Cancer is the second leading cause of death worldwide. Many research and development schemes have been developed to treat cancer over the years.
Recently, the combination of CAR-T cells and CRISPR for cancer immunotherapy has shown great promise.
This article reviews CAR-T cells and CRISPR technologies, explains the rationale behind combining them, and goes through the milestones associated with this innovation.
CAR-T cell technology explained.
CAR-T cell is the technology of engineering T lymphocytes to express chimeric antigen receptors (CARs). Chimeric receptors bear both antigen-binding and T-lymphocyte activity on a single receptor. The specificity and activation of CAR-T cells result from the ability of chimeric receptors to effectively recognize extracellularly a specific antigen target in tumor cells and the intracellular signaling pathway of TCR for the activation of T-cells.
These “living drugs” are made by collecting T cells from the patient and engineering them in the laboratory to produce CARs. These cells are then introduced to cancer patients (allogenic or autologous), expressing cytotoxic properties that help patients’ immune system survive the immunosuppressive tumor microenvironment and attack the cancerous cells with the target antigen on their surface.
The cytotoxic properties include interleukin triggering and cytokine production. When CAR-T cells are engineered to harbor either of CD4/CD8 receptors, anti-tumor effects are further enhanced.
Approved CAR T-cell products available on the market.
Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykti are drugs available in the market for treating hematologic malignancies.
Despite certain limitations (allogeneic vs. autologous transplantation-related drawbacks, cytokine release syndrome, toxicities, manufacturing issues, high cost, and more), CAR-T cell technology is a promising technology that has brought great joy to scientists, opening new roads for research and development of effective anti-cancer therapies.
CRISPR technology, in simple words.
The CRISPR-Cas9 system comprises the Cas9 enzyme and a designed RNA guide (gRNA) that targets specifically a genetic sequence. Once inside the nucleus, the gRNA pairs with the target, and Cas9 cuts downstream of the bound gRNA sequence. This activates the repair mechanisms in the cell, which corrects the cut and introduces a desired mutation to correct the initial defective gene. There are various types of CRISPR-Cas9 systems, such as nickases, which are less prone to errors but less effective than traditional CRISPR-Cas9 systems. The selection between the two depends on several factors.
CRISPR-Cas9 revolutionized accuracy, efficacy, low cost, and speed in gene targeting technologies and is meant to be routinely used in the future. The challenge till then is finding ways to eliminate “off-target” effects and tackle repair machinery errors that could result in unexpected mutations.
Teaming up CAR-T cells and CRISPR revolutionizes cancer immunotherapy.
CRISPR is an editing technology and CAR-T cells are cells can now be created with this technology. Teaming them up can revolutionize cancer immunotherapy, increasing efficiency, speed, and cost/effectiveness ratio.
Viral vectors were used for CAR T-cells generation. Using CRISPR technology instead of viral vectors, eliminates the associated viral vectors barriers, like random integration, insertional mutagenesis, stable expression, and immune rejection.
Additionally, with CRISPR, scientists can edit more than one receptor much faster. Also, CRISPR-edited CAR-T cells’ expansion, production, and storage are relatively easy.
Finally, there is the hope that CRISPR will broaden the applications of CAR-T cells beyond hematologic malignancies to solid tumors.
Milestones in the CAR-T cells and CRISPR combinational therapy.
The first trial that utilized CRISPR-cas9 modified T-cells (PD-1 edited T-cells) was conducted in 2016 to treat lung cancer. Although this was not a traditional CAR-T cell therapy, it provided deep knowledge and understanding of how to use CRISPR technology in humans.
By then, several trials combining the two technologies are under clinical development, with most targeting CD19 antigen.
Below we will talk about some trials that utilize the two technologies for the treatment of B-cell malignancies as well as the treatment of solid tumors.
- CRISPR-CAR-T cells for B-cell malignancies
CARBON-NCT04035434 clinical trial: At the 64th Annual Meeting of the American Society of Hematology (ASH) in New Orleans in December 2022 the advancements in hemophilia and hematology-related oncology were presented.
Among them were the promising results from a Phase-I clinical trial (CARBON- NCT04035434) on CTX110, a new asset developed wholly by CRISPR Therapeutics.
The results indicated interesting tolerability endpoints and a durable complete response for almost half of the cohort.
32 adult patients with B-cell malignancies were treated with the CRISPR-engineered CAR-T cell infusion. The key results showed that the infusion(s) was well tolerated; 67% of the cohort and 41% showed overall response rate (ORR) for certain doses and complete response (CR), respectively.
The duration of response (DOR) was almost 6 months for nearly half the cohort. Cytokine Release Syndrome (CRS) was the most common adverse event, and a (non-insignificant!) 22% of patients experienced serious adverse events (SAEs) because of the treatment. However, this study is still promising and ongoing and CTX110 will be evaluated in an expansion phase.
