Project description:The purpose of this experiment was two-fold. The first was to examine how the gene expression profile changes over time in C7R-CAR NK cells. C7R is a constitutively active IL-7 receptor that provides persistent activation of STAT5. The second was to examine the differential gene expression in C7R CAR NK cells or CAR NK treated with exogenous IL-15 (exIL15) after 2 weeks of stimulation.
Project description:The purpose of this experiment to examine how the gene expression profile and differential gene expression in NK cells, TIGITKO NK cells, TIGITKOGD2.CAR NK cells or GD2.CAR NK cells changes after 72 hours of neuroblastoma tumor exposure.
Project description:Human Immune Gene Expression Profile in activated and expanded NK cells, TIGITKO NK cells, GD2.CAR NK cells and TIGITKOGD2.CAR NK cells
Project description:Chimeric antigen receptor (CAR)-natural killer (NK) cell therapies hold promise for solid tumors but remain limited by poor tumor infiltration, persistence, and resistance within the tumor microenvironment (TME). To identify gain-of-function (GOF) targets that enhance CAR-NK efficacy, we performed an unbiased in vivo Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) activation (CRISPRa) screen, followed by a barcoded targeted in vivo open reading frame (ORF) screen in primary human CAR-NK cells. We identified, and robustly validated OR7A10, a G protein-coupled receptor (GPCR), as the top candidate. Engineering CAR-NKs with OR7A10 cDNA, a CRISPR-independent method with simple manufacturing strategy, enhanced proliferation, activation, degranulation, cytokine production, death ligand expression, chemokine receptor expression, cytotoxicity, persistence, metabolic fitness, and TME resistance, while reducing exhaustion in primary human NK cells derived from multiple peripheral blood and cord blood donors. OR7A10-GOF CAR-NKs displayed robust in vivo efficacy across multiple solid tumor models, achieving a 100% complete response in an orthotopic breast cancer model with long term tumor control and survival benefit. These findings establish OR7A10-engineered CAR-NKs as a highly potent and scalable off-the-shelf therapeutic for solid tumors.
Project description:Chimeric antigen receptor (CAR) engineering of NK cells is an active area of research with early-phase clinical studies showing an excellent safety profile with encouraging clinical responses. However, the transcriptional signatures that control the fate of CAR-NK cell after infusion and their association with tumor control remain poorly understood. Here, we performed single-cell RNA sequencing (scRNA-seq) to depict the evolution of various engineered CAR-NK cells from the ex vivo infusion products to the in vivo peak phase of tumor control and finally to the relapse phase. Single cell RNA sequencing (scRNA) has revolutionized high-thoughout systems-based analysis of cellular and functional heterogeneity, and dynamic changes in the immune response during the anti-tumor immune cell therapy . The goals of this work are to compare transcriptome profiling (RNA-seq) from both engrafted tumor cells and infused CAR-NK cells over time of treatment course to evaluate the kenetic of tumor cell response and effector functional change of CAR-NK cell. Our study represents the first detailed transcriptomic analysis of using CAR-NK cell therapy aganist Raji-engrafted mouse model. Collecting samples from different time points and organs, the data analysis reported here should privide an envision of the dynamic about how tumor response to immune cell therapy of using CAR-NK cells and also how immune effector fucntion of CAR-NK cell was modulated over time during the treatment courses.
Project description:Primary chimeric antigen receptor (CAR) natural killer (NK) cells show strong cytotoxic efficacy against acute myeloid leukemia (AML) in vivo. However, NK cell-mediated tumor killing is often impaired by tumor-mediated immune cell inactivation. Here, we report a novel strategy to overcome NK cell inhibition caused by the immune checkpoint NKG2A, which interacts with HLA-E expressed on AML blasts. We generated AML-specific CD33-directed CAR (CAR33)-KLRC1ko-NK cells with CRISPR/Cas9-based gene editing of the NKG2A-encoding KLRC1 gene. Single-cell multi-omic analyses revealed a higher proportion of activated cells in CAR33-NK- and CAR33-KLRC1ko-NK pools, which were preserved following AML-cell contact. This activated state of the CAR33-KLRC1ko-NK cells has been translated into improved antileukemic activity in vitro and in vivo against AML cell lines and primary blasts. This dual modification of primary NK cells has the potential to bypass the suppressive effect not only of AML but also in a broad range of other cancer identities.
