Single cell RNA sequencing to study the in vivo behaviour of allogeneic HSC-derived CAR-engineered NKT cells
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ABSTRACT: CAR-T cell therapy has achieved remarkable clinical outcomes, yet the autologous nature of FDA-approved CAR-T products present significant challenges in manufacturing, cost, and patient selection. Therefore, there is a growing demand for off-the-shelf cell therapy. Here we introduce an ex vivo feeder-free culture to differentiate gene-engineered HSCs into allogeneic NKT cells, as well as their CAR-armed and IL-15-enhanced derivatives (Allo15CAR-NKT cells). In order to study the epigenetic regulation of the generated cells, we performed DNA methylation sequencing on both IL-15-enhanced and non-IL-15-engineered AlloCAR-NKT cells. Conventional CAR-T cells were included as a control.
Project description:CAR-T cell therapy has achieved remarkable clinical outcomes, yet the autologous nature of FDA-approved CAR-T products present significant challenges in manufacturing, cost, and patient selection. Therefore, there is a growing demand for off-the-shelf cell therapy. Here we introduce an ex vivo feeder-free culture to differentiate gene-engineered HSCs into allogeneic NKT cells, as well as their CAR-armed and IL-15-enhanced derivatives (Allo15CAR-NKT cells). In order to study the epigenetic regulation of the generated cells, we performed DNA methylation sequencing on both IL-15-enhanced and non-IL-15-engineered AlloCAR-NKT cells. Conventional CAR-T cells were included as a control.
Project description:CAR-T cell therapy has achieved remarkable clinical outcomes, yet the autologous nature of FDA-approved CAR-T products present significant challenges in manufacturing, cost, and patient selection. Therefore, there is a growing demand for off-the-shelf cell therapy. Here we introduce an ex vivo feeder-free culture to differentiate gene-engineered HSCs into allogeneic NKT cells, as well as their CAR-armed and IL-15-enhanced derivatives (Allo15CAR-NKT cells). In order to study the epigenetic regulation of the generated cells, we performed DNA methylation sequencing on both IL-15-enhanced and non-IL-15-engineered AlloCAR-NKT cells. Conventional CAR-T cells were included as a control.
Project description:CAR-T cell therapy has achieved remarkable clinical outcomes, yet the autologous nature of FDA-approved CAR-T products present significant challenges in manufacturing, cost, and patient selection. Therefore, there is a growing demand for off-the-shelf cell therapy. Here we introduce an ex vivo feeder-free culture to differentiate gene-engineered HSCs into allogeneic NKT cells, as well as their CAR-armed and IL-15-enhanced derivatives (Allo15CAR-NKT cells). In order to study the epigenetic regulation of the generated cells, we performed DNA methylation sequencing on both IL-15-enhanced and non-IL-15-engineered AlloCAR-NKT cells. Conventional CAR-T cells were included as a control.
Project description:CAR-T cell therapy has achieved remarkable clinical outcomes, yet the autologous nature of FDA-approved CAR-T products present significant challenges in manufacturing, cost, and patient selection. Therefore, there is a growing demand for off-the-shelf cell therapy. Here we introduce an ex vivo feeder-free culture to differentiate gene-engineered HSCs into allogeneic NKT cells, as well as their CAR-armed and IL-15-enhanced derivatives (Allo15CAR-NKT cells). In order to study the epigenetic regulation of the generated cells, we performed DNA methylation sequencing on both IL-15-enhanced and non-IL-15-engineered AlloCAR-NKT cells. Conventional CAR-T cells were included as a control.
Project description:CAR-T cell therapy has achieved remarkable clinical outcomes, yet the autologous nature of FDA-approved CAR-T products present significant challenges in manufacturing, cost, and patient selection. Therefore, there is a growing demand for off-the-shelf cell therapy. Here we introduce an ex vivo feeder-free culture to differentiate gene-engineered HSCs into allogeneic NKT cells, as well as their CAR-armed and IL-15-enhanced derivatives (Allo15CAR-NKT cells). In order to study the epigenetic regulation of the generated cells, we performed DNA methylation sequencing on both IL-15-enhanced and non-IL-15-engineered AlloCAR-NKT cells. Conventional CAR-T cells were included as a control.
Project description:Current CAR-T cell therapy's clinical potential is hampered by its autologous nature that poses significant challenges in manufacturing, costs, and patient selection. This spurs demand for off-the-shelf therapies. Here we introduce an ex vivo feeder-free culture to differentiate gene-engineered HSCs into allogeneic NKT cells and their CAR-armed derivatives (AlloCAR-NKT cells). The AlloCAR-NKT cells are generated at high yield, purity, and robustness. These cells exhibit potent antitumor efficacy, showing effective tumor homing, clonal expansion, and persistence In vivo. Impressively, AlloCAR-NKT cells can alter the tumor microenvironment by selectively depleting immunosuppressive TAMs and MDSCs, and can antagonize tumor immune evasion by deploying CAR/TCR/NKR triple-targeting mechanisms. AlloCAR-NKT cells also demonstrate an appealing safety profile with low GvHD and CRS risks, and exhibit a stable “hypoimmunogenic” phenotype attributed to epigenetic and signaling regulations. The reported technology presents a scalable strategy for diverse allogeneic CAR-NKT cell products, with potential for clinical translation and commercialization against various cancers.
