Supervised by: Yuhui Zhou, BA (Hons). Yuhui is a 5th year medical student at the University of Cambridge. She gained a First class degree in her intercalated year studying Pathology. She has an interest in Cancer & Immunology and has been awarded a Wellcome Trust Biomedical Vacation Scholarship to study host responses to infection.
Recent oncological advancements have brought about the use of genetic engineering to specialize cancer treatments to individuals. Amongst the most promising are CAR-T (Chimeric Antigen Receptor T) cells, an up and coming immunotherapy that engineers a patient’s own immune system to fight against cancerous growths. CAR-T immunotherapy has shown particular success against blood-borne cancers such as leukemia and lymphoma, and oncologists are optimistic about the future of such treatments (1). But despite its advancements, there are still many ways CAR-T cells can be improved. Individually, both CRISPR and microbiome research have shown to drastically increase CAR-T efficacy, and are quickly becoming a focus of the oncological community (2, 3). Our study sought to give an overview of such applications, along with CAR-T cells themselves.
Millennia have passed since the first records of cancer research, but oncologists continue to be baffled by the complexities of the disease. As Dr. Siddhartha Mukherjee explains in his biography, Emperor of all Maladies, this “four-thousand year battle against cancer” continues to evolve as scientists innovate radical solutions in attempts to answer one of biology’s most imposing questions: Can cancer be cured (4)? William Stewart Halstead performed radical surgeries that, as effective as they were, deformed human anatomy (5). Sidney Farber reutilized antifolates to develop chemotherapy and Donald Pinkel used extreme radiation therapy to prevent cell replication (6, 7). Yet, despite increasing scales of research, studies still show more than 600,000 cancer-induced deaths in the United States alone, 20 million annual diagnoses worldwide, and an astronomical 10 million accounting for global fatalities every year (8). These high stakes quantify the pressure oncologists face in developing a new and effective treatment, or better yet, a “cure” altogether.
There are several misconceptions regarding cancer, amongst the most prevalent being that it is incurable (9). Innovations in human genome sequencing have shifted the trajectory of oncological treatment through the connection between immunology and cancer. Currently, one of the most promising treatments utilizes a patient’s immune T cells to protect against antigens present on tumors. This approach, better known as CAR-T cell therapy, has seen great success in attacking and destroying blood-borne cancers such as leukemia and lymphoma. CAR-T cells have the potential to revolutionize cancer treatment through both their clinical success and implications on research connecting immune function to oncology (10).
Oncologists turn also to other technologies such as gene editing and immune enhancement to improve the efficacy of CAR-T cell therapies. CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, (or CRISPR- associated nuclease 9, abbreviated as Cas9) is the most common gene editor due to its advances in treating cancers caused by genetic disorders (2). Additionally, an interesting emphasis has been placed on bacteria and microbiome health as studies have consistently concluded that our microbiome plays a critical role in our immune system, negatively and positively, depending on diet and other environmental factors (11). These alternative and complementary studies may individually improve the scope and efficacy of CAR-T cell therapy, as discussed below in more detail. Additionally, included is a review on the ethics of engineering human cells and altering genetic information, something other papers have often shied away from discussing.
CAR-T Cell Therapy and Research
CAR-T therapy has been effectively used for relapsed or refractory tumors, specifically hematological malignancies. Initially, the FDA approved anti-CD19 CAR-T therapy as it yielded impressive results but following it, high relapse rates and resistance in the patients occurred. This has led to further study aimed at solving such problems, along with additional upsets in the therapy. Currently, alterations have been made in the structure and manufacturing of CAR-T cells that have increased effectiveness and tenacity, particularly in the fourth-generation CAR-T cells. When combined with an immune modifier, the fourth-generation and next generation will not be limited because of cytotoxic effects; therefore, allowing it to flourish and overcome the tumor microenvironment, which has been a huge barrier in CAR-T cell therapy. As a result, studies have been made involving the ectodomain, transmembrane domain, and endodomain of the CAR structure in order to improve future development of CAR-T cell therapy.
One challenge was identifying targets to avoid “on target off tumor” effects that can cause damage to healthy tissues and potentially put the patient in a life-threatening situation. Research in “Recent Advances in CAR-T cell engineering” shows the difficulties for antigens like CD123 or CD33 and attempting to use them in treating malignancies of the myeloid lineage because it can be expressed in vital bone marrow stem cells, leading to myelosuppression since the stem cells are also killed (12). Ultimately, the fourth and upcoming-generation of CAR-T cells have been arranged to increase their ability to kill tumors, infiltrate solid tumor tissues, and modify the immune microenvironment (10).
