CARs, TRUCKs & Armoured CARs: How will Immunotherapy vehicles navigate roadblocks presented by solid tumors?
Chimeric antigen receptor (CAR) T cell therapy is a novel immunotherapy for treating cancer that works by boosting the body’s own immune system.1 Positive results in liquid cancers have caused excitement about the potential magnitude of this therapy as a cancer treatment.2 This background research paper will focus on the structure of a CAR T cell, the benefits, its current uses in liquid cancers, the problems solid tumors pose, and a brief overview of the approaches that are being taken to attempt to increase the success of the therapy in solid tumors.
CAR T cell therapy is a highly personalized treatment where a patient’s own T cells are genetically engineered to express a receptor specific to the antigen expressed on the cancerous cells.1,2,3,4 Usually, as a mechanism to evade the immune system, cancer cells decrease their expression of major histocompatibility complexes (MHC), thereby making it harder for native T cells to find and kill the cancer cells.1,2,3,4 In order to overcome cancer’s defense mechanism, CAR T cells are genetically engineered to be able to directly bind to the surface protein antigens expressed on the cancer cells instead of through the usual pathway of MHCs.1,2,3,4
To perform CAR T cell therapy, a sample of the patient’s blood is drawn and the T cells are extracted.1 Next, in a lab, the genetic information of the receptor for the antigen on the cancerous cells is inserted into the T cells usually through a viral vector, creating a Chimeric Antigen Receptor T cell.5 The receptor is usually made of two components: the single chain variable fragment of a monoclonal antibody and a transmembrane domain.1 When the CAR T cells are infused back into the patient’s bloodstream, they circulate the body looking for cancerous cells to destroy. It is important to note that the addition of costimulatory domains on the inside of the T cell’s membrane has been crucial in improving the efficacy of CAR T cells through increasing activation and stimulation of the T cells.1 First generation CAR’s only used a CD3 zeta costimulatory domain; second generation CAR’s used the aforementioned domain plus another domain, usually CD28 or 4-1BB; and, third generation CAR’s use the CD3 zeta domain and two other domains, usually CD28 and 4-1BB (see figure 1).1
CAR T cell therapy has the potential to have significant advantages over the conventional cancer treatments of chemotherapy, radiation, and surgery. Firstly, this therapy is bioengineered to target the antigens on the cancerous cells and hopefully leave the non cancerous cells alone.1,2,3,4 Secondly, this therapy can provide long lasting immune surveillance to prevent relapse.1,2,3,4 If the CAR T cells can stay around in the body for a long time, if the cancer returns, the cells are there and ready to act quickly. Thirdly, CAR T cell therapy is not restricted to whether a tumor is operable or inoperable and is much less invasive than surgical removal.
The first CAR was genetically engineered in 1987 and CAR T cell therapy was first successfully used in 2011 to cure a patient of leukemia.6 So far, CAR T cell therapy has had success in treating some lymphomas and leukemias.1,2,3,4 CAR T cell therapy is currently on the market for certain leukemias and lymphomas where it targets the CD19 antigen that is expressed on the cancerous cells.1 Even though the treatment is successful, one limitation is that the patient will no longer have any B cells left as B cells also express the same antigen.5 Other limitations to CAR T cell therapy currently include lack of long term persistence, cancer mutates to not express the targeted antigen, T cell exhaustion, and high risk of cytokine release syndrome and neurotoxicity.1
In addition, due to the highly personalized nature of CAR T cell therapy, it is extremely expensive, costing approximately $400,000 per person in Alberta.7 The hope is to one day make this therapy an ‘off the shelf’ therapy meaning it would not be personalized to each patient.5 The advantages of an ‘off the shelf’ therapy include a lower price and quicker access. One of the disadvantages of an ‘off the shelf’ therapy is that with a less personalized therapy comes a higher risk of graft vs. host disease and host vs. graft disease.5 This means that the body will either attack the CAR T cells causing them to fail or the CAR T cells will attack the body causing failure of healthy tissues.
