Abstract

CAR T-cell therapy is a type of immunotherapy that modifies T cells, allowing them to identify and eliminate cancerous cells. As T cells in the thymus keep reproducing, they turn into helper, cytotoxic, memory, and regulatory T cells. This paper examines the functions of these cells in CAR T-cell therapy. In addition, it explores the limitations of CAR T-cell therapy. To this end, an analysis of the reasons why the therapy might fail, why it is not yet a first-line form of cancer treatment, and the complexity of controls that patients must undergo for effective results is undertaken. This paper then discusses additional medications administered to patients as part of CAR T-cell therapy and how to mitigate their accompanying side effects. Lastly, future improvements to CAR T-cell therapy are addressed that may offer cancer patients hope for recovery. 

Background Information 

T cells are types of white blood cells in the human body that aid in immune system functioning. T cells start out in the bone marrow and mature in the thymus (Editors of Encyclopaedia Britannica). There are various types of T cells that promote proper immune system functioning. For instance, helper T cells are important to cell immunity because they are a type of white blood cell and lymphocyte that stimulates killer T cells, macrophages, and B cells to make immune responses (National Cancer Institute Staff). These helper T cells begin to initiate activity once antigens are presented. Upon antigen presentation, helper T cells then multiply and secrete cytokines that send for macrophages and cytotoxic T cells to the infected site. Cytotoxic T cells are also called CD8+ cells. Their primary functions consist of identifying and eliminating antigens (National Cancer Institute Staff). As a result, Cytotoxic T cells are instrumental in eliminating bacteria, infected cells, and as cancerous cells. Cytotoxic T cells achieve these results through a process called apoptosis. Apoptosis occurs when the death of cells transpire as a normal and controlled part of an organism’s growth or development (Venkatachalam et al.). To implement apoptosis, Cytotoxic T cells bind to antigens. The holes created through this binding process are made by excreted perforin. “Perforin is a glycoprotein responsible for pore formation in cell membranes of target cells. Perforin is able to polymerize and form a channel in target cell membrane” (Osinska et al.). This enzyme promotes apoptosis, resulting in cell death. 

Memory T cells are also a crucial dimension of the human immune system. “Memory T cells are antigen-experienced cells that mediate a faster and more potent response upon repeat encounter with antigen. These cells are long-lived and when developed following an infection can protect against subsequent infections with the same pathogen” (Miller et al.). Memory T cells appear after pathogen removal, and are among the most important T cells. They are responsible for antigens that are reintroduced to the body and building an immunological tolerance. These Memory T cells are also significant because they have the special ability to expand into a large number of Effector T cells upon exposure to familiar antigens. Effector T cells are defined as relatively short-lived activated cells that defend the body in an immune response (Editors of Encyclopedia Britannica). Indeed, it is the presence of such cells that can account for why vaccines create immunities in the human body. These cells also help explain why people can recover from illnesses such as the common cold much faster than if an antigen was introduced into the body for the first time. Lastly, Regulatory T cells “...are a specialized subpopulation of T cells that act to suppress immune response, thereby maintaining homeostasis and self-tolerance. It has been shown that Tregs are able to inhibit T cell proliferation and cytokine production and play a critical role in preventing autoimmunity” (Kondelkova et al.). Once Cytotoxic T cells and Helper T cells remove an antigen, they are prevented from undertaking further action by Regulatory T cells. Without the assistance of Regulatory T cells, T cells, and cytotoxic cells can harm and destroy healthy cells.

To implement CAR T-cell therapy, T cells are extracted from a patient’s blood and then taken to a laboratory. The way that T cells are extracted is through a blood filtration process called leukapheresis. Leukapheresis is defined as the removal of a patient’s white blood cells from the circulating blood (Totapally). T cells are then preserved by cooling samples to very low temperatures, through a process called cryopreservation. Cryopreservation is preservation (as of cells) by subjection to extremely low temperatures (Merriam-Webster Staff). Then the cells are shipped to a laboratory wherein technicians change these T Cells by adding a gene for a receptor. Viral vectors are used in this step to genetically encode T cells to recognize cancer cells and their antigens. 