Limitations and Potential Approaches of CAR-T Cell Therapy
As much as experimentation in CAR-T therapy has progressed, much of it concerns B cell leukemia or lymphoma. There are still many limitations that restrict further development and efficacy in solid tumors and hematological malignancies. Some major limitations include limited efficacy against solid tumors, limited persistence, antigen escape, “on-target off-tumor” effects, poor trafficking and tumor infiltration, the immunosuppressive microenvironment, inhibition and resistance in B cell malignancies, and life-threatening toxicities (13).
One of the most difficult limitations in CAR-T cell therapy is antigen escape, which is the development of tumor resistance to single antigen-targeting CAR constructs. At first, single antigen targeting CAR-T cells are able to cause high response rates, but malignant cells of those being treated with CAR-T cells later show partial or complete loss of target antigen expression. For example, 70-90% relapsed and/or refractory ALL (Acute lymphoblastic leukemia) patients had shown a response to CD19 targeted CAR-T cell therapy; however, recent studies revealed the formation of a disease resistance mechanism that had resulted in the loss of CD19 antigen in 30-70% of the patients after treatment. These patterns of resistance have also been found in the research of solid tumors. For instance, a CAR-T cell therapy case found that targeted IL13Ra2 in glioblastoma indicated a decrease in IL13Ra2 expression. As a result, methods for treating malignancies and solid tumors rely on targeting multiple antigens. The clinical trials that used dual-target CAR-T cells (CD19/CD22 or CD19/BCMA) have produced positive results (14).
Another, previously mentioned, is the “on-target off-tumor” effects in regards to targeting solid tumor antigens. Due to the tendency for solid tumor antigens to be expressed on normal tissues, careful antigen selection is necessary for CAR design to restrict “on-target off-tumor” toxicity. One future approach for overcoming this would be targeting tumor-restricted post-translational modifications, for example, solid tumor overexpressed truncated O-glycans, like Tn (GalNAca1-O-Ser/Thr) and sialyl-Tn (STn) (NeuAca2–6-GalNAca1-O-Ser/Thr).
CAR-T cell trafficking and tumor infiltration display the constraints in solid tumor CAR-T therapy since the immunosuppressive and physical tumor barriers limit the effectiveness and mobility of CAR-T cells. A strategy to improve this would be through the use of delivery routes other than systemic delivery as local administration either removes the need for CAR-T cell traffic to disease sites or restricts on-target off-tumor toxicities while the CAR-T cells on-target activity focuses on tumor cells that minimize interaction with normal tissues.
Several cell types, such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and regulatory T cells (Tregs), that control immunosuppression can infiltrate solid tumors in the tumor microenvironment. These infiltrate and the tumor cells direct the manufacturing of tumor facilitating cytokines, chemokines, and growth factors. Furthermore, immune checkpoint pathways can lead to a decrease in antitumor immunity. Since a primary reason for weak or no response to CAR-T cell therapy is insufficient T cell expansion and short-term T cell persistence, there are hypotheses that CAR-T cells and checkpoint blockades will be combined for immunotherapy. However, even this might be inadequate to cause infiltration of T cells.
CRISPR/Cas-9 genetic engineering has become a staple of modern biotechnology over the past few years, and it’s important to know what it actually is before analyzing its applications. Modern CRISPR technology consists of an endonuclease protein with programmable genetic information allowing for short guide RNA to alter its target DNA. Endonuclease protein is a form of enzyme commonly found in bacteria to protect against DNA viruses. The enzyme’s ability to both recognize and cleave target DNA made it the perfect candidate for genetic engineering. Scientists have since been able to program endonucleases to target CRISPR-associated genes on DNA strands, thus manipulating genetic information of eukaryotic cells. This process has been adapted by many biotechnology fields because of its efficiency and ease of use, including CAR-T immunotherapy (15).
Using CRISPR to Reduce CAR-T Cellular Exhaustion Against Tumors
Despite its immense success, CAR-T immunotherapy still has a long way to go before it can be used as a universal cancer treatment. Amongst the most notable barriers is enabling CAR-T cells to target all forms of cancer, tumors and blood-borne alike. While there’s clearly been formidable progress towards the latter, the tumor microenvironment (TME) has proven time and time again to suppress immune function, making it difficult for CAR-T cells to seek and destroy solid tumors. Its immunosuppressant capabilities cause decreased activity and longevity in CAR-T cells, labeling it as a target of ongoing research (16).
