In the 2022 AACR annual meeting, Michelle Monje et al.'s study on CAR-T for diffuse pontine glioma (DIPG) and diffuse midline gliomas (DMG) with H3K27M mutation The study has gained widespread attention and single cell sequencing technology has played an important role in this research. The paper was published online in the international academic journal Nature on February 7, 2022

CAR-T for the treatment of DIPG/ DMG with H3K27M mutation

CAR-T cell therapy technology is a widely popular cutting-edge cancer therapy, which is based on the principle of artificially adding receptors for specific cancer cell surface antigens to normal T cells so that the T cells can specifically bind to cancer cells and thus kill them through cell killing action.

Once CAR-T cell therapy technology was introduced, it has firstly played a great advantage in the treatment of hematological malignancies, and as of the end of March 2022, six CAR-T products have been approved for marketing by FDA and two CAR-T products have been approved for marketing by NMPA. CAR-T treatments for solid tumors are also currently in rapid development.

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DIPG/DMG type H3K27M is a highly aggressive, solitary, fatal tumor with few treatment options, an average life expectancy of 10 months after diagnosis, and a 5-year survival rate of less than 1%.

Michelle Monje's team found in previous studies that disialogangliosid (GD2) is highly expressed on diffuse midline glioma cells with H3K27M mutation, so they selected GD2 as a potential CAR-T cell therapeutic target for DIPG/DMG and conducted initial exploration in mice.

In this study, the researchers first screened several DIPG/DMG patients by intravenous injection of 1×10^6 Cells/Kg dose of GD2-CAR T cells, and finally selected four H3K27M mutant DIPG/DMG patients to receive GD2-CAR T infusion (three DIPG patients and one spinal DMG patient ), which was administered intracerebroventricularly.

a. MRI results showed significant tumor reduction in patient with DIPG #3

b. Patients with DIPG #3 showed significant physiological remission

Clinical results showed that three of the four patients achieved imaging and clinical benefit after intravenous administration of GD2-CAR T cells (one patient with spinal DMG experienced rapid tumor progression during CAR T cell preparation and was therefore removed from the study), patients lived significantly longer after diagnosis, and patients showed significant recovery in neurological function. However, what is the journey of GD2-CAR T cells into the human body and how do they interact with cancer cells and immune cells? These questions need to be answered by more detailed research methods.

Single-cell Sequencing Explains the Therapeutic Process of CAR-T Technology

In order to gain a deeper understanding of the mechanism of efficacy of GD2-CAR T-cell infusion, researchers conducted further analysis using single-cell sequencing technology. The researchers took cerebrospinal fluid (CSF) samples from four patients at different treatment stages, as well as CAR-positive and negative T cells isolated from CAR T-cell products, and performed single-cell RNA sequencing analysis. A total of 25598 cells from CSF and 40,000 cells from CAR T-cell products were sequenced.

Cell profiles of CSF samples based on single-cell RNA sequencing analysis

The cell profile of CSF samples based on RNA sequencing data showed that in addition to lymphocytes, myeloid cells, rare astrocytes and B cells, there was also a group of dual-spectrum cells in CSF samples (upper right panel, boxed in red), suggesting the possibility of myeloid cells phagocytosing T cells in patients.

Single-cell transcriptome analysis of different myeloid cell subpopulations

The investigators further performed a more detailed cellular profiling analysis for myeloid cells in the CSF samples after drug administration. The results revealed that myeloid cells could be divided into seven different cell populations, including monocytes, microglia and macrophages expressing various functional characteristics, as well as proliferating myeloid cells.

Proportion of myeloid cells in CSF samples at different treatment time periods

The analysis revealed the presence of myeloid cells characterized by interferon response at the time point of peak inflammation after intracerebroventricular administration, and this cell population showed a distinct immune activation profile. In contrast, a subpopulation of cerebrospinal fluid myeloid cells expressed a significant phagocytosis and lipid metabolism gene program at the late intraventricular and administration points, and these cells showed a strong immunosuppressive profile. All these results provide further evidence for the effectiveness of GD2-CAR T cells in treating H3K27M+ mutant DIPG/DMG.

In summary, single-cell sequencing technology further explored the process of GD2-CAR T treatment of H3K27M+ DIPG/DMG at the cellular level and gene expression level, and observed the heterogeneity of different treatment stages, different routes of administration and different patients, which is of guiding significance for understanding the mechanism of GD2-CAR T action and subsequent clinical studies.

Prospects of single-cell sequencing technology applied to solid tumor CAR-T

Currently, various studies using CAR-T technology for the treatment of gastric cancer, pancreatic cancer, colorectal cancer, melanoma, and liver cancer are in full swing. Single-cell sequencing technology can be used to investigate the proliferation and cloning of CAR-T cells before and after infusion, the changes in gene expression of each cell in the body at different stages before and after infusion, the relationship between cell types and cloning, proliferation and efficacy of each cell in the body before and after infusion, the actual effect and mechanism of CAR-T cells in vivo, and other aspects of cell therapy. It is believed that in the future, single cell sequencing technology will play more roles in CAR-T and other cell therapy technologies.

References

1 Majzner, R. G. et al. GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas. Nature. 603, 934–941 (2022).2 Mount, C. W. et al. Potent antitumor efficacy of anti-GD2 CAR Tcells in H3-K27M+ diffuse midline gliomas. Nat. Med. 24, 572–579 (2018).3 Cooney, T. et al. Contemporary survival endpoints: an International Diffuse Intrinsic Pontine Glioma Registry study. Neuro-Oncol. 19, 1279–1280 (2017).