This receptor for which T cells are encoded is called the chimeric antigen receptor, otherwise known as CAR (American Cancer Society Staff). CAR is comprised of three proteins. One of the proteins is used to recognize the antigens on cancer cells. The other two of these proteins are for signaling the T cell to activate when the first protein attaches to an antigen on the cancer cell (Portell). Antigens are found on the surface of specific cancer cells and are substances that the body recognizes as detrimental (National Cancer Institute Staff). When the protein CAR is combined with T cells it creates CAR T-cells. CAR T-cells then duplicate outside the patient’s body through a process known as mitosis. At this point, a quality check is undertaken so that laboratory technicians can ensure that CAR T-cells are able to locate and eliminate the antigens on cancer cells with extreme precision.

The production of CAR T-cells is a very delicate and careful process that requires multiple steps of precision. In the beginning of the process, leukapheresis is used to withdraw blood from the patient’s body, separate the leukocytes, and then reinsert the remaining blood into circulation. Then the next step is removing the cells from the anticoagulant-containing leukapheresis buffer. After counterflow centrifugal elutriation, which divides cells based on size and density and retains cell viability, lymphocytes can be enriched (Levine et al.). 

Scientists have found a way to purify autologous antigen-presenting cells (used to activate the cells) without making the process labor intensive. One of the solutions were beads engulfed by anti-CD3 and anti-CD28 monoclonal antibodies. Although anti-CD3 antibodies have been used alone or in combination with feeder cells and growth factors, such as IL-2, for many years, the activation and ex vivo expansion are subpar compared to beads coated with anti-CD3/anti-CD28 monoclonal antibodies or cell-based artificial APCs (aAPCs) (Levine et al.). 

Viral vectors are used by molecular biologists and act as an aid/mechanism that has the ability to transfer genetic material into the body’s cells. The viral vector encoding the chimeric antigen receptor is incubated with the T cells during the activation phase, and after a few days, the vector is removed from the culture by dilution and/or changing the media (McBride). Viral machinery is efficiently used by the body, in which it produces proteins that can attach to the body’s cells. It also is used to produce polypeptides that are necessary for viral replication. The steps for viral replication are attachment, penetration, uncoating, replication, assembly, and release. The viral vector attaches to the patient’s cells using viral machinery, and once within the cells, it releases RNA that contains genetic material. This genetic material encodes the CAR in the context of CAR T cell therapy.

The CAR expression is maintained while the patient’s cells divide and multiply in the bioreactor because the RNA is reverse-transcribed into DNA and permanently integrated into the genome of the patient’s cells. The patient’s cells then translate and express the CAR, which is expressed on the cell surface (Levine et al.). These CAR T-cells are now prepared to attack the cancerous antigens in the patient’s body.

The next step of CAR T-cell therapy implementation is known as lymphodepleting chemotherapy. Lymphodepleting chemotherapy decreases the number of T cells in one’s body to make room for the new CAR T-cells (Bristol-Myers Squibb Staff). This type of chemotherapy is implemented to reduce the level of white blood cells in the patient’s body so that it can accept the reprogrammed CAR T-cells. These CAR T-cells are then reintroduced to the patient through cell infusion. This allows CAR T-cells to traverse the body to search and destroy cells that carry the specific antigens for which the CAR T-cells were programmed to detect. Receiving lymphodepleting chemotherapy results in having a low count of white blood cells. This puts the patient at a high risk for infections and for this reason they are administered different types of treatment for side effect mitigation.

Other types of chemotherapy administered at this point in the CAR T-cell process may result in side effects similar to those described above. For example, bendamustine is one type of lymphodepleting chemotherapy that reduces the replication of cancer cells, making the cancer less active. The side effects for this part of the treatment are fatigue, low blood counts, and infusion reactions. Infusion reactions are a specific kind of hypersensitivity reaction that occurs during or soon after the delivery of the treatment (McBride). Fludarabine is a treatment that mainly is used to treat a specific type of Leukemia called chronic lymphocytic leukemia (CLL). This type of cancer occurs when the bone marrow makes too many lymphocytes, the side effects of this treatment also include low blood counts, as well as tingling, numbness, nausea, and poor appetite (Staff at HOPA). The third type of treatment for lymphodepleting chemotherapy is cyclophosphamide, which is mainly used to treat cancer of the ovaries, breast, lymph system, blood, and nerves, and multiple myeloma (Staff at Mayo Clinic). The most common side effects of cyclophosphamide include, but are not limited to, low blood counts, hair loss, bladder iration, and mouth soreness.

