-cell antigen CD19 are standard therapy for relapsed or refractory B-cell lymphoma and leukemia. CAR T cell therapy in solid tumors is limited due to an immunosuppressive tumor microenvironment and a lack of tumor-restricted antigens. We recently engineered an oncolytic virus (CF33) with high solid tumor affinity and specificity to deliver a nonsignaling truncated CD19 antigen (CD19t), allowing targeting by CD19-CAR T cells. Here, we tested this combination against pancreatic cancer. STUDY DESIGN: We engineered CF33 to express a CD19t (CF33-CD19t) target. Flow cytometry and ELISA were performed to quantify CD19t expression, immune activation, and killing by virus and CD19-CAR T cells against various pancreatic tumor cells. Subcutaneous pancreatic human xenograft tumor models were treated with virus, CAR T cells, or virus+CAR T cells. RESULTS: In vitro, CF33-CD19t infection of tumor cells resulted in >90% CD19t cell-surface expression. Coculturing CD19-CAR T cells with infected cells resulted in interleukin-2 and interferon gamma secretion, upregulation of T-cell activation markers, and synergistic cell killing. Combination therapy of virus+CAR T cells caused significant tumor regression (day 13): control (n = 16, 485 ± 20 mm 3 ), virus alone (n = 20, 254 ± 23 mm 3 , p = 0.0001), CAR T cells alone (n = 18, 466 ± 25 mm 3 , p = NS), and virus+CAR T cells (n = 16, 128 ± 14 mm 3 , p < 0.0001 vs control; p = 0.0003 vs virus). CONCLUSIONS: Engineered CF33-CD19t effectively infects and expresses CD19t in pancreatic tumors, triggering cell killing and increased immunogenic response by CD19-CAR T cells. Notably, CF33-CD19t can turn cold immunologic tumors hot, enabling solid tumors to be targetable by agents designed against liquid tumor antigens.

Progress, implications, and challenges in using humanized immune system mice in CAR-T therapy-Application evaluation and improvement.

In recent years, humanized immune system (HIS) mice have been gradually used as models for preclinical research in pharmacotherapies and cell therapies with major breakthroughs in tumor and other fields, better mimicking the human immune system and the tumor immune microenvironment, compared to traditional immunodeficient mice. To better promote the application of HIS mice in preclinical research, we selectively summarize the current prevalent and breakthrough research and evaluation of chimeric antigen receptor (CAR) -T cells in various antiviral and antitumor treatments. By exploring its application in preclinical research, we find that it can better reflect the actual clinical patient condition, with the advantages of providing high-efficiency detection indicators, even for progressive research and development. We believe that it has better clinical patient simulation and promotion for the updated design of CAR-T cell therapy than directly transplanted immunodeficient mice. The characteristics of the main models are proposed to improve the use defects of the existing models by reducing the limitation of antihost reaction, combining multiple models, and unifying sources and organoid substitution. Strategy study of relapse and toxicity after CAR-T treatment also provides more possibilities for application and development.

Risk factors and outcome of Chimeric Antigen Receptor T-Cell patients admitted to Pediatric Intensive Care Unit: CART-PICU study.

Introduction: Chimeric antigen receptor (CAR)T-cell CD19 therapy is an effective treatment for relapsed/refractory B-cell acute lymphoblastic leukemia. It can be associated with life-threatening toxicities which often require PICU admission. Purpose: to describe clinical characteristics, treatment and outcome of these patients. Methods: Prospective observational cohort study conducted in a tertiary pediatric hospital from 2016-2021. Children who received CAR-T admitted to PICU were included. We collected epidemiological, clinical characteristics, cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), treatment, length of stay and mortality. Results: CAR T-cells (4-1BB constructs) were infused in 59 patients. Twenty-four (40.7%) required PICU admission, length of stay was 4 days (IQR 3-6). Median age was 8.3 years (range 4-24). Patients admitted to PICU presented higher disease burden before infusion: 24% blasts in bone marrow (IQR 5-72) vs. 0 (0-6.9), p<0.001. No patients with <5% blasts were admitted to PICU. Main reasons for admissions were CRS (n=20, 83.3%) and ICANS (n=3, 12.5%). Fourteen patients (58.3%) required inotropic support, 14(58.3%) respiratory. Sixteen patients (66.6%) received tocilizumab, 10(41.6%) steroids, 6(25.0%) anakinra, and 5(20.8%) siltuximab. Ten patients (41.6%) presented neurotoxicity, six of them severe (ICANS 3-4). Two patients died at PICU (8.3%) because of refractory CRS-hemophagocytic lymphohistyocitosis (carHLH) syndrome. There were no significant differences in relapse rate after CAR-T in patients requiring PICU, it was more frequently CD19 negative (p=0.344). Discussion: PICU admission after CAR-T therapy was mainly due to CRS. Supportive treatment allowed effective management and high survival. Some patients presenting with carHLH, can suffer a fulminant course.

