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Unlocking the Power of Precision Medicine: An Introduction to CAR-T Single Cell Analysis

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Cell ang Gene Therapy

Unlocking the Power of Precision Medicine: An Introduction to CAR-T Single Cell Analysis

Introduction to CAR-T therapy

The concept of using genetically modified T cell therapy to target cancer cells for cancer treatment was first proposed in the late 1980s [1].

Researchers found that T cells, which are a type of white blood cell, could be used to target and destroy cancer cells in the same way that they target and destroy viruses and other pathogens. T cells could be collected from a person and genetically modified to express chimeric antigen receptors (CARs). These CARs allow the T cells to recognize and target specific proteins on the surface of cancer cells. The modified T cells are then expanded in the lab and reinfused back into the person’s body to trigger an immune response that releases cytokines and destroys the cancer cells.

The first CAR-T cell therapy (anti-CD19), Kymriah (tisagenlecleucel), was approved by the U.S. Food and Drug Administration (FDA) in 2017 for the treatment of acute lymphoblastic leukemia (ALL) [2]. Since then, field of CAR-T cell therapy has continued to grow and evolve, with several other CAR-T cell therapies approved for the treatment of different types of cancers.

There are typically four generations of CAR-T cell therapy, each representing a step forward in the development of this type of cancer treatment (Figure 1).

Figure 1. Four generations of CAR-T cells (figure taken from Zhang X., Zhu L., Chen, S., and Xiao Y. (2022) [3]; https://creativecommons.org/licenses/by/4.0/). The first generation of CAR-T cell is the original form of a single CAR inserted into a patient’s T-cells. From the second generation, two or more antigen-recognition domains are added. The third generation includes an additional co-stimulatory domain and fourth generation has additional safety features with improved efficacy.

While CAR-T cell therapy has shown great promise in the treatment of cancer, there are also a number of concerns and challenges associated with these treatments from design (Figure 2) and non-scientifically.

Figure 2. Limitation of CAR-T therapy from design (figure taken from Sterner, R.C., and Sterner, R.M. (2021) [4]; https://creativecommons.org/licenses/by/4.0/). Scientific limitations illustrated in Figure 2 include 1) non-sensitive and unspecific targeting of tumor cells from the designed CAR-T cells, 2) difficult for CAR-T cells to reach the cancer cells in solid tumors, 3) complicated reactions from tumor microenvironments with other immune cells and 4) toxicities triggered by CAR-T cells like cytokine-release syndrome or neuro-inflammations.

In addition to illustrated in Figure 2, cost, manufacturing capabilities, duration of response, potential resistance to the therapy remain challenging factors for CAR-T treatments.

Single Cell Applications in CAR-T Cell Therapy: The Power of Bioinformatics

The use of single cell analysis in CAR-T cell therapy research has become increasingly important in recent years as scientists strive to better understand the behaviour and function of these genetically modified immune cells and their interactions within the complex system.

Enhancing CAR-T cell specificity and identify novel CAR-T cell targets

By analysing the molecular and functional characteristics of individual cells, single cell analysis determines the levels of expression of specific antigens. This information can be used to improve the specificity of CAR-T cell therapies, identify potential new targets and reduce the risk of off-target effects.

There have been several studies published that demonstrate the potential of single cell analysis to improve CAR-T cell specificity. Irfan Bandey and the team [5] used mathematical modelling and single-cell profiling to analyse the difference between killer and non-killer CAR-T cells. The study identified CD137 as a costimulatory molecule that is upregulated in killer T cells and distinguishes them from the CD107a+(degranulating) subset of CAR-T cells. The study also showed that the addition of CD137 ligand (CD137L) to CAR-T cells improved their ability to kill cancer cells and reduced cell exhaustion. In trans, the CAR-T cell treatment results were improved in leukemia and refractory ovarian cancer models in mice. These findings could potentially lead to improved therapeutic strategies for the treatment of cancer.

Study tumor microenvironments

An important way in which single cell analysis is being used in CAR-T cell therapy is through the study of biology mechanisms. These algorithms analyse the behaviour of individual T-cells in the patient’s body to understand how they interact with cancer cells. This information can then be used to monitor the progress of the treatment.

Nicholas Haradhvala and team [6] studied clinical response to CAR-T cell therapy in patients with high-grade B cell lymphoma. The study involved single-cell transcriptome sequencing of pre- and post-treatment samples collected from 32 patients who received either of two CD19 CAR-T products (axicabtagene ciloleucel or tisagenlecleucel). The study found that the expansion of a rare group of proliferative memory-like CD8 clones was a signature of tisagenlecleucel response, while axicabtagene ciloleucel responders displayed more heterogeneous populations. The study also detected elevations in CAR-T regulatory cells (CAR-Tregs) among non-responders to axicabtagene ciloleucel, which were capable of suppressing conventional CAR-T cell expansion and driving late relapses in an in vivo model.

This study provides new insights into the temporal dynamics of effective responses to CAR-T therapy, the distinct molecular phenotypes of CAR-T cells with different designs, and the potential for even small increases in CAR-Tregs to drive relapse. These findings could help inform the development of new CAR-T cell therapies that are more effective and have fewer relapse rates.