During lymphodepleting chemotherapy and CAR T-cell therapy, the patient is treated with certain medications that can help to limit adverse consequences. The first medication is consumed orally and is called allopurinol. Allopurinol helps with kidney damage, and the patient will be on this medication for at least one week during the beginning of lymphodepleting chemotherapy. Fortunately, antibiotic medications help with averting potential infections and are typically administered after treatment has been received. In turn, this regimen can lower the potential for infections. Anti-seizure medicines are also given; the duration of which is approximately thirty days and typically starts upon commencement of CAR T-cell therapy treatment. Antiviral and antifungal medications are the last type of medications that are both simple and do not contain many side effects. Antiviral medications can help prevent the reactivation of simplex herpes virus (HSV). Antifungal medications help prevent infection with a fungus after the CAR T-cell therapy treatment (Staff at Bristol-Myers Squibb). 

Limitations of CAR T-Cell Therapy

Overview

There are several reasons that can help explain why CAR T-cell therapy may fail. One reason is related to the intrinsic factors of tumors. In order for CAR T-cell therapy to retain its efficacy, CAR T-cells must bind onto the CD 19 epitopes of cancer cells. Epitopes are the part of the antigen upon which antibodies can attach themselves (Mahmoudi Gomari et al.). A lack of CD 19 on the cell surface is present in 10-20% of patients with the Acute lymphocytic leukemia (ALL) cancer subtype. Additionally, CD 19 is absent in 27% of lymphocyte cancer patients. Host-related factors also pose limitations for CAR T-cell therapy. The premise behind this limitation is that a patient’s previous treatments can alter the effectiveness of CAR T-cell therapy treatment. This phenomenon is known as CAR T-cell exhaustion (Tang et al.). Unfortunately, there is still a relative lack of data concerning why CAR T-cell exhaustion occurs, or which treatments can alter the effectiveness of CAR T-cell therapy. 

Inadequacy of CAR T-cell therapy may pose additional obstacles to effective CAR T-cell therapeutic interventions. Inadequacies can occur when the structure of the CAR T-cells affect response outcomes. If the CAR T-cell is not shaped in an appropriate way to attack a given antigen, the CAR T-cell can become unusable and inefficient. This inadequacy can be evident in certain types of cancers. “Although treatment with CAR T-cells has produced remarkable clinical responses with certain subsets of B cell leukemia or lymphoma, many challenges limit the therapeutic efficacy of CAR T-cells in solid tumors and hematological malignancies” (Sterner). 

Obstacles To Widespread CAR T-Cell Therapy Usage

T cells are collected during a pause in cancer treatment when oncologists have determined a lull in cancer cell activity. If cancer cells are active, then increased symptoms can occur when the T cells are extracted from the patient (American Cancer Society Staff). For certain types of cancer, this can be the most difficult part of the process. In light of this, only a certain number of patients are eligible for CAR T-cell therapy. Specifically, CAR T-cell therapy is recommended after at least three forms of cancer treatments have previously been undertaken on the patient. People who are up to 25-years-old and have the type of cancer called ‘Leukemia B Cell ALL’ are eligible for CAR T-cell therapy (National Cancer Institute Staff). Moreover, adults who have specific types of lymphoma are also eligible for this therapy. These types of lymphoma are B cell lymphoma, primary mediastinal B cell lymphoma, or mantle cell lymphoma (National Cancer Institute Staff). To date, there are approximately 200 adults who receive CAR T-cell therapy per year in the UK alone (Cancer Research UK Staff). 

Antigen Escape

Antigen escape is one of the most difficult limitations for CAR T-cell therapy. In the first instance of the infusion therapy process, it can seem that single antigen targeting would have a guaranteed effective rate of binding since it delivers high responses once presented to the body. In most cases when receiving CAR T-cell therapy, the patient’s malignant cells will exhibit a loss of target antigen expression that is either partial or total. 

