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: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:Peripheral blood NK cells were cultured into either control or cytokine-induced memory-like NK cells as described in PMID: 32614951. After differentiation, cells were labeled with hashtag antibodies and cite-seq antibodies and control and memory-like NK cells were combined and loaded onto the 10x Genomics Chromium Instrument a the McDonnell Genome Institute at Washington University School of Medicine. The following samples were analyzed to develop a classification tool for unbiased identification of memory-like NK cells in a bulk single-cell RNA-seq population.
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: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:We performed transcriptome sequencing on Neo-2/15 stimulated CAR NK cells,to shed light on the function and phenotype changes of CAR-NK cells stimulated by IL-2 and Neo-2/15.
Project description:Cord blood (CB)-derived chimeric antigen receptor (CAR)-natural killer (NK) cells targeting CD19 has been shown to be effective against B cell malignancies. While human CD56+ NK cells can be expanded in vitro, it is also known that NK cells can be differentiated from hematopoietic progenitor cells. It is still unclear whether CAR-NK cells are originated from mature NK cells or NK progenitor cells in CB. Here we found that CAR-NK cells are predominantly derived from the CD56- NK progenitor cells. We first found that substantial numbers of CD19 CAR-NK cells were produced from the CD56- CB mononuclear cells after in vitro culture for two weeks. Single cell RNAseq analysis of CD56-CD3-CD14-CD19- CB mononuclear cells revealed that those cells were subdivided into three subpopulations based on the expression of CD34 and HLA-DR. NK cells were predominantly produced from the CD34-HLA-DR- cells. In addition, in the CD34-HLA-DR- cells, only CD7+ cells could differentiate into NK cells. These results indicate that CD56-CD7+CD34- HLA-DR-lineage marker (Lin)- cells are the major origin of human CB-derived CAR-NK cells, indicating that we need to develop methods to enhance the quality and quantity of NK cells produced from these NK cell progenitor cells.
Project description:Chimeric antigen receptor (CAR)-NK therapy has emerged as a highly promising alternative to CAR-T cell therapy, due to its remarkable efficacy and safety in hematological tumors. However, the efficacy of CAR-NK cells currently remains limited in solid tumors. Here, we successfully developed CISH locus-specific integrated CAR-NK cells using a non-viral mini-circular single-stranded DNA (mcssDNA)-based CRISPR/Cas9 targeted genome editing (mcssDNA/CRISPR/Cas9) technology via a single-step electroporation procedure. The developed CISH-knockout (CISH-KO) CAR-NK cells exhibited the enhanced proliferation and cytotoxicity against hepatocellular carcinoma (HCC) compared with conventional lentivirus-transduced CAR-NK (LV-CAR-NK) cells. Single-cell RNA sequencing analysis revealed an important subset of adaptive NK cells that were highly enriched in CISH-KO CAR-NK cells compared to LV-CAR-NK cells and showed the up-regulated JAK/STAT, energy metabolism and activating receptor signaling. Furthermore, non-viral CISH locus-specific integrated IL-15-armored CAR-NK cells were generated and achieved persistent tumor regression and a significant survival advantage in a lung metastatic mouse model of HCC. Collectively, our results demonstrate that non-viral CISH locus-specific integrated CAR-NK cells can be generated by a simplified, single-step manufacturing procedure and achieve potent efficacy against HCC, thus providing an innovative technology for CAR-NK cell therapy against solid tumors.