A recent study under the American Society for Clinical Investigation (ASCI) sought to solve this problem by eliminating the TGF-ꞵ receptor II (TGFBR2) that regulates the TME and directly binds to TGF-ꞵ (17, 18). Previous research shows that transforming growth factor-ꞵ (TGF-ꞵ) leads to tumor development and cytotoxicity suppression (19, 20, 21, 22). On the basis of this information, the ASCI study sought to connect CAR-T expression to levels of TGF-ꞵ1 in the TME. They discovered that TGF-ꞵ1 decreases CAR-T cell cytotoxicity by activating the TGF-ꞵ receptor. This leads to the dreaded cellular exhaustion that makes it so difficult to engineer tumor-fighting CAR-T cells. With the help of CRISPR, the ASCI study was able to directly edit out the TGFBR2 gene, eliminating these effects and allowing CAR-T cells to be more effective in solid tumor suppression (16).
CRISPR technology has made great progress in overcoming challenges posed by tumorous cancers to CAR-T immunotherapy. By reducing cellular exhaustion, CAR-T cells have the ability to fight for longer, improving both their immediate rates of success and lasting effects. But despite the headway this research has made, more trials are needed before any conclusion can be made. Leading researchers affirm that the TGF-ꞵ receptor signals through many types of cells in the TME to suppress T cell activation (23). The ACSI model doesn’t account for other factors in the TME that might be affected by a lack of TGF-ꞵRII, so it is imperative to collect more information before beginning clinical trials.
With CRISPR technology becoming increasingly mainstream, new opportunities are arising to enhance CAR-T cell function and reduce cellular exhaustion. Although there is certainly more research to account for, CAR-T cells are well on their way to fight not only against blood-borne cancers but tumors as well.
Engineering Cells to Stop CAR-T Activation Inhibition
Another study in 2019 under Shanghai Jiao Tong University sought to enhance CAR-T cell cytotoxicity, particularly against PD-L1 expressing cancer cells. They observed how programmed cell death protein 1 (PD-1) interacted with activated T cells and ligands located on target tumors to inhibit CAR-T cell cytotoxicity. To solve this issue, researchers used CRISPR technology to inhibit the PD-1 receptor. Further experimentation clearly exhibited a greater CAR-T response against cancerous cells expressing PD-L1 after genetic engineering (24).
Similar to the ASCI research mentioned above, the Shanghai Jiao Tong University study based their research off of only a limited model. The study focused on human triple-negative breast cancer cells instead of a variety of PD-L1-expression, so more laboratory experimentation and modeling is necessary before further action.
Ethical Limitations of CRISPR Engineering
With genetic engineering becoming an accepted part of the biomedical field, it’s important to identify the ethical limitations necessary to dictate its applications. A common model to determine biomedical ethicality is based on minimizing risk and maximizing benefit. CRISPR and other forms of genetic engineering have the potential to revolutionize biomedical therapies, and thus save lives. But no matter how beneficial a treatment may be, there are some applications that must be limited to minimize the risk factor involved. In order to maintain the risk-benefit ratio, researchers must identify how much CRISPR should be used, who should have access to it, and if their applications meet international guidelines (25).
In the case of CAR-T cell therapy, CRISPR has been able to eliminate many issues limiting its efficacy, but it is still important to ensure that the benefits outweigh any risk. Using the ASCI experiment as an example, the study neglected various factors making up the TME, leaving risk for unfavorable results in clinical trials. If further research shows that eliminating the TGFBR2 gene may inflict more harm on the patient than it does good as a treatment, such would be an unethical application.
CRISPR has the potential to be a revolutionary medical tool, but it can just as easily fall into unethical territory. With the field evolving so rapidly, regulation should be decided based on maximizing the risk-benefit ratio to ensure the technology is used properly. Strict experimental guidelines must also be met before any decisions can be made about accessibility of CRISPR-based treatments (25).
Humans are host to more than 10 times more bacterial cells than human cells. In other words, humans are mainly composed of bacteria cells (26). Investigating the symbiosis between human and bacteria has become a ‘trend’ in developing medical treatments. Most of the bacteria in the human body is concentrated in the small intestines. In fact, humans have 1.8 kilograms of bacteria in our gut (the human to bacteria ratio is an estimated 100 trillion bacteria cells per one human cell). Studies have found that the relationship between our bacteria and gut is not only mutualistic but critical for human health and, most importantly for this study, for the immune system (27).