Malignant cells are cancerous and will spread and divide. This is particularly problematic because there are a vast number of such cells. As the cell becomes more immune to the chimeric antigen receptor, the less successful the engineered CAR T-cell will be on the patient. ‘Antigen Escape’ is the term used to describe this phenomenon. Tumor cells can avoid being killed by expressing alternate variants of the target antigen that do not include the extracellular epitopes recognized by CAR T cells (Magzener and Mackall). The majority of patients with acute lymphocytic leukemia have been resistant to CD 19. In 30-70 percent of patients who experience recurrent disease following treatment, recent follow-up studies imply development of a common disease resistance mechanism, including downregulation/loss of CD 19 antigen (Sterner).

The malignant cells of a sizable fraction of patients treated with these CAR T-cells show either partial or complete loss of target antigen expression, despite the fact that single antigen targeting of CAR T-cells initially have the potential to produce high response rates. This phenomenon is commonly referred to as an antigen escape process. “For example, although 70-90% of relapsed and/or refractory ALL patients show durable responses to CD19 targeted CAR T-cell therapy, recent follow-up data suggest development of a common disease resistance mechanism, including downregulation/loss of CD19 antigen in 30–70% of patients who have recurrent disease after treatment” (Sterner). Unfortunately, B-cell maturation antigen (BCMA) expression has been shown to be downregulated or lost in multiple myeloma patients who have received CAR T-cells that target such antigens.

Solid tumors have also shown similar antigen escape resistance patterns. “For example, a CAR T-cell therapy case report that targeted IL13Ra2 in glioblastoma suggested that tumor recurrences displayed decreased IL13Ra2 expression” (Brown et al.). In response, several tactics have been deployed to target numerous antigens that lower recurrence rates in CAR T-cell therapy of both hematological malignancies and solid tumors. These methodologies use either dual CAR constructions or tandem CARs, which are single CAR constructs with two single-chain fragment variables that target several tumor antigens at the same time. “Clinically, it appears that both of these strategies may result in prolonged durable remission rates, and there are several CD19 and CD20 or CD 19 and CD22 clinical trials” (Sterner). Preliminary findings from clinical trials that use dual-targeted CAR T-cells (CD19/CD22 or CD19/BCMA) have been positive. Specifically, these effective trials have included adult patients with ALL and diffuse large B cell lymphoma. “Furthermore, preliminary results of BCMA/CD19 targeted CARs in the treatment of multiple myeloma suggest BCMA/CD19 targeted CARs are highly efficacious with favorable safety profiles” (Sterner). 

Several tandem CARs have been explored in preclinical models in solid tumors. These tumors include HER2 and IL13Ra2 in glioblastoma and HER2 and MUC1 in breast cancer (Sterner). Fortunately, dual targeting has produced greater anti-tumor responses in both cases when compared to single targeted treatments. “In the glioblastoma study, CARs targeting HER2 and IL13Ra2 led to improved anti-tumor activity and decreased antigen escape when compared against two other dual-targeting therapies” (Hegde et al.). Such studies reinforce the need to improve target antigen selection. This, in turn, can increase not only antitumor responses but also antigen escape mechanisms and ultimately avoid tumor recurrence.

Attachment of Antibody and Antigen 

Single chain fragment variables (scFv) are created by fusing the variable heavy and light chains of a monoclonal antibody through the implementation of a peptide linker (Sterner). ScFvs play a crucial role in the process of an immune response and are produced from a phage display. This phage display-based production process happens when antibodies are combined on a strand of DNA. The DNA strand is then covered by a protein coat made from the virus. Lastly, these antibody genes combine to make a hat-like structure, which is then placed on top of the virus coat (Sterner). Each antibody is unique and binds to a specific disease target, and this is why they are very effective and play a significant role in the immune response process. “The scFv impacts CAR function beyond simply recognizing and binding the target epitope” (Sterner).

The binding of the antigen to the antibody is not a simple process and requires a copious amount of specificity. There are multiple interactions through various role players in the process of binding. Interactions between the variable heavy chain and variable light chain incorporated with the complementarity-determining regions’ relative positions alter the affinity and antigenicity of the CAR for its target epitope. An epitope is a molecular region on the surface of an antigen that is able to elicit an immune response and combine with the specific antibody produced by such a response (Merriam-Webster Staff).

As an important concept in the binding process, affinity refers to the total strength of an antibody’s attachment to an antigen. The CARs antigen binding affinity must be sufficiently high to identify antigens on tumor cells, initiate CAR signaling, and activate T cells (Sterner). When combined, these aforementioned processes comprise the reason why the binding of the CAR to its target antigen can be optimized to its fullest potential. 

