Biology subjects

Sadelain, M.

Publications and source records attributed to Sadelain, M..

7 recordsLinked to original sources

bioRxiv2026

Development of a Fully Non-Viral 1XX-enhanced BCMA CAR-T Cell Therapy for Multiple Myeloma

Multiple myeloma (MM) is a clonal plasma cell malignancy characterized by bone marrow infiltration, monoclonal immunoglobulin production, and microenvironmental dysregulation that leads to systemic organ damage. The advent of B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T-cell therapy has induced unprecedented responses and durability for patients with relapsed/refractory MM. These outcomes are rarely observed with prior salvage strategies, although relapse remains the predominant long-term challenge for most patients. The two currently approved BCMA CAR-T cell products use viral vectors to semi-randomly insert the CAR gene, which results in heterogeneous genomic composition and variability in efficacy, safety, and product consistency. To address these challenges, we integrated targeted CRISPR genome engineering with precise CAR transgene insertion at the T-cell receptor alpha constant (TRAC) locus, 1XX CAR signaling architecture to enhance potency and durability, and non-viral manufacturing with a single-stranded DNA repair template to improve efficiency and yield. This approach confers physiological CAR expression, reduces insertional mutagenesis, and improves persistence by mitigating tonic signaling and exhaustion. Our GMP manufacturing process consistently achieved high CAR integration (37.7-72.7%) and yields across all full-scale runs and met predefined release criteria for identity, purity, safety, and quality. In NSG mouse models of MM, the UCCT-BCMA-1 product exhibited exceptionally potent tumor control, CAR-T cell expansion 100-1000-fold greater than that of lentiviral constructs, and durable clearance of myeloma cells after multiple rechallenges. These findings establish a CRISPR-edited, fully non-viral manufacturing platform for next-generation 1XX-BCMA CAR-T therapies with enhanced persistence, safety, and efficacy. One Sentence SummaryCRISPR-engineered, TRAC-targeted 1XX-BCMA CAR-T therapy with improved safety, potency, and persistence in relapsed and refractory multiple myeloma.

cancer biology↗

bioRxiv2024

Dendritic cells accelerate CAR T cells in irradiated tumors through chimeric synapses

The persistence of adoptively transferred T cells is vital for anti-tumor efficacy. Chimeric antigen receptor (CAR) T cells can persist indefinitely when delivered to patients with B cell cancers and can confer long-term remission. For patients with solid tumors, however, sustaining CAR T cell activity remains a major challenge. This has been attributed in part to the immune microenvironment within solid tumors, though the contribution of specific immune subsets to resistance to CAR T cells is not clear. Here we resolve how the immunology of irradiated tumors dramatically enhances persistence and efficacy of CAR T cells targeted to advanced lung metastases in a syngeneic mouse model. Remarkably, CAR T cell persistence depended critically on dendritic cells (DC) that underwent trogocytic "antigen-dressing" of tumor target antigens and stimulated CAR T cells through the chimeric receptor. Furthermore, tumor irradiation increased antigen-dressing onto DCs. In the absence of functional DCs, CAR T cell activity in irradiated tumor was short-lived and tumors relapsed. These findings establish a critical mechanism through which DCs maintain the CAR T cell pool in irradiated tumors, thus supporting translation of this approach to advance CAR T cell therapy for solid tumors.

immunology↗

bioRxiv2024

Senolytic CAR T cells reverse aging-associated defects in intestinal regeneration and fitness

Intestinal stem cells (ISCs) drive the rapid regeneration of the gut epithelium to maintain organismal homeostasis. Aging, however, significantly reduces intestinal regenerative capacity. While cellular senescence is a key feature of the aging process, little is known about the in vivo effects of senescent cells on intestinal fitness. Here, we identify the accumulation of senescent cells in the aging gut and, by harnessing senolytic CAR T cells to eliminate them, we uncover their detrimental impact on epithelial integrity and overall intestinal homeostasis in natural aging, injury and colitis. Ablation of intestinal senescent cells with senolytic CAR T cells in vivo or in vitro is sufficient to promote the regenerative potential of aged ISCs. This intervention improves epithelial integrity and mucosal immune function. Overall, these results highlight the ability of senolytic CAR T cells to rejuvenate the intestinal niche and demonstrate the potential of targeted cell therapies to promote tissue regeneration in aging organisms.

cell biology↗

bioRxiv2024

CAR-engineered lymphocyte persistence is governed by a FAS ligand/FAS auto-regulatory circuit

