Overcoming the Limitations of CAR-T Cell Therapy with CRISPR

In recent years, chimeric antigen receptor (CAR)-T cell therapy has emerged as a ground-breaking treatment for various cancers, offering new hope to patients with previously untreatable diseases.¹ However, despite its remarkable success, CAR-T cell therapy has its limitations, including issues related to specificity, durability, and off-target effects. Enter CRISPR, the revolutionary gene-editing tool, which holds the promise to address these limitations and enhance the effectiveness of CAR-T cell therapy. In this article, we will discuss the limitations of CAR-T cell therapy and explore how CRISPR technology is being harnessed to usher in a new era of personalized cancer treatment.

Understanding CAR-T cell therapy

CAR-T cell therapy is a form of adoptive immune therapy that involves the modification of a patient's own T cells to express chimeric antigen receptors. These bioengineered receptors are then capable of targeting specific antigens and eliciting an immune response against specific cancer cells.

The production of CAR T cells is a bespoke process, involving harvesting autologous leukocytes from the patient via leukapheresis and subsequent enrichment and proliferation of these ex vivo cells in vitro. The enriched cells are then gene edited using viral transduction with lentiviruses or retroviruses that encode the CAR sequence introducing the antigen receptor to the T cell. The gene-edited T cells expressing the CARs on their surface are then proliferated in bioreactors and reinfused into the patient. These CARs enable T cells to recognize and attack cancer cells with precision, offering a highly targeted approach to cancer treatment.

The limitations of CAR-T cell therapy

The application of CAR-T cell therapy has had much clinical success, especially in the case of hematological malignancies such as acute lymphoblastic leukemia (ALL) with CAR T cells recognizing the CD19 antigen present on B cells and resulting in patient remission.² But despite this, the approach has several limitations that need to be addressed.

Lack of specificity

CARs are engineered to recognize specific antigens present on the surface of cancer cells. However, some healthy cells may also express these antigens at lower levels, leading to off-target effects. This lack of specificity can result in adverse side effects and limit the therapy's overall safety—for example, triggering cytokine release syndrome (CRS)—an acute inflammatory syndrome that can lead to organ dysfunction.³

Durability of response

In some cases, CAR-T cell therapy initially achieves a complete response, but the cancer may eventually relapse due to the loss of CAR-T cell persistence or the emergence of cancer cells with reduced antigen expression. This limitation necessitates the development of strategies to enhance the long-term effectiveness of CAR-T cell therapy.

Heterogeneity of solid tumors

CAR-T cell therapy has shown less success in treating solid tumors compared to hematological cancers as solid tumors often display greater heterogeneity, making it challenging for CAR T cells to target all cancer cells effectively. CAR T cells must also persist in the toxic tumor microenvironment of solid-state tumors. Addressing this limitation requires innovative approaches to improve CAR-T cell therapy's efficacy in solid tumor treatment.

Manufacturing bottleneck

The bespoke, multi-step process of manufacture can impede the application of CAR-T cell therapy, leading to patients succumbing to advanced disease progression while waiting for treatment.

Enter CRISPR—a potential gamechanger for CAR-T cell therapy

CRISPR is a revolutionary gene-editing tool that allows scientists to precisely modify the DNA of living organisms, including human cells. Requiring only a short guide RNA (sgRNA) and Cas9 endonuclease, CRISPR can be used to introduce targeted and specific changes to the genome more easily than previous gene-editing technologies such as Zinc Fingers and TALENs. The potency of CRISPR in integrating the CAR cassette into T cells was highlighted in a 2017 study that used CRISPR gene editing to insert CARs into the TRAC locus of T cells to produce CAR T cells that stably expressed the CAR on their surface and outperformed traditionally edited cells in a mouse model of leukemia.⁴

CRISPR technology offers unparalleled precision and versatility in manipulating genes, making it a powerful tool for potentially overcoming the limitations of CAR-T cell therapy. Researchers are exploring ways to use CRISPR to engineer CAR T cells with improved antigen recognition, the ability to withstand the immunosuppressive signals in the tumor microenvironment, as well as modify genes responsible for T-cell exhaustion to improve longevity in the body.⁵ Here are some examples of how CRISPR is being used to improve CAR-T cell therapy:

Reducing side effects

In 2021, researchers conducted a pilot study using CRISPR-edited CAR T cells with a knock-out of GM-CSF and found that the CRISPR-edited CAR T cells reduced the occurrence and severity of cytokine release syndrome in patients with non-Hodgkin’s lymphomas and multiple myelomas, while still providing a safe and effective anti-cancer therapy.⁶

CRISPR for enhanced solid tumor targeting

The heterogeneity and toxic microenvironment of solid tumors presents a unique challenge in CAR-T cell therapy, but CRISPR gene editing has also shown to be effective in creating robust CAR T cells capable of surviving the environment of solid-state tumors with increased functionality. A 2020 study demonstrated that CAR T cells edited with CRISPR to no longer express TGBF, the receptor for TGF-β, had better tumor elimination efficacy in cell lines and tumor models.⁷

Producing allogeneic CAR T cells with CRISPR

The reliance on autologous T cells in CAR-T cell therapy is the major limitation to their manufacture and application, for example due to poor proliferation of T cells in vitro or lack of harvestable T cells in patients who have undergone chemotherapy. Therefore, the generation of universal, allogeneic CAR T cells harvested from a healthy donor and then edited to elude host immune response has become an important area of CRISPR research.

In 2022, researchers at Bioray Laboratories and Zheizhang University released data from a Phase I clinical trial using CRISPR-modified allogeneic CAR T cells targeting the CD19 protein of certain leukemia and lymphomas. This study demonstrated a high success rate in the treatment of B cell non-Hodgkin lymphoma with 7 out of 8 participants entering remission.⁸ Applying CRISPR gene editing to generate universal allogeneic CAR T cells overcomes many of the limitations of the current autologous production process and offers a faster, more-effective anti-cancer therapeutic.

CRISPR: The future of CAR-T cell therapy

CAR-T cell therapy has revolutionized the treatment of certain types of blood cancers, offering new hope to patients facing previously untreatable diseases. But there remain several limitations related to specificity, durability, solid tumor targeting, and manufacturing. However, CRISPR technology holds tremendous potential to overcome these limitations, producing CAR-T cell therapies with increased functionality and efficacy while increasing safety and mitigating side effects. CRISPR also has the potential for personalized medicine, and the possibility of engineering universal, allogeneic CAR T cells that are customized to an individual’s cancer, promising improved treatment outcomes and reduced side effects.

Key Takeaways

• CAR-T cell therapy is a revolutionary cancer treatment that engineers T cells harvested from a patient to express chimeric antigen receptors to target specific antigens and elicit an immune response against cancer cells
• Despite the proven efficacy of CAR-T cell therapy, there are a number of limitations that restrict wider applications. These include issues with specificity, durability, the ability to target solid tumors, and a manufacturing bottleneck that can delay treatment
• CRISPR gene-editing technology has the potential to overcome the limitations of CAR-T cell therapy by increasing functionality and efficacy of the treatment, improving safety and mitigating side effects, while offering the potential to produce universal, allogenic CAR T cells

References

1. Mikkilineni, L., Kochenderfer, J, N. Chimeric antigen receptor T-cell therapies for multiple myeloma. Blood 2017; 130 (24): 2594–2602. 

2. Sheykhhasan, M., Manoochehri, H. & Dama, P. Use of CAR T-cell for acute lymphoblastic leukemia (ALL) treatment: a review study. Cancer Gene Ther 29, 1080–1096 (2022). 

3. Sterner, R.C., Sterner, R.M. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 11, 69 (2021). 

4. Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature. 2017;543:113–7.

5. Dimitri, A., Herbst, F. & Fraietta, J.A. Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing. Mol Cancer 21, 78 (2022). 

6. Yi, Y., Chai, X., Zheng, L. et al. CRISPR-edited CART with GM-CSF knockout and auto secretion of IL6 and IL1 blockers in patients with hematologic malignancy. Cell Discov 7, 27 (2021).

7. Tang, J. et al. TGF-β inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors. JCI Insight. 2020 Feb 27;5(4):e133977. 

8. Zhang, J., Hu, Y., Yang, J. et al. Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL. Nature 609, 369–374 (2022).