CRISPR-Engineered Immunotherapies Show Promise

Cancer is a complex disease caused by uncontrolled cell growth resulting from genetic and epigenetic changes to the genomic sequence. While there have been significant advances in treatment strategies and options, including chemotherapy, radiotherapy, and surgery, disease recurrence or resistance to therapy often occurs, leading to poor prognosis and disease outcomes for patients.

A newer approach to cancer treatment is immunotherapy—where the patient’s own immune system is harnessed to prevent, control, and eliminate cancer. CRISPR-based gene editing is playing an important role in the development of this new treatment option—the flexibility and versatility of the system allows for the production of engineered immune cells such as therapeutic CAR-T and TCR cells, inhibition of immune checkpoint signaling pathways, or the ability to screen for novel immunotherapy targets. This article looks at how CRISPR-based gene editing is playing a role in the various applications of immunotherapy treatment options.

Harnessing the immune system to fight cancer

One of the hallmarks of cancer is evasion of the body’s immune response—tumors can circumvent numerous antitumor responses by evading recognition by immune cells or secreting extracellular compounds into the tumor microenvironment to distort surrounding immune cell function. Immune cells are inherently powerful at controlling and eliminating cancer cells, and so harnessing the precision, dynamic adaptation, and memory of the immune system is a potential game-changer for cancer treatment. The aim of immunotherapies is to reduce tumor-induced immunosuppression or enhance the anti-tumor immune response. One approach is adoptive T cell therapy, where immune cells are extracted from the patient, genetically engineered ex vivo to enhance recognition and killing of cancer cells, and then reintroduced back into the patient.

Generation of CAR-T cells with CRISPR

An exciting application of CRISPR-based gene editing is in the generation of CAR-T cells that contain an artificial chimeric antigen receptor (CAR), consisting of an intracellular chimeric signaling domain and an extracellular single-chain variable fragment that specifically recognizes cancer-specific antigens without the requirement for major histocompatibility complex (MHC). CAR-T cell therapy has been successfully used to treat blood-based malignancies, with several CD19-directed CAR-T cells receiving FDA approval to treat B-cell leukemias and lymphomas.¹

However, the production of autologous CAR-T cells is time consuming, costly, and can be challenging in patients with low T cell populations. The creation of universal, allogeneic CAR-T cells, available immediately off-the-shelf and in sufficient quantities for re-infusion, would improve availability of CAR-T cell therapy and be a major benefit for those patients with rapidly progressing disease. The generation of universal CAR-T cells would require both transduction of CAR as well as knockout of the TCR and HLA molecules to prevent alloreactivity (e.g., host versus graft response).

Compared to other gene-editing technologies such as ZFN and TALENs, CRISPR-Cas9 has more applications in the generation of universal CAR-T cells due to the ability to perform multiplex gene editing, thereby targeting multiple gene loci simultaneously. By incorporating multiple guide RNAs in a CAR lentiviral vector, Ren et al was able to create CAR-T cells with edits at multiple genomic loci simultaneously, thereby generating a CAR-T cell with endogenous TCR and HLA knocked out, as well as other inhibitory molecules to improve CAR-T cell efficacy in a one-shot approach.² The safety and efficiency of this approach in vivo still needs to be tested and there are several clinical trials ongoing, but this is an exciting application of CRISPR in immunotherapy and shows potential promise in the treatment of cancers.

Enhancing CAR-T cell function with CRISPR

The immunosuppressive tumor microenvironment and exhaustion of effector immune cells can result in limited success of CAR-T cell therapy in some patients. As well as the generation of CAR-T cells, CRISPR has also been used to enhance CAR-T cell function by disrupting several co-inhibitory molecules. For example, depletion of the programmed cell death 1 (PD-1) receptor improves the ability of CAR-T cells to destroy tumor cells.³ CRISPR has also been used to knockout expression of other genes to improve efficacy of CAR-T cells, including granulocyte-macrophage stimulating factor (GM-CSF) and endogenous TGF-b II receptor.^4,5

T cell receptor (TCR) engineering with CRISPR

CAR-T cells have had great success in the treatment of blood-based malignancies but have played a limited role in the treatment of solid tumors, possibly due to tumor heterogeneity or lack of tumor-specific antigens. Another adoptive T cell therapy approach is to use TCR-T cells—unlike CAR-T cells that bind naturally occurring antigens on the surface of cancer cells, TCR-T cells recognize cancer antigens presented on MHC via a cell surface T cell receptor (TCR). This approach enables a greater number of antigens to be targeted, therefore enhancing the personalization of treatment, which could lead to better outcomes for patients. But a major issue with the generation of TCR-T cells is the presence of endogenous TCR on recipient T cells, which can reduce the expression of transgenic TCR due to competition with CD3 and mixed dimer formation between endogenous and transgenic TCR.

To solve this problem, CRISPR can be utilized to introduce a tumor-specific TCR sequence as well as knock out endogenous TCRs. The first in-human, Phase 1 clinical trial using this approach was reported in 2020 to demonstrate the safety and feasibility of multiplex CRISPR-Cas9-based gene editing of T cells in three patients with advanced, refractory cancer.⁶ Autologous T cells were engineered with CRISPR to express a TCR-specific for NY-ESO-1 and LAGE-1, and to knock out endogenous TCR, along with PD-1. The authors demonstrated successful edits at all three genomic loci and engineered T cells were successfully trafficked to the bone marrow in all patients where they persisted for up to nine months.