Project description:Chimeric antigen receptor (CAR)-engineered T cell therapy holds promise for targeting myeloid malignancies including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, autologous approaches pose significant challenges in manufacturing and patient selection, emphasizing the need for off-the-shelf cell products. In this study, we systematically characterized primary AML and MDS patient samples, identifying a unique therapeutic opportunity for CAR-engineered invariant natural killer T (CAR-NKT) cell therapy. Utilizing a clinically guided culture method, we generated allogeneic IL-15-enhanced CD33-targeting CAR-NKT (Allo15CAR33-NKT) cells through HSPC engineering and an ex vivo, feeder-free HSPC differentiation culture. These cells demonstrated potent antitumor efficacy against blast cells via multiple tumor-targeting mechanisms. In vivo, Allo15CAR33-NKT cells exhibited effective tumor homing, expansion, and persistence, characterized by high effector function and low exhaustion propensity. Furthermore, these cells synergized with hypomethylating agent (HMA) treatment, which upregulated CD1d and NK ligand expression on tumor cells, enhancing susceptibility to Allo15CAR33-NKT cell-mediated killing. Notably, Allo15CAR33-NKT cells displayed minimal off-tumor effects against hematopoietic precursors, and low risks of graft-versus-host disease and cytokine release syndrome, highlighting their substantial therapeutic potential for myeloid malignancies.
Project description:Semi-invariant natural killer T (NKT) cells are thymus-derived innate lymphocytes that modulate microbial and tumour immunity as well as autoimmune diseases. These immunoregulatory properties of NKT cells are acquired during their development. Much has been learnt regarding the molecular and cellular cues that promote NKT cell development, yet how these cells are maintained in the thymus and the periphery and how they acquire functional competence are incompletely understood. We found that IL-15 induced several Bcl-2 family survival factors in thymic and splenic NKT cells in vitro. Yet, IL15-mediated thymic and peripheral NKT cell survival critically depended on Bcl-xL expression. Additionally, IL-15 regulated thymic developmental stage 2 (ST2) to ST3 lineage progression and terminal NKT cell differentiation. Global gene expression analyses and validation revealed that IL-15 regulated Tbx21 (T-bet) expression in thymic ST3 NKT cells. The loss of IL15-dependent T-bet expression resulted in poor expression of IFN-γ and several NK cell receptors in NKT cells. Taken together, our findings reveal a critical role for IL-15 in NKT cell survival, which is mediated by Bcl-xL, and effector differentiation, which is regulated by T-bet. Gene expression was measured in NKT cells sorted from pooled thymi of wild-type (3 replicates) or IL-15 deficient (2 replicates) mice.
Project description:Vα24-invariant natural killer T cells (NKTs) have antitumor properties that can be enhanced by transgenic expression of tumor-specific receptors. Here, we report the results of the first-in-human clinical evaluation of autologous NKTs co-expressing a GD2-specific chimeric antigen receptor with interleukin (IL)15 (GD2-CAR.15) in 12 children with neuroblastoma (NB) treated on four dose levels (NCT03294954). Objectives included assessing safety, antitumor activity, and immune response. No dose-limiting toxicities occurred, and one patient had grade 2 cytokine release syndrome resolved by tocilizumab. The overall response rate was 25% (3/12) and disease control rate was 58% (7/12) including four patients with stable disease, two partial responses, and one complete response. CD62L+ NKT frequency in infused products correlated with CAR-NKT expansion in patients and was higher in responders than non-responders (71% vs 35.3%, p=0.002). Singe-cell RNA sequencing analysis identified B cell translocation gene 1 (BTG1) as one of the top upregulated genes in GD2-CAR.15-NKTs after in vitro serial tumor challenge. Genetic gain- and loss-of-function experiments revealed that BTG1 is a key driver of hyporesponsiveness in exhausted NKT and T cells. Crucially, NKTs co-expressing GD2-CAR.15 and BTG1-specific shRNA eradicated metastatic NB in mice. These results indicate that CAR-NKTs are safe, produce objective responses in NB patients, and that targeting BTG1 can enhance their therapeutic potency.
Project description:Vα24-invariant natural killer T cells (NKTs) have antitumor properties that can be enhanced by transgenic expression of tumor-specific receptors. Here, we report the results of the first-in-human clinical evaluation of autologous NKTs co-expressing a GD2-specific chimeric antigen receptor with interleukin (IL)15 (GD2-CAR.15) in 12 children with neuroblastoma (NB) treated on four dose levels (NCT03294954). Objectives included assessing safety, antitumor activity, and immune response. No dose-limiting toxicities occurred, and one patient had grade 2 cytokine release syndrome resolved by tocilizumab. The overall response rate was 25% (3/12) and disease control rate was 58% (7/12) including four patients with stable disease, two partial responses, and one complete response. CD62L+ NKT frequency in infused products correlated with CAR-NKT expansion in patients and was higher in responders than non-responders (71% vs 35.3%, p=0.002). Singe-cell RNA sequencing analysis identified B cell translocation gene 1 (BTG1) as one of the top upregulated genes in GD2-CAR.15-NKTs after in vitro serial tumor challenge. Genetic gain- and loss-of-function experiments revealed that BTG1 is a key driver of hyporesponsiveness in exhausted NKT and T cells. Crucially, NKTs co-expressing GD2-CAR.15 and BTG1-specific shRNA eradicated metastatic NB in mice. These results indicate that CAR-NKTs are safe, produce objective responses in NB patients, and that targeting BTG1 can enhance their therapeutic potency.