Chimeric antigen receptor (CAR)-engineered T and NK cells can cause durable remission of B-cell malignancies; however, limited persistence restrains the full potential of these therapies in many patients. The FAS ligand (FAS-L)/FAS pathway governs naturally-occurring lymphocyte homeostasis, yet knowledge of which cells express FAS-L in patients and whether these sources compromise CAR persistence remains incomplete. Here, we constructed a single-cell atlas of diverse cancer types to identify cellular subsets expressing FASLG, the gene encoding FAS-L. We discovered that FASLG is limited primarily to endogenous T cells, NK cells, and CAR-T cells while tumor and stromal cells express minimal FASLG. To establish whether CAR-T/NK cell survival is regulated through FAS-L, we performed competitive fitness assays using lymphocytes modified with or without a FAS dominant negative receptor ({Delta}FAS). Following adoptive transfer, {Delta}FAS-expressing CAR-T and CAR-NK cells became enriched across multiple tissues, a phenomenon that mechanistically was reverted through FASLG knockout. By contrast, FASLG was dispensable for CAR-mediated tumor killing. In multiple models, {Delta}FAS co-expression by CAR-T and CAR-NK enhanced antitumor efficacy compared with CAR cells alone. Together, these findings reveal that CAR-engineered lymphocyte persistence is governed by a FAS-L/FAS auto-regulatory circuit.

immunology↗

bioRxiv2023

Targeting HLA-E Positive Cancers with a Novel NKG2A/C Switch Receptor

HLA-E is overexpressed by approximately 80% of solid tumors, including malignant glioblastoma, and is emerging as a major checkpoint for NKG2A+ CD8+ T cells and NK cells in the tumor microenvironment and circulation. This axis operates side-by-side with PD-L1 to shut down effector responses by T and NK cells. Here, we engineered a novel chimeric A/C switch receptor, combining the strong HLA-E binding affinity of the NKG2A receptor ectodomain with the activating signaling of the NKG2C receptor endodomain. We found that A/C Switch-transduced NK and T cells displayed superior and specific cytotoxic function when challenged with tumor cells exhibiting medium to high HLA-E expression. Furthermore, A/C Switch-expressing human T cells demonstrated enhanced anti-tumor function in a xenograft model of glioblastoma. Importantly, the activity of the modified T cells was governed by an equilibrium between A/C Switch transduction level and HLA-E expression, creating a therapeutic window to safeguard against on-target off-tumor toxicities. Indeed, normal cells remained insensitive to A/C Switch engineered T cells even after pre-treatment with IFN-{gamma} to induce HLA-E expression. We propose that this novel A/C switch receptor may operate alone to control tumor cells expressing high levels of HLA-E or in combination with other engineered specificities to overcome the suppressive NKG2A/HLA-E checkpoint.

immunology↗

bioRxiv2023

Genetic ablation of adhesion ligands averts rejection of allogeneic immune cells

Allogeneic cell therapies hold promise for broad clinical implementation, but face limitations due to potential rejection by the recipient immune system. Silencing of beta-2-microglobulin (B2M) expression is commonly employed to evade T cell-mediated rejection, although absence of B2M triggers missing-self responses by recipient natural killer (NK) cells. Here, we demonstrate that deletion of the adhesion ligands CD54 and CD58 on targets cells robustly dampens NK cell reactivity across all sub-populations. Genetic deletion of CD54 and CD58 in B2M-deficient allogeneic chimeric antigen receptor (CAR) T and multi-edited induced pluripotent stem cell (iPSC)-derived NK cells reduces their susceptibility to rejection by NK cells in vitro and in vivo without affecting their anti-tumor effector potential. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection of allogeneic immune cells for immunotherapy.

immunology↗

bioRxiv2022

Balancing activation and costimulation of CAR tunes signaling dynamics and enhances therapeutic potency

BackgroundPrimary human T cells engineered with chimeric antigen receptors (CARs) ex vivo can be adoptively transferred to treat cancer. CD19-targeting CAR with CD28 costimulatory domain and CD3{zeta} activation domain have been approved by the US FDA for treating B cell malignancies. MethodsHere we generated mutation of immunorecpetor tyrosine-based activation motifs (ITAMs) in CD3{zeta}, namely 1XX CAR, which altered the balance of activation and costimulation. Next we investigated whether 1XX design could enhance therapeutic potency against solid tumors. We constructed both CD19- and AXL-specific 1XX CARs and compared their in vitro and in vivo functions with their WT counterparts. ResultsEven though 1XX CARs decreased cytotoxicity against tumor cells in vitro, they showed better anti-tumor efficacy in both pancreatic and melanoma mouse models. Detailed analysis revealed that 1XX CAR-T cells proliferated more in response to antigen stimulation in vitro, persisted longer in vivo and had higher percentage of central memory cells. As 1XX modification directly calibrates CAR activation potential, we utilized fluorescence resonance energy transfer (FRET)-based biosensor to monitor signaling dynamics downstream of CARs. Decreased ITAM numbers in 1XX resulted in similar ZAP70 activation, while 1XX induced higher Ca2+ elevation and faster Erk activation than WT CAR, which may contribute to the better therapeutic potency of 1XX. ConclusionsOur results established the surpiosity of 1XX against two targets in different solid tumor models and shed light on the underlying molecular mechanism of CAR signaling, paving the way for the clinical application of 1XX CARs against solid tumors.

immunology↗