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Monitoring CAR T cell generation with a CD8-targeted lentiviral vector by single-cell transcriptomics.

ABSTRACT: Quantifying gene expression in individual cells can substantially improve our understanding about complex genetically engineered cell products such as chimeric antigen receptor (CAR) T cells. Here we designed a single-cell RNA sequencing (scRNA-seq) approach to monitor the delivery of a CD19-CAR gene via lentiviral vectors (LVs), i.e., the conventional vesicular stomatitis virus (VSV)-LV and the CD8-targeted CD8-LV. LV-exposed human donor peripheral blood mononuclear cells (PBMCs) were evaluated for a panel of 400 immune response-related genes including LV-specific probes. The resulting data revealed a trimodal expression for the CAR and CD8A, demanding a careful distribution-based identification of CAR T cells and CD8+ lymphocytes in scRNA-seq analysis. The fraction of T cells expressing high CAR levels was in concordance with flow cytometry results. More than 97% of the cells hit by CD8-LV expressed the CD8A gene. Remarkably, the majority of the potential off-target cells were in fact on-target cells, resulting in a target cell selectivity of more than 99%. Beyond that, differential gene expression analysis revealed the upregulation of restriction factors in CAR-negative cells, thus explaining their protection from CAR gene transfer. In summary, we provide a workflow and subsetting approach for scRNA-seq enabling reliable distinction between transduced and untransduced cells during CAR T cell generation.

SUBMITTER: Charitidis FT

PROVIDER: S-EPMC8546366 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Clinical success with chimeric antigen receptor (CAR)- based immunotherapy for leukemia has been accompanied by the associated finding that antigen-escape variants of the disease are responsible for relapse. To target hematologic malignancies with a chimeric antigen receptor (CAR) that targets two antigens with a single vector, and thus potentially lessen the chance of leukemic escape mutations, a tandem-CAR approach was investigated.Antigen binding domains from the FMC63 (anti-CD19) and Leu16 (anti-CD20) antibodies were linked in differing configurations to transmembrane and T cell signaling domains to create tandem-CARs. Expression on the surface of primary human T cells was induced by transduction with a single lentiviral vector (LV) encoding the tandem-CAR. Tandem-CARs were compared to single antigen targeting CARs in vitro and in vivo, and to an admixture of transduced cells expressing each CAR in vivo in immunodeficient (NSG) disease-bearing mice.Tandem constructs efficient killed the Raji leukemia cell line both in vitro and in vivo. Tandem CARs generated less cytokine than the CD20 CAR, but similar to CD19 CARs, on their own. In co-culture experiments at low effector to target ratios with both single- and tandem- CAR-T cells, a rapid down-modulation of full-length CD19 expression was seen on leukemia targets. There also was a partial down-modulation of CD22, and to a lesser degree, of CD20. Our data also highlight the extreme sensitivity of the NALM-6 cell line to general lymphocyte-mediated cytotoxicity. While single and tandem constructs were effective in vivo in a standard setting, in a high-disease burden setting, the tandem CAR proved both effective and less toxic than an admixture of transduced T cell populations expressing single CARs.Tandem CARs are equally effective in standard disease models to single antigen specificity CARs, and may be both more effective and less toxic in a higher disease burden setting. This may be due to optimized cell killing with more moderate cytokine production. The rapid co-modulation of CD19, CD20, and CD22 may account for the ability to rapidly evolve escape mutants by selecting for leukemic clones that not require these target antigens for continued expansion.

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Monitoring CAR T cell generation with a CD8-targeted lentiviral vector by single-cell transcriptomics.

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