A GMP-Compliant Procedure for the Generation of Gene-Modified T cells.

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies. Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

Hypomethylation-induced regulatory programs in T cells unveiled by transcriptomic analyses.

Regulatory T cells (Tregs) are essential mediators of tolerance mitigating aberrant immune responses. While naturally occurring Treg (nTreg) development and function are directed by epigenetic events, induced Treg (iTreg) identity and mechanisms of action remain elusive. Mirroring the epigenetic circuits of nTregs, we and others have used hypomethylation agents (HAs) to ex vivo convert T cells into iTregs (HA-iTregs) and further showed that the suppressive properties of the HA-iTregs are predominantly confined in an emergent population, which de novo expresses the immunomodulatory molecule HLA-G, consequently providing a surface marker for isolation of the suppressive HA-iTreg compartment (G+ cells). We isolated the HA-induced G+ cells and their G- counterparts and employed high-throughput RNA-sequencing (RNA-seq) analyses to uncover the G+-specific transcriptomic changes guiding T cells toward a regulatory trajectory upon their exposure to HA. We found a distinct transcriptional upregulation of G+ cells accompanied by enrichment of immune-response-related pathways. Although single-cell RNA-seq profiling revealed regulatory G+ cells to have molecular features akin to nTregs, when assessed in conjunction with the comparative transcriptomic analysis and profiling of secreted cytokines against the non-suppressive G- cells, FOXP3 and other T-helper signatures appear to play a minor role in their suppressive phenotype. We found an ectopic expression of IDO-1 and CCL17/22 in G+ cells, denoting that in vitro exposure of T cells to HA may well unlock myeloid suppressor genes. This report provides transcriptional data shaping the molecular identity of a highly purified and potent HA-iTreg population and hints toward ectopic myeloid-specific molecular mechanisms mediating HA-iTreg function.

Study protocol: Phase I/II trial of induced HLA-G+ regulatory T cells in patients undergoing allogeneic hematopoietic cell transplantation from an HLA-matched sibling donor.

Regulatory T-cell (Treg) immunotherapy has emerged as a promising and highly effective strategy to combat graft-versus-host disease (GvHD) after allogeneic hematopoietic cell transplantation (allo-HCT). Both naturally occurring Treg and induced Treg populations have been successfully evaluated in trials illustrating the feasibility, safety, and efficacy required for clinical translation. Using a non-mobilized leukapheresis, we have developed a good manufacturing practice (GMP)-compatible induced Treg product, termed iG-Tregs, that is enriched in cells expressing the potent immunosuppressive human leucocyte antigen-G molecule (HLA-G+). To assess the safety and the maximum tolerable dose (MTD) of iG-Tregs, we conduct a phase I-II, two-center, interventional, dose escalation (3 + 3 design), open-label study in adult patients undergoing allo-HCT from an HLA-matched sibling donor, which serves also as the donor for iG-Treg manufacturing. Herein, we present the clinical protocol with a detailed description of the study rationale and design as well as thoroughly explain every step from patient screening, product manufacturing, infusion, and participant follow-up to data collection, management, and analysis (registered EUDRACT-2021-006367-26).

Combining a CAR and a chimeric costimulatory receptor enhances T cell sensitivity to low antigen density and promotes persistence.

Despite the high remission rates achieved using T cells bearing a chimeric antigen receptor (CAR) against hematogical malignancies, there is still a considerable proportion of patients who eventually experience tumor relapse. Clinical studies have established that mechanisms of treatment failure include the down-regulation of target antigen expression and the limited persistence of effective CAR T cells. We hypothesized that dual targeting mediated by a CAR and a chimeric costimulatory receptor (CCR) could simultaneously enhance T cell cytotoxicity and improve durability. Concomitant high-affinity engagement of a CD38-binding CCR enhanced the cytotoxicity of BCMA-CAR and CD19-CAR T cells by increasing their functional binding avidity. In comparison to second-generation BCMA-CAR or CD19-CAR T cells, double-targeted CAR + CD38-CCR T cells exhibited increased sensitivity to recognize and lyse tumor variants of multiple myeloma and acute lymphoblastic leukemia with low antigen density in vitro. In addition, complimentary costimulation by 4-1BB and CD28 endodomains provided by the CAR and CCR combination conferred increased cytokine secretion and expansion and improved persistence in vivo. The cumulatively improved properties of CAR + CCR T cells enabled the in vivo eradication of antigen-low tumor clones, which were otherwise resistant to treatment with conventional CAR T cells. Therefore, multiplexing targeting and costimulation through the combination of a CAR and a CCR is a powerful strategy to improve the clinical outcomes of CAR T cells by enhancing cytotoxic efficacy and persistence, thus preventing relapses of tumor clones with low target antigen density.