Single-cell Sequencing Combined with CAR-T to Explore Tumor Therapies

Single-cell Sequencing and CAR-T

Chimeric Antigen Receptor (CAR) is a recombinant transmembrane molecule consisting of an extracellular segment (a single-chain antibody variable region that binds antigens), a transmembrane region, and an intracellular segment (an intracellular signaling domain responsible for transmitting activation signals), and it is an artificially constructed receptor that recognizes specific antigens. T cells expressing CAR molecules are called chimeric antigen receptor T cells (CAR-T cells), which are prepared by transferring the CAR gene into T cells in vitro via a variety of vectors. CAR-T cells can track and recognize the specific receptors they carry on their surface and guide T cells to kill cancer cells.

Single-cell sequencing refers to sequencing technology to obtain genetic information of individual cells, i.e., extraction and amplification of genetic information of genome, transcriptome, epigenome and other genetic information and high-throughput sequencing analysis at the level of individual cells. The field of single-cell genomics has in recent years revealed numerous clues to the complexity of biological systems. The combination of high-resolution single-cell sequencing and CAR T therapy can help us to further explore the efficacy of CAR-T cell therapy, mechanism of action, and develop new targeted drugs and combination therapies to optimize and improve the scope of clinical application of CAR-T cell therapy.

Unveiling Novel Targets for AML Therapy by Transcriptome Profiling

The research team employed advanced methodologies, including single-cell RNA sequencing (scRNA-seq), single-cell T-cell receptor sequencing (scTCR-seq), and flow cytometry, to meticulously isolate CAR-T cells. These cells were obtained from blood and bone marrow samples of 10 patients diagnosed with relapsed refractory childhood acute B-lymphoblastic leukemia, up to five years post-treatment with CD19 CAR-T cells.

The investigation involved a comprehensive comparison between patients exhibiting persistent CAR-T cells and those experiencing outcomes below expectations. Notably, the study revealed that enduring CAR-T cells exhibited a distinctive CD4/CD8 double-negative phenotype and a memory state resembling failure, characterized by a unique transcriptional profile. This enduring profile was notably prevalent in all pediatric patients responding favorably to long-term therapy, and interestingly, it was also identified in two adult patients with chronic lymphocytic leukemia. The findings shed light on a universal transcriptional signature associated with the presence of clinically effective and durable CD19 CAR-T cells.

Study overview and workflow. (Anderson et al., 2023)

The authors generated single-cell transcriptome profiles containing more than 28,000 healthy and malignant myeloid cells and 500,000 healthy cells based on 15 scRNA-seq datasets from AML patients and 11 scRNA-seq datasets from 9 healthy human tissues (brain, lungs, lymph nodes, heart, skin, liver, kidneys, colon, and esophagus) and performed cell surface molecular expression analysis on these data were analyzed for cell surface molecular expression to find antigens that are expressed on the surface of malignant tumor cells but are least expressed in healthy human cells, including T cells. After rigorous screening, two new targets for CAR-T cells that can be used to treat AML were identified: CSF1R and CD86.

A scRNA-seq-based screening approach identifies CSF1R and CD86 as potential CAR targets in AML. (Gottschlich et al., 2023)

References:

1. Anderson, Nathaniel D et al. "Transcriptional signatures associated with persisting CD19 CAR-T cells in children with leukemia." Nature medicine vol. 29,7 (2023): 1700-1709.
2. Gottschlich, A., Thomas, M., Grünmeier, R. et al. Single-cell transcriptomic atlas-guided development of CAR-T cells for the treatment of acute myeloid leukemia. Nat Biotechnol 41, 1618–1632 (2023).