The Microbiome and Cancer
The gut microbiome plays a critical role in a person’s immune health mostly due to bacteria that help protein, vitamin, and nutrient synthesis which promotes a healthy immune system and eubiosis, a healthy relationship between the host and biofilm. Rainer J Klement and Valerio Pazienza reveal in their paper the correlation between microbacterium and cancer through their association with short chain fatty acids and different kinds of diets (30). The study concludes that a diet rich in proteins and starved of processed foods will stimulate eubiosis. Eubiosis, they claim, is directly related to the prevention and death of cancer cells. Discussing the Paleolithic, Ketogenic, Low-Carbohydrate, Mediterranean diets as well as fasting, Klement and Pazienza’s review concludes that the diets that potentially prevent cancer have such effects because of its promotion of an eubiotic microbiome. Although the research is limited and contains some conflicting outcomes depending on diet and study groups, all of the research highlights the importance of continuing to study the microbiome as a potential source of prevention of cancer (11).
Immunotherapy, the Microbiome, and Cancer
Immunotherapy has emerged in oncology as a treatment that manipulates the immune system to prevent metastasis and/or tumor growth. The microbiome is now being studied as a complement to immunotherapy as it is proven that being able to improve the efficacy of cancer immunotherapy is directly affected by the microbiome “both locally and systemically” (28). Immunotherapy, in the case of hematologic malignancies, uses the immune checkpoint inhibitors (ICIs) to block the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1) and, of course, the programmed cell death 1 ligand (PD-L1). An obstacle, however, in this research is immunotherapy resistance. Nevertheless, the promising research suggests that the gut microbiome’s effect on the peripheral immune system can be key to improving immunotherapy efficacy (28).
Additionally, the study states that improving immunological response through the gut microbiota may improve the immune and cellular responses, and most importantly, it is estimated that it will also improve immunotherapies, such as CAR-T cell therapy. Since T cells are part of the immune system, diversifying the microbiota can improve immune response which leads to the conclusion that diversifying the microbiota will improve the immune system’s T cells and therefore improve efficacy of CAR-T cell therapy (28).
When the immune system works optimally, T cells locate antigens and kill virally infected cells as well as tumors. Often, the problem with T cells is that they are not able to locate the latter due to its practically identical structure to any other body cell. Tumors, in this case, are able to grow and metastasize because T cells have not recognized them as a threat to the body. As explained previously, CAR-T cell therapy engineers the T cells’ TCR (T Cell Receptor) to locate tumor cells (through APCs, or antigens presenting cells) and then they are able to kill the tumor. Other co-receptors are required to stabilize the antigen peptide, the enzyme that will break down the antigen (28).
How to Improve Immune Response and its Effects on Demographics
Although information correlating the relationship between the microbiome is incredibly limited in correlating the microbiome to CAR-T cell therapy, the connection is inevitable due to the direct effect that the microbiome has on the immune system, as stated above. According to a Harvard Health study, T cell’s, the protectors of the immune system, will be strengthened by avoiding smoking, exercising regularly, getting adequate sleep, minimizing stress, and eating healthfully. Strengthened in this case relates to increasing the T cell count in the body. Interestingly, the demographic with the lowest T cell count are those who cannot afford a variety of healthy foods, are malnourished, or have deficiencies; their cells will not be able to protect them against viruses (27).
The CDC provides that the highest cancer deaths in America are mourned by the black community from 2014-2018. After that come white deaths followed by hispanic, American Indian and Alaska Natives, and lastly Asian and Pacific Islanders. These statistics however consist of diagnosed cancer deaths, they do not account for all the thousands of cancer patients who are undiagnosed due to the costs that come with receiving a medical treatment, meaning that most minorities would go undiagnosed and the rates of death would affect the previous statistics (29).
Both of these studies relate diet and cancer deaths due to the malnourishment of minorities, whether that be because of a lack of education or financial limitations. The studies show that a weak immune system will affect the body’s capability to attack cancer due to the lower efficacy of immune cells to recognize and kill tumors. Studies have repeatedly proven that improving diversity in the microbiota increases the efficacy and number of T Cells which gives the promising hypothesis that it will help T Cell therapy as well.
Using the Microbiome to Improve CAR-T Treatment and Outcome
CAR-T cell therapy has many limitations, one of which includes limited resistance. Limited resistance is often attributed to the weak immune system many cancer patients have that will decrease their T Cell count. Tumors, as stated before, suppress the immune function so conducting T cell therapy will not be as effective since the body is focused on fighting against the tumor, unless the patient’s immune system is exceptionally strong. T cell therapy, in most cases, won’t be as effective if a patient’s immune system is not taken care of (11).