Chimeric antigen receptor T cells therapy in solid tumors

Chimeric antigen receptor T cells therapy (CAR-T therapy) is a class of ACT therapy. Chimeric antigen receptor (CAR) is an engineered synthetic receptor of CAR-T, which give T cells the ability to recognize tumor antigens in a human leukocyte antigen-independent (HLA-independent) manner and enables them to recognize more extensive target antigens than natural T cell surface receptor (TCR), resulting in tumor destruction. CAR-T is composed of an extracellular single-chain variable fragment (scFv) of antibody, which serves as the targeting moiety, hinge region, transmembrane spacer, and intracellular signaling domain(s). CAR-T has been developing in many generations, which differ according to costimulatory domains. CAR-T therapy has several limitations that reduce its wide availability in immunotherapy which we can summarize in antigen escape that shows either partial or complete loss of target antigen expression, so multiplexing CAR-T cells are promoted to enhance targeting of tumor profiles. In addition, the large diversity in the tumor microenvironment also plays a major role in limiting this kind of treatment. Therefore, engineered CAR-T cells can evoke immunostimulatory signals that rebalance the tumor microenvironment. Using CAR-T therapy in treating the solid tumor is mainly restricted by the difficulty of CAR-T cells infiltrating the tumor site, so local administration was developed to improve the quality of treatment. The most severe toxicity after CAR-T therapy is on-target/on-tumor toxicity, such as cytokine release syndrome (CRS). Another type of toxicity is on-target/off-tumor toxicity which originates from the binding of CAR-T cells to target antigen that has shared expression on normal cells leading to damage in healthy cells and organs. Toxicity management should become a focus of implementation to permit management beyond specialized centers (AU)

Applications of virus-specific T cell therapies post-BMT.

Hematopoietic stem cell transplantation (HSCT) has been used as a curative standard of care for moderate to severe primary immunodeficiency disorders as well as relapsed hematologic malignancies for over 50 years [1,2]. However, chronic and refractory viral infections remain a leading cause of morbidity and mortality in the immune deficient period following HSCT, where use of available antiviral pharmacotherapies is limited by toxicity and emerging resistance [3]. Adoptive immunotherapy using virus-specific T cells (VSTs) has been explored for over 2 decades [4,5] in patients post-HSCT and has been shown prior phase I-II studies to be safe and effective for treatment or preventions of viral infections including cytomegalovirus, Epstein-Barr virus, BK virus, and adenovirus with minimal toxicity and low risk of graft vs host disease [6-9]. This review summarizes methodologies to generate VSTs the clinical results utilizing VST therapeutics and the challenges and future directions for the field.

[Role of allogeneic hematopoietic cell transplantation after anti-CD19 CAR T-cell treatment: Guidelines from the SFGM-TC]. / Place de l'allogreffe de cellules souches hématopoïétiques après traitement par CAR T-cell anti-CD19 : recommandations de la SFGM-TC.

The role of allogeneic hematopoietic cell transplantation (allo-HCT) after CAR T- treatment cells in hematologic malignancies is currently controversial. Prolonged remissions after several years of follow-up suggest that there is a curative effect of CAR T-cells therapy, whereas allo-HCT was previously considered the only curative treatment in relapse situation. The aim of this harmonization workshop is to detail the existing data in the literature on the feasibility of allo-HCT after CAR T-cells and to propose to consider allograft in selected patients with B-acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL). In B-ALL, various intrinsic factors (inherent to the patient, to the disease, to the type of CAR T-cells) and especially various post CAR T-cells criteria (early expansion kinetics, residual disease at D28, early loss of B-cell aplasia) should lead to consider performing allo-HCT before the occurrence of a relapse. In DLBCL, although there are risk factors for relapse at diagnosis and prior to CAR T-cells therapy, response assessed by PET-CT at three months is critical and allo-HCT cannot currently be recommended in cases of complete or partial remission. In any case, if the age is appropriate for allogeneic transplantation, HLA typing should be performed before CAR T-cells treatment in order not to delay the allo-HCT project if needed.

Engineering time-controlled immunotherapy.

Designer lymphocytes expand the dynamic range of possibilities for treating disease.

Synthetic cytokine circuits that drive T cells into immune-excluded tumors.

Chimeric antigen receptor (CAR) T cells are ineffective against solid tumors with immunosuppressive microenvironments. To overcome suppression, we engineered circuits in which tumor-specific synNotch receptors locally induce production of the cytokine IL-2. These circuits potently enhance CAR T cell infiltration and clearance of immune-excluded tumors, without systemic toxicity. The most effective IL-2 induction circuit acts in an autocrine and T cell receptor (TCR)- or CAR-independent manner, bypassing suppression mechanisms including consumption of IL-2 or inhibition of TCR signaling. These engineered cells establish a foothold in the target tumors, with synthetic Notch-induced IL-2 production enabling initiation of CAR-mediated T cell expansion and cell killing. Thus, it is possible to reconstitute synthetic T cell circuits that activate the outputs ultimately required for an antitumor response, but in a manner that evades key points of tumor suppression.

Multidimensional control of therapeutic human cell function with synthetic gene circuits.