CAR T-Cell Trafficking 

CAR T-cell trafficking is another limitation for CAR T-cell Therapy. Hematological malignancies are cancers that begin in the bone marrow or that are incorporated with the immune system in some capacity. “Compared to hematological malignancies, solid tumor CAR T-cell therapy is limited by the ability of CAR T-cells to traffic to and infiltrate solid tumors as the immunosuppressive (partial or complete weakening the immune system) tumor microenvironment and physical tumor barriers such as the tumor stroma (structure keeping the tumor tissues together) limit the penetration and mobility of CAR T-cells (Sterner).”

The reason that CAR T-cell trafficking can be considered as a limitation is because there is a deep dependency on the CAR T-cells to traffic and navigate their path to affected cells. The way to address this problem is to utilize delivery routes, rather than a systemic delivery process. Systemic delivery, a method of distribution that directly enters the bloodstream to reach and impact cells throughout the body, is a procedure that has an impact on the entire body (Law Insider Staff).

Tumor penetration is the time and action of a substance making its way into the tumor. Tumor penetration is limited by the tumor stroma which is a physical barrier in the CAR T-cell therapy process. For the CAR T-cell to pass through the tumor, it needs to express heparanase. Heparanase is an enzyme that is excreted by cancerous cells and can degrade the heparin sulfate proteoglycan (HSPG). HSPG needs to be degraded before entering the tumor. CAR T-cells that express harperenase may provide an amplified filtration process and antitumor activity. In the future, when dealing with complex solid tumors, medical researchers need to increase efficiency standards so that this process can become better organized and systematic. 

Immunosuppressive microenvironments contain many immune cells that are designed to combat antigens in the human body. These immune cells include natural killer cells and CD8+ T cells, which have tumor-suppressive properties, as well as certain tumor-promoting cells with immunosuppressive properties (Yingying Xing et al.). This microenvironment is critical because it is responsible for fighting cancers and viruses in the body, thereby boosting immunity.

Side Effects of CAR T-cell Therapy

There are possible side effects to every form of cancer treatment, and some effects are more common than others. The common side effects are high fever and chills, hypoxia, severe nausea, vomiting, and/or diarrhea, feeling dizzy or lightheaded, headaches, tachycardia, fatigue, andathralga, and myalgia (American Cancer Society Staff).  

The most common side effect is CRS, which translates to Cytokine Release Syndrome. CRS occurs when the immune system reacts more strongly than necessary to an infection or an immunotherapy treatment. The majority of patients experience it. According to Rutgers Health, 70-90% of patients experience CRS, but the upside to this is that it only lasts for up to a week.  Patients liken the feeling of having CRS to a severe flu, joint pains, body aches, and fatigue (Rutgers Cancer Institute of Technology Staff).

These side effects occur because the body has been trying to fight back antigens using the engineered CAR T-cells, which then causes an immune response. There are also similar side effects for lymphoma patients receiving CAR T-cell therapy. To start out, patients receive a low dosage of chemotherapy, which can have additional side effects such as exhaustion and cytopenia, to prepare the body for CAR T-cell therapy. Due to the fact that this treatment may affect the brain, CAR T cell-related encephalopathy syndrome (CRES) can occur. Defined as a form of disease that alters brain function or structure, CRES’ side effects tend to be clinically less frequent but more severe. Patients who are diagnosed with CRES may feel disoriented, agitated, unaware, or have trouble identifying their relatives and friends (Carter).

Side Effect Management 

There are ways that the fevers, breathing, and body aches, along with CRS, can be managed at the hospital. For CRS, which is the most severe side effect, there are multiple agents that can be used depending on symptom severity. One example is tocilizumab; tocilizumab blocks the inflammatory proteins which helps joint pain and swelling of the patient’s body.  Corticosteroids are another example; corticosteroids are medications that are man-made that closely imitate the hormone cortisol, which is naturally produced by the adrenal glands. Cortisol is important to the body as it releases glucose so the patient can stay on high alert (Staff of Cleveland Clinic). If the patient is experiencing severe hypoxia, they may receive oxygen therapy. This generalized form of using oxygen as a medical treatment through a pressurized chamber can increase the amount of oxygen that one’s blood can carry. 

