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Analysis of CD70 wild type or CD70 knock out CD70-specific nanoCAR T cells [RNA-Seq]

ABSTRACT: Analysis of CD70 wild type or CD70 knock out CD70-specific nanoCAR T cells [RNA-Seq]

PROVIDER: PRJNA1105004 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR28818774_1.fastq.gz	Fastqsanger.gz
	SRR28818774_2.fastq.gz	Fastqsanger.gz
	SRR28818775_1.fastq.gz	Fastqsanger.gz
	SRR28818775_2.fastq.gz	Fastqsanger.gz
	SRR28818776_1.fastq.gz	Fastqsanger.gz

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Analysis of CD70 wild type or CD70 knock out CD70-specific nanoCAR T cells

Project description:Analysis of CD70 wild type or CD70 knock out CD70-specific nanoCAR T cells

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Analysis of CD70 wild type or CD70 knock out CD70-specific nanoCAR T cells [RNA-Seq]

Project description:CD70 is an attractive target for chimeric antigen receptor (CAR) T-cell therapy for the treatment of both solid and liquid malignancies. However, the functionality of CD70-specific CAR T-cells is modest. We optimized a CD70-specific VHH-based CAR (nanoCAR). We evaluated the nanoCARs in clinically relevant models in vitro, using co-cultures of CD70-specific nanoCAR T-cells with malignant rhabdoid tumor organoids, and in vivo using a diffuse large B-cell lymphoma (DLBCL) patient-derived xenograft (PDX) model. Whereas the nanoCAR T-cells were highly efficient in organoid co-cultures, they showed only modest efficacy in the PDX model. Fratricide was not causing this loss in efficacy but rather CD70 interaction in cis with the nanoCAR, as validated by imaging flowcytometry, induces exhaustion. Knocking out CD70 expression in nanoCAR T-cells using CRISPR/Cas9, resulted in dramatically enhanced functionality in the DLBCL PDX model. Through single-cell transcriptomics, we obtained evidence that CD70 knock out (KO) CD70-specific nanoCAR T-cells were protected from antigen-induced exhaustion. In addition, we were able to demonstrate that WT CD70-specific nanoCAR T-cells already exhibit signs of exhaustion shortly after production. Their gene signature strongly overlapped with gene signatures of exhausted CAR T-cells. On the other hand, the gene signature of KO nanoCAR T-cells overlapped with the gene signature of CAR T-cell infusion products that led to complete responses in chronic lymphatic leukemia patients. Our data show that CARs targeting endogenous T-cell antigens, negatively affect CAR T-cell functionality by inducing an exhausted state, which can be overcome by knocking out the specific target, in this case CD70.

2024-06-17 | GSE265937 | GEO

Analysis of CD70 wild type or CD70 knock out CD70-specific nanoCAR T cells

Project description:CD70 is an attractive target for chimeric antigen receptor (CAR) T cell therapy as treatment for both solid and liquid malignancies. However, functionality of CD70-specific CAR T cells is only modest. Here, we optimized a CD70-specific VHH based CAR (nanoCAR). We evaluated the nanoCARs in clinically relevant models in vitro, using co-cultures of CD70-specific nanoCAR T cells with malignant rhabdoid tumor organoids, and in vivo by using a diffuse large B cell lymphoma (DLBCL) patient-derived xenograft (PDX) model. Whereas the nanoCAR T cells were highly efficient in organoid co-cultures, they showed only modest efficacy in the PDX model. Knocking out CD70 expression in the nanoCAR T cells resulted in dramatically enhanced functionality in the PDX model, suggesting that CD70 interaction in cis with the nanoCAR induces exhaustion. Through single-cell transcriptomics, we obtained evidence that CD70 KO CD70-specific nanoCAR T cells are protected from antigen-induced exhaustion. Our data show that CARs targeted to endogenous T cell antigens, negatively affect CAR T cell functionality by inducing an exhausted state, which can be overcome by knocking out the specific target, in this case CD70.

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Wild type and EGR2 knock-out mouse alveolar macrophages

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Expression data from hippocampus of Phf8 knock out and wild type mice

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3xMBT pull-down from wild-type and Rkm1 knock-out yeast

Project description:We have presented a protocol that utilizes the triple malignant brain tumor domains of L3MBTL1 (3xMBT) to enrich proteins modified with mono- and di-methylated lysine from cell lysate. Cells in culture are grown with amino acids containing light or heavy stable isotopic labels. Methylated proteins are enriched by incubating cell lysates with 3xMBT, or with the binding-null D355N mutant as a negative control. Quantitative liquid chromatography and tandem mass spectrometry (LC-MS/MS) is then used to identify proteins that are specifically enriched by 3xMBT pull-down. Addition of a third isotopic label allows comparison of protein methylation between biological conditions. As an example yeast cells were prepared in SILAC media under three conditions. In two independent experiments either light or heavy cells are Rkm1 knock-out, while the other two are wild-type. 3xMBT was used to enrich methylated proteins from the light/heavy cells, with 3xMBT_D355N as a negative control using the medium cell lysate. Proteins were separated into three fractions by SDS-PAGE and analyzed by LC-MS/MS using an Orbitrap Velos. Data was analyzed with MaxQuant version 1.3.0.5 using default parameters except that mono- and di-methylation of lysine were included as variable modifications, the modification-specific false-discovery was set to 10%, and minimum peptide count for quantitative data set to 1. The amount of ribosomal protein Rpl23, a known substrate of Rkm1, is greatly decreased in 3xMBT pull-down from Rkm1 knock-out cells relative to wild-type. The level of heat shock protein SSA4 is also greatly reduced, suggesting that it may also be methylated by Rkm1.

2013-11-11 | PXD000360 | Pride

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