19(T2)28z1xx TRAC CAR-T cells clinical trial: Another trial that started on February 23, 2023, is the phase I NTC05757700 from Memorial Sloan Kettering Cancer Center with Takeda as a collaborator to test the efficacy of 19(T2)28z1xx TRAC CAR-T cells in B-cell lymphoma. Here CAR-T cells are boosted with the insertion of 1XX molecule and the TRAC location, which offers CAR positioning at a specific location in the chromosome. This way, the activity of CAR is more “controlled” and long-lasting.
Interim phase I results were announced in the December 2022 Annual Meeting of the American Society of Hematology, showing a safe and effective treatment at low doses with a complete response rate of 69% in a median follow-up of 155 days.
- CRISPR-CAR-T cells for solid tumors
CRISPR Therapeutics has been developing proprietary CAR-T cells/ CRISPR-Cas9 edited products, like CD70 — targeting CTX130 for types of cutaneous T-cell lymphoma (CTCL) — which accepted an FDA Regenarative Medicine Advanced Therapy (RMAT) designation.
CAR-T cells/ CRISPR-Cas9 edited products are tested in clinical trials for solid tumors: the relapsed or refractory renal cell carcinoma (RCC), CD38-targeting CAR-T product in the pre-clinical stage, and CTX112 (a next-generation CTX110).
Additionally, Dr. Krishanu Saha, Professor of Biomedical Engineering at the University of Wisconsin-Madison, and his team engineered T cells to target a sugar molecule in neuroblastoma solid tumor cells. The results were very promising in mouse models, and the development of a non-viral CAR-T product is among the next steps.
- PACT CAR-T cells clinical trial for solid or non-solid types of tumors
Another trial that combines the two technologies is the Phase 1a CAR-T cell/ CRISPR clinical trial NCT03970382 (suspended for business reasons) by PACT Pharma. The development of autologous, PACT CAR-T cells through a rather massive screening and engineering pipeline that generated custom-made, high-precision antibody vehicles to attack many different solid or non-solid types of tumors was a breakthrough and brought the first spark of excitement.
That was a personalized treatment approach at its finest; the first step was screening the patients in the cohort by identifying unique antigens (neo-epitopes) from the tumor. A complex pipeline was applied following the biopsy collection, isolation of peripheral blood mononuclear cells (PBMCs), and extraction of mutations (from whole exome and RNA sequencing data), resulting in the ultimate selection of TCRs (T cell chimeric receptors) that recognize a median of 5 unique non-somatic mutations.
The neoTCRs were inserted into the CD4 and CD8 T cells of healthy donors using the CRISPR method. Finally, up to three neoTCRs per patient were selected for clinical application.
The primary endpoints of the clinical trial were the feasibility of neoTCR identification and CRISPR-edited CAR-T cell production, and secondary endpoints were the objective response rate, duration of response, and overall survival. All sixteen infused patients tolerated the product well, and the safety profile was good. But, regarding efficacy, five showed no change in the disease status, whereas eleven had a disease progression. Despite meeting the primary endpoints, there is still a long way to go regarding efficacy and duration.
What holds the future for the CAR T cells – CRISPR/Cas9 “marriage”?
Researchers and pharma (or biotech companies) focus on safe and effective immunotherapy, and precisely engineered CAR-T cells hold the promise to get there. There are high expectations for the CRISPR/Cas9 and CAR-T combination to flourish.
The limitations and challenges, especially in clinical translation, show the difficulty and risk of the venture, calling for cautious steps from the few key players.
Despite the obstacles, there are already applications in solid tumor therapies. The future of immunotherapy is promising and lies in this highly precise, personalized approach.
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Causes of death – Our World in Data
Milestones in Cancer Research and Discovery – NCI
Driving the next wave of innovation in CAR T-cell therapies | McKinsey
CAR T Cells: Engineering Immune Cells to Treat Cancer – NCI
CRISPR CAR-T cells: Edited T Cells Are Revolutionizing Cancer Treatment
Chinese scientists to pioneer first human CRISPR trial | Nature
CRISPR CAR-T Insights | April | Airfinity
https://stemcellres.biomedcentral.com/articles/10.1186/s13287-021-02510-7/figures/2
Editorial Article: CRISPR-generated CAR T-cells: A new frontier in the treatment of solid tumors
PACT Pharma culls lead asset, diverts 54 staffers to new venture
Neoantigen TCR T-cell Therapy Shows Early Proof-of-Concept in Solid Tumors
Non-viral precision T cell receptor replacement for personalized cell therapy | Nature
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