Synthetic gene circuits that precisely control human cell function could expand the capabilities of gene- and cell-based therapies. However, platforms for developing circuits in primary human cells that drive robust functional changes in vivo and have compositions suitable for clinical use are lacking. Here, we developed synthetic zinc finger transcription regulators (synZiFTRs), which are compact and based largely on human-derived proteins. As a proof of principle, we engineered gene switches and circuits that allow precise, user-defined control over therapeutically relevant genes in primary T cells using orthogonal, US Food and Drug Administration-approved small-molecule inducers. Our circuits can instruct T cells to sequentially activate multiple cellular programs such as proliferation and antitumor activity to drive synergistic therapeutic responses. This platform should accelerate the development and clinical translation of synthetic gene circuits in diverse human cell types and contexts.

The future of engineered immune cell therapies.

Immune cells are being engineered to recognize and respond to disease states, acting as a "living drug" when transferred into patients. Therapies based on engineered immune cells are now a clinical reality, with multiple engineered T cell therapies approved for treatment of hematologic malignancies. Ongoing preclinical and clinical studies are testing diverse strategies to modify the fate and function of immune cells for applications in cancer, infectious disease, and beyond. Here, we discuss current progress in treating human disease with immune cell therapeutics, emerging strategies for immune cell engineering, and challenges facing the field, with a particular emphasis on the treatment of cancer, where the most effort has been applied to date.

The emerging era of cell engineering: Harnessing the modularity of cells to program complex biological function.

A new era of biological engineering is emerging in which living cells are used as building blocks to address therapeutic challenges. These efforts are distinct from traditional molecular engineering-their focus is not on optimizing individual genes and proteins as therapeutics, but rather on using molecular components as modules to reprogram how cells make decisions and communicate to achieve higher-order physiological functions in vivo. This cell-centric approach is enabled by a growing tool kit of components that can synthetically control core cell-level functional outputs, such as where in the body a cell should go, what other cells it should interact with, and what messages it should transmit or receive. The power of cell engineering has been clinically validated by the development of immune cells designed to kill cancer. This same tool kit for rewiring cell connectivity is beginning to be used to engineer cell therapies for a host of other diseases and to program the self-organization of tissues and organs. By forcing the conceptual distillation of complex biological functions into a finite set of instructions that operate at the cell level, these efforts also shed light on the fundamental hierarchical logic that links molecular components to higher-order physiological function.

Therapeutic Potential of Ex Vivo Expanded γδ T Cells against Osteosarcoma Cells.

Immunotherapy is an attractive therapeutic strategy for the treatment of osteosarcoma (OS). The unique features of γδ T cells have made them popular for cancer immunotherapy. Here, we expanded γδ T cells using human peripheral blood mononuclear cells (PBMCs) and investigated their therapeutic potential against OS cells. PBMCs from healthy donors were cultured for 10 days with CON medium (unstimulated control); EX media, CON with recombinant human interleukin-2 (rhIL-2) and zoledronate; and EX28 media, CON with rhIL-2, zoledronate, and CD3/CD28 activator. The expanded γδ T cells were isolated by magnetic cell separation or fluorescence-activated cell sorting, cultured with two OS cell lines (KHOS/NP and MG-63) at various cell ratios with or without doxorubicin or ifosfamide, and analyzed for cytotoxicity and cytokine secretion. The number of CD3+γδTCR+Vγ9+ triple-positive γδ T cells and concentrations of IFN-γ and TNF-α were highest in the rhIL-2 (100 IU) and zoledronate (1 µM) supplemented culture conditions. The CD3/CD28 agonist did not show any additional effects on γδ T cell expansion. The expanded γδ T cells exhibited potent in vitro cytotoxicity against OS in a ratio- and time-dependent manner. The γδ T cells may enhance the effect of chemotherapeutic agents against OS and may be a new treatment strategy, including chemo-immunotherapy, for OS.

Identification of genomic signatures in bone marrow associated with clinical response of CD19 CAR T-cell therapy.

CD19 CAR T-cell immunotherapy is a breakthrough treatment for B cell malignancies, but relapse and lack of response remain a challenge. The bone marrow microenvironment is a key factor in therapy resistance, however, little research has been reported concerning the relationship between transcriptomic profile of bone marrow prior to lymphodepleting preconditioning and clinical response following CD19 CAR T-cell therapy. Here, we applied comprehensive bioinformatic methods (PCA, GO, GSEA, GSVA, PAM-tools) to identify clinical CD19 CAR T-cell remission-related genomic signatures. In patients achieving a complete response (CR) transcriptomic profiles of bone marrow prior to lymphodepletion showed genes mainly involved in T cell activation. The bone marrow of CR patients also showed a higher activity in early T cell function, chemokine, and interleukin signaling pathways. However, non-responding patients showed higher activity in cell cycle checkpoint pathways. In addition, a 14-gene signature was identified as a remission-marker. Our study indicated the indexes of the bone marrow microenvironment have a close relationship with clinical remission. Enhancing T cell activation pathways (chemokine, interleukin, etc.) in the bone marrow before CAR T-cell infusion may create a pro-inflammatory environment which improves the efficacy of CAR T-cell therapy.