Unfortunately, CAR T cell therapy for solid tumors has had much more limited success.1,2,3,4 The primary reasons for lack of success of CARs in solid tumors include lack of suitable targets (lots of the antigens seem to be expressed on healthy tissue as well), physical barriers for T cells to enter the tumor, immunosuppressive tumor microenvironment, and decreased fitness of the T cells, in part due to a phenomenon called T cell exhaustion.1,2,3,4 As a result of these problems, a common occurrence with CAR T cell therapy in solid tumors is on target off tumor toxicity meaning that the CAR binds to the antigen which exists on other healthy tissues, thus damaging the tissue and leaving the tumor unaffected.3
There are currently multiple trials underway focusing on altering CAR T cell therapy, so that cancer cells in solid tumors are more responsive to treatment. This includes, but is not limited to: the use of dual antigen targeting, natural killer (NK) cells instead of T cells, the use of drugs and cytokines along with the therapy, additional signals for activating T cells, inhibiting molecules that deactivate T cells (immune checkpoint inhibitors ie. PD-1/PD-L1), localized delivery of CAR’s, fourth generation CAR’s (TRUCKS), armoured CARs, on and off/safety switches, deleting certain molecules in the T cells, adapting T cells so they are activated/attracted by molecules present in the tumor, increasing T cell metabolism, and targeting the stromal cells of the tumor.1,2,3,4,8 Within each of these approaches, there are a variety of ways currently being explored to tackle each approach which will be further explained in the rest of this research.
While CAR T cell therapy has the potential to revolutionize the face of cancer treatment, there are many barriers that continue to lie in the way due to the complexity of the interactions between the cancerous cells and the T cells.
(If figure 1 is not appearing above, please use the following link to view figure 1: https://docs.google.com/document/d/19TGOPSxlj45Sl2iqnt-28yyMIClU-Fj8ZxxVlqD3V9o/edit?usp=sharing)
Having demonstrated efficacy in liquid cancers, how can CAR T cell therapy be adapted and altered to be effective and viable in the challenging tumor microenvironment (TME) that exists within solid tumors?
In order to overcome the TME barrier of solid tumors, CAR T cell therapy will require a combination of alterations to the CAR, use of additional drugs, and combining CAR T cell therapy with other immunotherapies.
A literary search was performed using PubMed to identify publications from the last four years. Keywords used included CAR T cell therapy, checkpoint blockade, cytokines, stroma targeting, tumor microenvironment, and solid tumor. From these review articles, a categorization of barriers preventing effective CAR T cell therapy in solid tumors was created. From a combination of review and primary articles, a categorization of strategies to overcome the TME barrier was created as well as a list detailing some past and current clinical and preclinical studies.
Published findings were then supplemented through virtual consultation with two experts in the field of CAR T cell therapy, Dr. Taku Kambayashi at University of Pennsylvania and Dr. Persis Amrolia at University College London. Research was performed under the guidance of Dr. Nicole Prokopishyn, University of Calgary.
Barriers to Effective CAR T Cell Therapy
The multiple barriers to effective CAR T cell therapy in solid tumors can be categorized into four main categories (Table 1): i) the antigen dilemma, ii) the immunosuppressive tumor microenvironment (TME), iii) T cell exhaustion, and iv) tumor penetration.2
The antigen dilemma refers to the problem of having so far found a lack of tumor specific antigens which can be targeted with CAR T cell therapy, especially antigens that are not found elsewhere in the body. This problem has led to a phenomenon known as on target off tumor toxicity which means that the antigen being targeted is also present on healthy body tissues and the CAR T cells attack the healthy body tissue instead of attacking the tumor.2,3,4
Tumor Microenvironment (TME)
The TME is known to be hypoxic, acidic and low in nutrients.4 The TME lacks oxygen causing cells in the TME to use anaerobic respiration resulting in a buildup of lactic acid decreasing the pH.9 The acidic environment prevents optimal function of T cells. The TME also lacks the amino acid arginine which is a critical amino acid for the proteins made by CAR T cells that allow for cell proliferation.10 Finally, the TME contains many immunosuppressive factors such as the cytokines tumor growth factor beta and interleukin 4,11 which further decreases the efficacy of the CAR T cells. As a result, CAR T cells have a hard time being effective and remaining viable in the TME as they are overwhelmed by a breadth of factors working against them.