CRISPR-mediated inhibition of immune checkpoint signaling pathways

Despite the presence of CD8+ tumour infiltrating lymphocytes (TILs) in solid tumors, they often fail to destroy tumor cells—the activation of inhibitory molecules such as PD-1 and CTLA-4 that modulate immune response is thought to lead to T cell exhaustion. The use of immune cell checkpoint inhibitors, such as anti-CTLA-4, has previously shown great promise in the treatment of various cancers.

CRISPR-Cas9-based gene editing has been applied to knockout CTLA-4 in cytotoxic T lymphocytes (CTLs) —disruption of CTLA-4 expression with CRISPR results in CTLs with pronounced anti-tumour effects in vivo, and also displayed increased TNF-α and IFN-γ secretion demonstrating that inhibition of CRLA-4 could significantly improve antitumour activity of CTLs.⁷ Similarly, disruption of PD-1 with CRISPR in CTLs also enhanced antitumor activity—the safety and feasibility of this approach was recently shown in a clinical trial in patients with non-small cell lung cancer showing that PD-1 disrupted T cells were safe and efficacious.⁸

The next generation of immunotherapy is close

The CRISPR-Cas9 gene-editing system has proven to be an efficient and flexible genome-editing tool and has been rapidly adopted, facilitating gene editing in a wide range of cell types and organisms. CRISPR has revolutionized cancer screening and treatment options and is increasingly used in immunotherapy, which aims to harness the immune system to fight cancer. As well as being used in the creation of therapeutic immune cells, such as CAR-T, TCR-T cells, and engineered CTLs, CRISPR is also demonstrating its worth as a powerful, large-scale functional genomic screening tool, enabling the identification of novel immunotherapy targets.

There are now several completed clinical trials showing the safety and feasibility of CRISPR-Cas9 for immunotherapy applications, as well as several that are ongoing. New advances to the CRISPR system, such as prime editing and base editing, could improve precision and specificity and may offer even safer in-human gene editing by helping to overcome some of the concerns regarding off-target effects. Patients are already benefitting from immunotherapies in the clinic, and CRISPR-engineered immunotherapies could be set to improve patient outcomes still further.

Key Takeaways

The CRISPR system (or Clustered Regularly Interspaced Short Palindromic Repeats) was originally part of a microbial immune system used to defend against foreign DNA and has since been repurposed into a simple but very powerful gene-editing tool

• Like other gene-editing technologies, such as ZFNs and TALENs, CRISPR-Cas9 relies on the introduction of a double-strand break (DSB) into the target site to activate endogenous cellular DNA repair pathways, either non-homologous end joining (NHEJ) or homologous recombination (HR).
• The NHEJ pathway is error-prone and often causes frameshift mutations or indels at the target site, so is therefore useful for creating gene knockouts. Alternatively, in the presence of a donor template sequence, a DSB can trigger HR, which integrates the exogenous DNA at the target site and so facilitates a sequence change.
• The CRISPR system consists of a short RNA molecule (single guide RNA, or sgRNA) and Cas9 endonuclease. The Cas9 protein recognizes the 3’-NGG-5’ PAM site (Protospacer Adjacent Motif), which occurs frequently throughout the genome—if the sequence adjacent to the PAM site shares homology with a 20-nucleotide portion of the sgRNA, then the endonuclease activity of Cas9 is activated and a DSB is introduced at the target loci
• Due to the RNA-based nature of the system, CRISPR is flexible, scalable, and user-friendly—reprogramming to recognize a new target site requires just a change to the 20-nucleotide portion sgRNA. Multiple sgRNA can be introduced simultaneously, thereby facilitating multiplex editing

References

1. Ou, X., Ma, Q., Yin, W., Ma, X., & He, Z. (2021). CRISPR/Cas9 Gene-Editing in Cancer Immunotherapy: Promoting the Present Revolution in Cancer Therapy and Exploring More. Frontiers in cell and developmental biology, 9, 674467. 

2. Ren J, Zhang X, Liu X, et al. (2017) A versatile system for rapid multiplex genome-edited CAR T cell generation. Oncotarget. 8(10):17002-17011. 

3. Rupp, L. J., Schumann, K., Roybal, K. T., Gate, R. E., Ye, C. J., Lim, W. A., et al. (2017). CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci. Rep. 7:737.

4. Sterner, R. M., Sakemura, R., Cox, M. J., Yang, N., Khadka, R. H., Forsman, C. L., et al. (2019). GM-CSF inhibition reduces cytokine release syndrome and neuroinflammation but enhances CAR-T cell function in xenografts. Blood 133, 697–709. 

5. Tang, N., Cheng, C., Zhang, X., Qiao, M., Li, N., Mu, W., et al. (2020). TGF-(inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors. JCI Insight5:e133977.

6. Stadtmauer, E. A., Fraietta, J. A., Davis, M. M., Cohen, A. D., Weber, K. L., Lancaster, E., et al. (2020). CRISPR-engineered T cells in patients with refractory cancer. Science (New York, N.Y.) 367:eaba7365.

7. Zhang, W., Shi, L., Zhao, Z., Du, P., Ye, X., Li, D., et al. (2019). Disruption of CTLA-4 expression on peripheral blood CD8 + T cell enhances anti-tumor efficacy in bladder cancer. Cancer Chemother. Pharmacol. 83, 911–920. 

8. Lu, Y., Xue, J., Deng, T. et al. (2020) Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat Med 26, 732–740.