Understand and monitor CAR-T functions for treatment prognosis

Cancer is a highly heterogeneous disease, with different cells within a single tumor having different molecular signatures that is associated to treatment response. Single cell analysis can help to identify and understand these differences, which can be used to personalize CAR-T cell therapies.

For example, it might monitor the proliferation and survival of individual T-cells in the patient’s body to determine the effectiveness of the treatment. If the T-cells are not surviving or proliferating as expected, the treatment plan can be adjusted to address the issue. It can also be used to understand the biology of the cancer to prepare for future treatments.

The prognostic algorithms use data from individual cancer cells to predict the likelihood of the disease progressing and to guide treatment decisions. For example, a prognosis algorithm might analyse the expression levels of specific genes in individual cancer cells to determine the aggressiveness of the cancer.

Zinaida Good and team [7] used single-cell proteomic profiling to identify biomarkers associated with resistance and toxicity in patients with large B cell lymphoma treated with CD19-specific CAR-T cell therapies. The study found that an increase in CD4+ Helios+ CAR-T cells on day 7 after infusion was associated with progressive disease and less severe neurotoxicity. Furthermore, this population had characteristics of T regulatory (Treg) cells, and that a model combining the expansion of this subset with lactate dehydrogenase levels predicted durable clinical response. These results suggest that the expansion of CAR-TReg cells may be a novel biomarker of response and toxicity after CAR-T cell therapy.

Overall, by using single cell analysis, scientists improved the understanding of the diversity of CAR-T cells, how they respond to different treatments, and how they interact with other cells in the body, including cancer cells and normal healthy cells. It has the high potential pushing the CAR-T cell therapy to the next level.


References

1. Barros LRC, Couto SCF, da Silva Santurio D, Paixão EA, Cardoso F, da Silva VJ, Klinger P, Ribeiro PDAC, Rós FA, Oliveira TGM, Rego EM, Ramos RN, Rocha V. Systematic Review of Available CAR-T Cell Trials around the World. Cancers (Basel). 2022 May 27;14(11):2667. doi: 10.3390/cancers14112667. PMID: 35681646; PMCID: PMC9179563.

2. Braendstrup P, Levine BL, Ruella M. The long road to the first FDA-approved gene therapy: chimeric antigen receptor T cells targeting CD19. Cytotherapy. 2020 Feb;22(2):57-69. doi: 10.1016/j.jcyt.2019.12.004. Epub 2020 Feb 1. PMID: 32014447; PMCID: PMC7036015.

3. Zhang, Xiaomin and Zhu, Lingling and Zhang, Hui and Chen, Shanshan and Xiao, Yang. CAR-T Cell Therapy in Hematological Malignancies: Current Opportunities and Challenges. Frontiers in Immunology. 2022. Doi: 10.3389/fimmu.2022.927153

4. Sterner, R.C., Sterner, R.M. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J.11, 69 (2021). https://doi.org/10.1038/s41408-021-00459-7

5. Bandey IN, Adolacion JRT, Romain G, Paniagua MM, An X, Saeedi A, Liadi I, You Z, Rajanayake RB, Hwu P, Singh H, Cooper LJ, Varadarajan N. Designed improvement to T-cell immunotherapy by multidimensional single cell profiling. J Immunother Cancer. 2021 Mar;9(3):e001877. doi: 10.1136/jitc-2020-001877. PMID: 33722906; PMCID: PMC7970283.

6. Nicholas J. Haradhvala, Mark B. Leick, Katie Maurer, Satyen H. Gohil, Rebecca C. Larson, Ning Yao, Kathleen M. E. Gallagher, Katelin Katsis, Matthe J. Frigault, Jackson Southard, Shuqiang Li, Michael C. Kann, Harrison Silva, Max Jan, Kahn Rhrissorrakrai, Filippo Utro, Chaya Levovitz, Raquel A. Jacobs, Kara Slowik, Brian P. Danysh, Kenneth J. Livak, Laxmi Parida, Judith Ferry, Caron Jacobson, Catherine J. Wu, Gad Getz, Marcela V. Maus. Distinct cellular dynamics associated with response to CAR-T therapy for refractory B-cell lymphoma. Nature Medicine doi: 1038/s41591-022-01959-0

7. Good Z, Spiegel JY, Sahaf B, Malipatlolla MB, Ehlinger ZJ, Kurra S, Desai MH, Reynolds WD, Wong Lin A, Vandris P, Wu F, Prabhu S, Hamilton MP, Tamaresis JS, Hanson PJ, Patel S, Feldman SA, Frank MJ, Baird JH, Muffly L, Claire GK, Craig J, Kong KA, Wagh D, Coller J, Bendall SC, Tibshirani RJ, Plevritis SK, Miklos DB, Mackall CL. Post-infusion CAR TReg cells identify patients resistant to CD19-CAR therapy. Nat Med. 2022 Sep;28(9):1860-1871. doi: 10.1038/s41591-022-01960-7. Epub 2022 Sep 12. PMID: 36097223.

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