T Cell Exhaustion
The third problem is T cell exhaustion which refers to the idea that CAR T cells have a lack of persistence and proliferation which can be due to chronic antigen exposure or exposure to immunosuppressive factors inhibiting T cell function.2,3,4 Lack of persistence is a major problem in CAR T cell therapy because one of the main ideas behind CAR T cell therapy is that the cells can stay around after the cancer is gone to prevent a major relapse.2,3,4
The last major problem is the lack of tumor penetration with CAR T cells. CAR T cells have a hard time finding the tumor and then physically getting into the tumor due to its unique structure.2,3,4 This problem furthers the issue of on target off tumor toxicity because if a CAR T cell has a hard time finding the antigens in the tumor, it will more likely bind to the antigens on the healthy body tissue. If the CAR T cell cannot get inside the tumor, the therapy will not work as the CAR T cells will not be able to reach the tumor cells.
To combat each barrier, there are currently multiple approaches being explored. Some of these examples of these approaches are outlined in table 1.
The rest of this research will primarily focus on the strategies being investigated to overcome the TME barrier. The TME barrier was chosen as a focus because it contains the most variety of strategies being explored.
Methods to Overcome Barriers in the Tumor Microenvironment
Within the barrier of the TME, there are four main groups of strategies to solve the problem (table 2): i) addition/deletion of signal pathways, ii) metabolic alteration of T cells, iii) targeting structure and stroma of tumors, and iv) cytokines. Each of these strategies have multiple mechanisms within them.
Addition/Deletion of Signal Pathways
Addition/deletion of signal pathways refers to altering interactions between the CAR T cells and different proteins present in the TME. One of the main strategies being explored using this idea is combining the already existent immunotherapy of checkpoint blockade with CAR T cell therapy (see figure 2).2,3,4,12 The body has many mechanisms in place to prevent itself attacking healthy body tissues, one of which is checkpoint inhibitors.4,12 This is where there is a protein on the T cell that matches the protein on the healthy cell and when the proteins bind to each other, the T cell is inhibited.12 Cancer upregulates checkpoint inhibitors as one of its many survival strategies. In checkpoint blockade, the proteins are prevented from binding and inhibiting the T cell.12 One common checkpoint inhibitor target for checkpoint blockade therapy is PD-1/PD-L1.12 There are two main ways to adapt this therapy for CAR T cells. Firstly, the two treatments are delivered independently of each other in a similar time frame.12 Secondly, the checkpoint blockade therapy could be integrated into the CAR T cell through a multitude of ways. One possibility is that the CAR T cell releases anti PD-L1 antibodies which would bind to the inhibitory protein on the tumor cells blocking the pathway.12 Another way is through the use of gene editing technology, the CAR T cells could be built to not express the PD 1 protein which would also block the pathway.12 The other possibility is to alter the pathway inside the T cell once the proteins bind together so that the intracellular pathway is deactivated or actually stimulates activation of the T cell.12
Another signal pathway that is showing potential is the addition of signal pathways that allow for costimulation.2 This means that certain proteins found in the TME have receptors on the CAR T cell and when the proteins bind to the receptor, the CAR T cell is further activated which has proved effective in preclinical models of pancreatic cancer.13
A very similar approach is combatting immunosuppressive soluble factors such as cytokines like interleukin-4 by blocking the immunosuppressive factors from binding to the T cells.11
Metabolic alteration of T cells allows for CAR T cells to be better suited to the harsh environment of the TME by altering their metabolism to work in coordination with the nutrients available. One method currently being used is altering the proteins in the costimulatory domains of CAR T cells.9 For example, in liquid cancers, CAR T cells with CD28 and 4-1BB (third generation CAR) have been shown to be more effective than a CAR with only those proteins because the metabolic pathway was quicker and more efficient.9
Another method under investigation is the addition of different chemicals to the culture in which the CAR T cells are incubated in before being put back in the patient.9 There is evidence to suggest that certain cytokines in the incubation tray cause CAR T cells to have a more effective metabolism.9
In order to combat the low arginine environment present in the TME, scientist have added genes into the cell coding for two enzymes, human argininosuccinate synthase and ornithine transcarbamylase, which allow the CAR T cells to use the limited arginine more efficiently.10 As mentioned before, cell proliferation in CAR T cell therapy is key to allowing an effective and widespread response.
