Transforming Cancer Treatment: CRISPR-Cas9 Revolutionizes CAR-T Cell Therapy (Part 24- CRISPR in Gene Editing and Beyond)

Welcome to the 24th part of the 40-part series on applications of CRISPR in Gene Editing and Beyond.

Links to the previous parts: Parts 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22 and 23.

Adoptive cell therapy, abbreviated as ACT, is a type of immunotherapy which seeks to harness immune cells, particularly T cells, for fighting cancer. Various adoptive cell therapies are:

(i) Tumor-infiltrating lymphocyte (TIL) therapy: It is one of the first types of ACT that is aimed at isolating naturally occurring T cells that have already infiltrated patients’ tumors, then activating and expanding them in vitro. After the T cells have been activated in large numbers, they are reintroduced into the patient's body, where they can locate and eliminate cancerous cells (Fig 1a).

(ii) Engineered TCR Therapy: The engineered T-cell receptor (TCR) therapy involves attaching new T cell receptors to the T cells’ surfaces, enabling them to target specific cancer antigens. It is done by cloning the TCR genes of specific tumor-infiltrating lymphocytes and transferring them to the isolated T cells. The result is the engineered T cells with defined specificity for tumor cells (Fig 1b).

However, the reliance of engineered TCR-T cells on major histocompatibility molecules (MHCs) to recognize tumor antigens is a major limitation of this therapy, as tumor cells are capable of downregulating MHC expression to evade immune surveillance.

(iii) CAR-T cell therapy: This technique involves equipping the patient’s extracted T cells with a synthetic receptor known as chimeric antigen receptor, abbreviated as CAR. A key advantage of CARs is their ability to bind to cancer cells by targeting the naturally occurring antigens present on the surface of cancer cells without the need for antigen presentation by MHCs (Mollanoori et al., 2020; Al Saber et al., 2021).

Few FDA approved CAR-T cell therapies are:

·      CD19-specific axicabtagene ciloleucel or Yescarta for the treatment of diffuse large B cell lymphoma (DLBCL)

·      Tisagenlecleucel or Kymriah for the treatment of CD19+ diffuse large B-cell lymphoma and relapsed or refractory acute lymphoblastic leukemia (ALL)

·      Brexucabtagene autoleucel or Tecartus for treating mantle cell lymphoma (MCL) and relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL)

·      Lisocabtagene maraleucel or Breyanzi for treating relapsed and refractory large B cell lymphoma.

·      Idecabtagene vicleucel or Abecma for treating relapsed or refractory multiple myeloma.

Chimeric Antigen Receptor in CAR-T cells: Chimeric antigen receptor or CAR consists of 3 parts:

(i) an extracellular antigen recognition domain of the single-chain Fragment variant (scFv), derived from a monoclonal antibody molecule specific to a unique antigen present on the target tumor cells. The scFv is produced by fusing variable domains of the IgG antibody’s heavy chain (VH) and the light chain (VL) through a short flexible peptide linker. Variable domains are chosen because they are responsible for forming an antigen-binding site. The scFv identifies the target antigen expressed on the surface of tumor cells without the need for antigen presentation by MHCs (Fig 2).

(ii) a transmembrane domain, which is connected to the scFv through a flexible linker. The main function of the transmembrane domain is to anchor the CAR in the T cell membrane.

(iii) and an intracellular T cell activation domain that includes costimulatory molecules. After the CAR scFv binds to the tumor antigen, the intracellular activation domain initiates T cell proliferation and activation signaling, ultimately leading to the cytotoxicity of the tumor cells.

The gene editing tools such as Zinc finger nucleases (ZFN), Transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 can be used to precisely insert the CAR construct into the correct position in the genome of the T cells so that it is expressed efficiently at sufficient levels to generate engineered CAR-T cells. However, as already discussed in previous chapters, CRISPR-Cas9 is easier to construct and a more efficient gene editing tool with few off-target effects. In addition, the previous studies also suggest CRISPR-Cas9 is a better tool for gene editing in T cells and can edit multiple genes simultaneously using several guide RNAs (Ghaffari et al., 2021; Quazi, 2022).