These are some of the different approaches to treat CRS. Every patient does not receive nor need the same amount of doses and it is determined solely on the condition of the patient, and the severity of the CRS grading. These medications given to help these side effects are not over-the-counter, and overdosing can be deadly. This is why doctors working with patients after CAR T-cell therapy need to watch diligently and make sure that patients receive the optimal resources needed. 

CRS gradings can vary on a scale of four levels. Moreover, the type of management approaches that the doctors take on the patient are decided based on a given CRS grading. CRS grade level 1 means that a patient has flu-like symptoms, a body temperature of 100.4 degrees or higher, and nausea. Doctors would treat this with a broad spectrum antibiotic, and antipyretics (medications that reduce fever). 

CRS grade level 2 has worse symptoms as the patient starts to experience hypoxia, which then is controlled by a low-flow nasal cannula. This medical device is used to deliver increased oxygen to a patient and can be adjusted to different settings. On its lowest setting, this antidote delivers 4-6 liters of oxygen per minute. The patient is also typically transferred to an Intensive Care Unit, given a low dose vasopressor, and administered 8 mg/kg of tocilizumab, which helps in blocking inflammatory proteins. 

CRS grade level 3 manifests when the patient experiences hypotension, which requires the patient to have one vasopressor. The hypoxia level is worse than level 2 as the patient is required to have a high-flow nasal cannula. The dose of tocilizumab is repeated and a low dose of corticosteroids (anti inflammatory medicine) can be given as well. 

CRS grade level 4 has every CRS grade level condition mentioned above to extreme measures. Since the level of hypoxia is severe, a continuous positive airway pressure (CPAP) device may be implemented. The CPAP device can create positive airway pressure through a machine attached to a mask for the patient to wear. The level of hypotension that the patient is experiencing requires multiple vasopressors. High doses of corticosteroids are given to the patient along with the doctor considering any further individual treatment.

The Future of CAR T-cell Therapy

Scientists are finding new ways to efficiently infuse the patient’s CAR T-cells through the use of allogeneic CAR T-cells. This process involves extracting T-cells from human donors. The influx of allogeneic CAR T-cell donors has led to an immediate availability of cryopreserved batches for patients. When T-cells are taken out of the body for a long period of time, the cells lose their ability to replicate. This means that there will likely be a major decrease in effectiveness, causing the CAR-T therapy to fail (Staff at Penn Medicine). This is why having an immediate availability of donations is a great advantage. In general, the newer the cells are, the more efficiently they will work in the body. Using an allogeneic approach also means that CAR T-cells are quickly available for use, although patients who are located further away from major metropolitan areas (where clinical trials are offered) can have a longer waiting time. 

Advantages of using allogeneic CAR T-cells from donors are the ability to standardize CAR T-cell production, increased time for multiple cell modifications, redosing or recombination of CAR T-cells directed against different targets, and a decreased cost that results from using an industrialized process. As of the publication date of this paper, the cost of CAR T-cell therapy can typically range from $373,000-$475,000 and, in some cases, can exceed one million dollars (Staff at Penn Medicine). Since each patient must have a customized treatment, the engineering of T-cells is an expensive and time-consuming process. Shortening the manufacturing process will increase the therapy’s affordability and patient accessibility (Staff at Penn Medicine).

The reason why the use of allogeneic CAR T-cells has not been implemented yet is because it may cause life-threatening graft viruses and can potentially be eliminated from the host’s immune system (Depil). Fortunately, this conflict is an active area of research as the potential of this finding can be successful in treating cancer. Many oncologists hope to see this as a frontline treatment option one day. 

Conclusion

Cancer patients who have failed immunotherapy and chemotherapy now have another option in the form of CAR T-cell therapy. With the use of CAR T-cell therapy, cancer patients can utilize and enhance their immune systems to their advantage. The fact that CAR T-cell therapy is highly personalized in nature offers an immense benefit to patients. This is because CAR T-cell therapy only attacks antigens using engineered CARs. In doing so, it can cause minimal harm to the healthy cells of the body. With increased research and affordability, this revolutionary treatment can provide millions of cancer patients with a glimmer of hope for a successful and healthy recovery. 

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