Project description:Chimeric antigen receptor (CAR)-natural killer (NK) cell therapies hold promise for solid tumors but remain limited by poor tumor infiltration, persistence, and resistance within the tumor microenvironment (TME). To identify gain-of-function (GOF) targets that enhance CAR-NK efficacy, we performed an unbiased in vivo Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) activation (CRISPRa) screen, followed by a barcoded targeted in vivo open reading frame (ORF) screen in primary human CAR-NK cells. We identified, and robustly validated OR7A10, a G protein-coupled receptor (GPCR), as the top candidate. Engineering CAR-NKs with OR7A10 cDNA, a CRISPR-independent method with simple manufacturing strategy, enhanced proliferation, activation, degranulation, cytokine production, death ligand expression, chemokine receptor expression, cytotoxicity, persistence, metabolic fitness, and TME resistance, while reducing exhaustion in primary human NK cells derived from multiple peripheral blood and cord blood donors. OR7A10-GOF CAR-NKs displayed robust in vivo efficacy across multiple solid tumor models, achieving a 100% complete response in an orthotopic breast cancer model with long term tumor control and survival benefit. These findings establish OR7A10-engineered CAR-NKs as a highly potent and scalable off-the-shelf therapeutic for solid tumors.
Project description:Chimeric antigen receptor (CAR)-natural killer (NK) cell therapies hold promise for solid tumors but remain limited by poor tumor infiltration, persistence, and resistance within the tumor microenvironment (TME). To identify gain-of-function (GOF) targets that enhance CAR-NK efficacy, we performed an unbiased in vivo Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) activation (CRISPRa) screen, followed by a barcoded targeted in vivo open reading frame (ORF) screen in primary human CAR-NK cells. We identified, and robustly validated OR7A10, a G protein-coupled receptor (GPCR), as the top candidate. Engineering CAR-NKs with OR7A10 cDNA, a CRISPR-independent method with simple manufacturing strategy, enhanced proliferation, activation, degranulation, cytokine production, death ligand expression, chemokine receptor expression, cytotoxicity, persistence, metabolic fitness, and TME resistance, while reducing exhaustion in primary human NK cells derived from multiple peripheral blood and cord blood donors. OR7A10-GOF CAR-NKs displayed robust in vivo efficacy across multiple solid tumor models, achieving a 100% complete response in an orthotopic breast cancer model with long term tumor control and survival benefit. These findings establish OR7A10-engineered CAR-NKs as a highly potent and scalable off-the-shelf therapeutic for solid tumors.
Project description:Chimeric antigen receptor (CAR)-natural killer (NK) cell therapies hold promise for solid tumors but remain limited by poor tumor infiltration, persistence, and resistance within the tumor microenvironment (TME). To identify gain-of-function (GOF) targets that enhance CAR-NK efficacy, we performed an unbiased in vivo Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) activation (CRISPRa) screen, followed by a barcoded targeted in vivo open reading frame (ORF) screen in primary human CAR-NK cells. We identified, and robustly validated OR7A10, a G protein-coupled receptor (GPCR), as the top candidate. Engineering CAR-NKs with OR7A10 cDNA, a CRISPR-independent method with simple manufacturing strategy, enhanced proliferation, activation, degranulation, cytokine production, death ligand expression, chemokine receptor expression, cytotoxicity, persistence, metabolic fitness, and TME resistance, while reducing exhaustion in primary human NK cells derived from multiple peripheral blood and cord blood donors. OR7A10-GOF CAR-NKs displayed robust in vivo efficacy across multiple solid tumor models, achieving a 100% complete response in an orthotopic breast cancer model with long term tumor control and survival benefit. These findings establish OR7A10-engineered CAR-NKs as a highly potent and scalable off-the-shelf therapeutic for solid tumors.
Project description:Chimeric antigen receptor (CAR) T cell therapy is a promising immunotherapy against cancer. Although there is a growing interest in other cell types, a comparison of CAR immune effector cells in challenging solid tumor contexts is lacking. Here, we compare mouse and human NKG2D-CAR expressing T cells, NK cells and macrophages against glioblastoma, the most aggressive primary brain tumor. In vitro we show that T cell cancer killing is CAR-dependent, whereas intrinsic cytotoxicity overrules CAR-dependence for NK cells and CAR macrophages reduce glioma cells in co-culture assays. In orthotopic immunocompetent glioma mouse models, systemically administered CAR T cells demonstrate superior accumulation in the tumor and each immune cell type induces distinct changes in the tumor microenvironment. An otherwise low therapeutic efficacy is significantly enhanced by co-expression of pro-inflammatory cytokines in all CAR immune effector cells, underscoring the necessity for multifaceted cell engineering strategies to overcome the immunosuppressive solid tumor microenvironment.