Autologous vs. allogenic CAR-T cell therapy

Autologous CAR-T cell therapy harnesses the power of T cells isolated from the cancer patient. The isolated T cells are modified to target cancer cells and then infused back into the same patient. This process takes several weeks to manufacture CAR-T cells, and a delay in treatment availability can have dire consequences, particularly in patients with highly proliferative diseases. Even in some conditions, lymphocyte repertoire is depleted due to which isolating T cells from the patient is not possible.

The challenges of autologous CAR T therapy can be overcomed by engineering T cells derived from healthy donors/volunteers instead of patients. These are also called “off-the-shelf” CAR T cells. Off-the-shelf or allogenic CAR-T cells can be made in advance and administered immediately when the patient needs. Unlike autologous therapies that directly treat one patient, allogeneic therapies can treat multiple patients.

But there are certain issues with allogenic CAR-T cell therapy as the patient’s own immune system may recognize the transferred CAR-T cells as foreign, causing serious life-threatening reactions. This rejection is caused by the interaction of T cell receptors (TCR) and human leukocyte antigen class I (HLA I) that are expressed on both the CAR-T and patient’s T cells.

The endogenous TCR on transferred CAR-T cells can identify alloantigens in HLA mismatched recipients, thereby leading to Graft-versus-host disease (GVHD), thus impeding the anti-tumor activity of CAR-T cells. On the other hand, if the patient's immune system detects foreign HLA molecules on the CAR-T cells, it can reject them. This can also interfere with the effectiveness of CAR-T cell therapy.

CRISPR in gene editing of “off the shell” allogenic CAR-T cells

There are different ways to avoid GVHD when designing allogeneic CAR-T cells, the most widely used strategy being the generation of CAR-T cells that abrogate the expression of endogenous TCRs. It can be done using genome editing tools CRISPR-Cas9 (Ureña-Bailénet al., 2020; Razeghian et al., 2021; Dimitri et al., 2022).

TCR is a membrane-bound protein complex that consists of α and β chains and is expressed on the surface of all T cells. The HLA class I molecules appear ubiquitously on the surface of cells throughout the body and consist of an α chain that is stabilized by β2-microglobulin (β2M). The allogeneic rejection arises when the TCR on transferred CAR-T cells recognizes the HLA I complex on the recipient’s cells as foreign and attacks them. Similarly, the TCRs of the recipient’s T cells recognize the HLA complex on donor CAR-T cells as foreign and attack them.

To remove these rejection barriers, disrupting the α or/and β chains of allogenic CAR-T cells TCR through CRISPR-Cas9 can prevent them from recognizing and attacking the recipient T cells. Similarly, the disruption of β2M through CRISPR-Cas9 can be used to prevent maturation and expression of HLA class I molecules on CAR-T cell surface. This will prevent the patient's own T cells from recognizing the donor allogenic CAR-T cells as foreign and attacking them.

CRISPR in disruption of Immune checkpoints

In addition to disrupting endogenous TCR and β2-microglobulin expression in CAR-T cells, disruption of immune checkpoint inhibitor genes is also required. In normal conditions, after cellular immunity is activated and has successfully neutralized the target, the immune checkpoints regulate T-cell over-activation to prevent them from attacking the cells indiscriminately. However, cancer cells use the similar ways to protect themselves from being attacked by T cells by stimulating immune checkpoints. When the checkpoint on the cancer cells and partner proteins on T cells bind together, they send an “off” signal to the T cells and prevent them from destroying the cancer cells. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and PD-1 are key immune checkpoints. For instance, CTLA‐4 expressed by activated T cells, after binding to the B7 molecule present on antigen-presenting/cancer cells, mediates immunosuppression by transmitting inhibitory signals to T-cells. PD-1 is another checkpoint protein present on T cells that usually acts as an “off switch” by attaching to a protein PD-L1 present on the other cells. The PD1-PDL1 interaction signals the activated T cells from not attacking those cells in the body and leaving them. Some cancer cells stimulate the expression of significant amounts of PD-L1, which helps them evade immune attacks. To counter this, drugs called therapeutic monoclonal antibodies that target PD-1 (nivolumab and pembrolizumab) and CTLA-4 (e.g., ipilimumab) are administered with CAR T-cell therapy for cancer treatment. Unfortunately, systemic blockade of immune checkpoints can also cause immune-related adverse effects throughout the body.