Using the microbiome to improve CAR-T cell therapy is an extremely novel combination but may be a promising additive to the treatment, especially for those who are immunosuppressed. Focusing on dietetics for patients undergoing such treatment may be an effective and easy way to prevent the limitations from halting the treatment. Adding probiotics to one’s diet is proven to improve homeostasis, yet probiotics is a field that is not developed enough to accurately correlate data with oncology. Additionally, as promising as the proposal to study the microbiome and CAR-T cell therapy in relation to efficacy and prevention of cancer and metastasis is, it is quite expensive to test out all the different factors that play out in such a study as it is an additive to the already expensive CAR-T cell therapy. Yet, regardless of the results, it is already proven how much of an advantage the immune system can be put in if the colon is in a eubiotic state (11).
Fourth generation CAR-T cells have clearly made headway towards becoming one of the leading cancer treatments, a goal they may very well reach with the help of genetic engineering (CRISPR/Cas-9) and microbiome-focused applications. CAR-T (Chimeric Antigen Receptor T Cells) have been particularly successful against blood-borne cancers, although there are also many limitations to take into consideration such as CAR-T induced cytotoxicity, B cell cancers, resistance, and limited longevity (10). Efficacy against solid tumors has been a specific point of weakness because of the tumor microenvironment (TME)’s immunosuppressant tendencies. Advances in CRISPR technology have the potential to alleviate this issue by editing out genes that code for such capabilities, as well as improve the longevity of CAR-T cells (16). The microbiome may also play a role in the TME’s effect on CAR-T. Consisting of beneficial bacteria particularly in the gut, microbiome health has been connected to both improvements in immune system activity and decreasing the risk of cancer. Novel treatments have even been proposed that combine CAR-T and microbiome treatments to improve both efficacy and longevity of the treatment as a whole. With so much promising research, CAR-T cells, CRISPR, and the microbiome have the potential to revolutionize oncology.
Many literary reviews have focused on accumulating data about CAR-T cells as a whole or individual applications of CRISPR or the microbiome. ‘Engineering CAR-T Cells for the Next Generation of Cancer Therapy,’ a paper under the Parker Institute for Cancer Immunotherapy and the Cancer Research Institute, provided a thorough accumulation of ongoing CAR-T research (10). Another study titled ‘Use of Cell and Genome Modification Technologies to Generate Improved “Off-the-Shelf” CAR-T and CAR NK Cells’ gave an outline of CRISPR/Cas-9 applications while ‘Gut Microbiome and CAR-T Therapy’ connected the microbiome as another feasible treatment (2, 11). In contrast, our paper focused on accumulating an overview of CAR-T cells and some of the most promising applications of the microbiome and genetic engineering. We discussed the ethics of these novel therapies and how to determine limitations on future innovations in this field of CAR-T immunotherapy. In taking a general approach, many of our explanations may be oversimplified, leaving the possibility for misinterpretations and gaps in understanding.
This study sought to provide an overview on CAR-T cells along with their CRISPR and microbiome applications. With regard for ethical limitations, we intended to shed light on the boundaries of immunotherapeutic research that may be overlooked in other papers of similar nature. Our approach also calls into question the effects of combining applications of CAR-T cells, CRISPR, and the microbiome into one. We hope that future studies can answer our question and bring about promising results in improving CAR-T efficacy.
CAR-T cell therapy, though effective against relapsed or refractory tumors, is currently limited to only certain cancers due to many difficulties that lead to failure of the T cell either discovering or destroying the malignant areas. To add, there are many limitations—such as cell-associated toxicities, limited efficacy against solid tumors, inhibition and resistance in B cell malignancies, antigen escape, limited persistence, poor trafficking and tumor infiltration, “on-target off-tumor”, and the immunosuppressive microenvironment—that hinder further development in solving these problems, which is why utilization of technology and a deeper understanding of tumors is necessary for advances in CAR-T therapy. As of now, CRISPR genetic engineering has been applied in CAR-T cell research as a means of better identifying the target DNA more precisely. Although more clinical trials are required to support this research, CRISPR technology has shown to improve CAR-T cells’ successes and persistence by reducing cellular exhaustion. The genetic engineering, CRISPR, used in CAR-T cell therapy has shown the possibilities that it wields not just for cancer treatment, but medicine in general.
For the immune system to have the capabilities of fighting malignancies, a healthy gut microbiome is necessary. Studies in Klement and Pazienza’s review state that eubiosis has the ability to prevent and kill cancer cells; furthermore, certain diets can bolster a eubiotic microbiome. By combining the microbiome with CAR-T cell therapy, treatment for immunosuppressed patients may benefit, since it inhibits the limitations from stopping the treatment. Additionally, the microbiome has direct relation to the effectiveness and prevention of cancer. Even though further research in this field has potential, it will be quite costly to conduct. Overall, CAR-T cell therapy, with the aid of additional research and other fields of science, has potential to break through the challenges it currently faces.
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