CRISPR-Cas9 technology can overcome this challenge by disrupting single or multiple immune checkpoint genes of allogenic CAR T-cells. Thus, avoiding the need to administrate antibodies that target immune checkpoints systemically (Xia et al., 2019; Wu and Cao, 2019; Ou et al., 2021). According to studies, CAR-T cells with disrupted immune checkpoint genes show stronger responses to PD-L1 and B7-expressing cancer cells, resulting in increased anti-tumor activity and prevention of cancer relapse.

An additional challenge faced by CAR-T cells is the immunosuppressive tumor microenvironment (TME) after infiltrating the solid tumor. TME is composed of various chemicals like transforming growth factor-beta (TGF-β), produced by TME and cancerous cells. TGF-β suppresses T-cell function by inhibiting T-cell activation, proliferation, and effector mechanisms. By knocking out the TGF-β receptor II gene in CAR-T cells using CRISPR-Cas9 technology, tumor eradication can become more efficient.

Thus, multiple genes are required to be knocked out to boost the therapeutic potential of CAR-T cells, and disrupting multiple genes is easier to do with CRISPR-Cas technology than with other gene editing tools like ZFNs and TALENs.

The next part 25 of the series, is about the "Therapeutic applications of CRISPR-Cas genome editing in viral infections."

If you liked this article and want to know more about more about applications of CRISPR in gene editing and beyond, click the below links:

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References:

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, 1179.

Wu, H. Y., & Cao, C. Y. (2019). The application of CRISPR-Cas9 genome editing tool in cancer immunotherapy. Briefings in functional genomics, 18(2), 129-132.

Xia, A. L., He, Q. F., Wang, J. C., Zhu, J., Sha, Y. Q., Sun, B., & Lu, X. J. (2019). Applications and advances of CRISPR-Cas9 in cancer immunotherapy. Journal of medical genetics, 56(1), 4-9.

Ghaffari, S., Khalili, N., & Rezaei, N. (2021). CRISPR/Cas9 revitalizes adoptive T-cell therapy for cancer immunotherapy. Journal of Experimental & Clinical Cancer Research, 40(1), 269.

Quazi, S. (2022). Elucidation of CRISPR-Cas9 application in novel cellular immunotherapy. Molecular Biology Reports, 49(7), 7069-7077.

Al Saber, M., Biswas, P., Dey, D., Kaium, M. A., Islam, M. A., Tripty, M. I. A., ... & Kim, B. (2021). A comprehensive review of recent advancements in cancer immunotherapy and generation of CAR T cell by CRISPR-Cas9. Processes, 10(1), 16.

Mollanoori, H., Shahraki, H., Rahmati, Y., & Teimourian, S. (2018). CRISPR/Cas9 and CAR-T cell, collaboration of two revolutionary technologies in cancer immunotherapy, an instruction for successful cancer treatment. Human immunology, 79(12), 876-882.

Razeghian, E., Nasution, M. K., Rahman, H. S., Gardanova, Z. R., Abdelbasset, W. K., Aravindhan, S., ... & Khiavi, F. M. (2021). A deep insight into CRISPR/Cas9 application in CAR-T cell-based tumor immunotherapies. Stem Cell Research & Therapy, 12(1), 1-17.

Ureña-Bailén, G., Lamsfus-Calle, A., Daniel-Moreno, A., Raju, J., Schlegel, P., Seitz, C., ... & Mezger, M. (2020). CRISPR/Cas9 technology: towards a new generation of improved CAR-T cells for anticancer therapies. Briefings in functional genomics, 19(3), 191-200.

Dimitri, A., Herbst, F., & Fraietta, J. A. (2022). Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing. Molecular Cancer, 21(1), 78.

Kang, K., Song, Y., Kim, I., & Kim, T. J. (2022). Therapeutic applications of the CRISPR-Cas system. Bioengineering, 9(9), 477.

Happy learning!