The use of click chemistry is also being applied to CAR T cells by binding a drug to increase metabolism to the membrane of the T cell.14 One example of a drug was avasimibe which when attached to the CAR T cell increased cholesterol in the membrane and resulted in a superior anti-tumor response.14
Targeting the Structure and Stroma of the Tumor
Another part of the TME that is hostile for CAR T cells is the structure and stroma of the tumor.2 There are currently two main solutions to this problem. The vessels can be remodelled or the stroma of the tumor can be directly targeted.15 Through certain cytokines such as interleukin-12 and antiangiogenic drugs, the poor vasculature of the tumor can be remodelled so that the CAR T cells will have an easier time navigating through the blood vessels.15 The stroma of the TME is full of other immune cells that work against T cells such as T cell regulatory cells and tumor associated macrophages.15,16 By targeting these cells in the stroma, there will be less inhibitory feedback to CAR T cells in the TME.15,16 The vasculature of the tumor can also be greatly improved through direct targeting of certain proteins on the blood vessels.15
Use of Cytokines
Cytokines are important proteins in the immune system that have a variety of roles including summoning other cells, activating cells, and killing tumors and viruses. One method of using cytokines is combining the popular cytokine immunotherapy with CAR T cell therapy independently of one another, but in the same time frame.17 Interleukin-2 is a common cytokine that is used alongside CAR T cell therapy as it enhances T cell activation and proliferation.17
These two immunotherapies can be combined into one through the use of an armoured CAR T cell (see figure 3) which is essentially a CAR T cell that is programmed to release cytokines such as interleukin-12.18 This would allow for cytokines to be released in the area they are needed, the tumor, instead of around the whole body helping to increase the potency of CAR T cell therapy.18
Chemokines are a type of cytokine common in the TME that helps enhance tumor growth.19 Certain receptors on chemokines could be targeted by CAR T cells to decrease the hostility of the TME by decreasing the amount of chemokines present.19
The use of cytokines in activating CAR T cells is becoming increasingly popular through the use of cytokine switch receptors/T cells Redirected for antigen‐Unrestricted Cytokine‐initiated Killing (TRUCKS).8,17 When TRUCKS bind to a tumor antigen, they are programmed to release certain cytokines such as interleukin-12 which will summon other immune cells such as tumor infiltrating leukocytes to help eradicate the tumor.8,17
Preclinical and Clinical Trials Results
Table 3 lists some of the past and current preclinical and clinical trials. In the research conducted, no clinical trials targeting the structure and stroma of the tumor were found.
This study has demonstrated that there are a variety of barriers to CAR T cell therapy for solid tumors, but there are also multiple trials and studies in progress to overcome these barriers that show promising solutions. Due to the many problems currently present with CAR T cell therapy in solid tumors, effective CAR T cell therapy would involve combining many solutions so as to address all the different problems.
To overcome the TME barrier, many different solutions have been put forward and have begun testing; however, there are very few clinical trials that focus on overcoming the TME barrier. There are multiple clinical trials working on the antigen dilemma.1 One of the reasons for the lack of clinical trials for the TME could be that clinical trials will have a hard time being effective if there is no suitable antigen to be targeted. Therefore, it is important to work towards solving the antigen dilemma and then other solutions can be incorporated into the therapy to make CAR T cell therapy the most effective it can possibly be.
Many of these solutions involve the use of combination therapy which involves combining another immunotherapy into CAR T cell therapy. This can be particularly effective since the other immunotherapy’s efficacy is enhanced through being directed at the tumor which could result in potentially less side effects in healthy tissue.
The research showed a discrepancy in the efficacy of second versus third generation CARs. One study20 showed that third generation CARs provided no benefit over second generation CARs while another study21 showed superior efficacy in third generation CARs.
One of the key limitations of this research study is that there is the possibility that the research missed a key strategy/study for CAR T cell therapy in solid tumors due to the vastness of this field.
The next steps in developing CAR T cell therapy for solid tumors involves taking the preclinical trials into larger animal models and then humans. Once certain solutions prove to be effective in humans, multiple solutions may need to be combined together to create a standard of care CAR T cell therapy for a specific solid tumor.
One of the major findings of this research is that inhibitory cellular pathways in T cells play a major role in preventing effective CAR T cell therapy in solid tumors. Therefore, future studies should consider exploring the role of technologies such as Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR Associated Protein 9 (CRISPR/Cas-9) to delete gene sequences that allow for inhibitory signals.
(If figure 2 does not appear above, please use the following link to view: https://docs.google.com/document/d/1U7BIWI5KVMF59hjnBAT6PXjDKU2tPOQQ9vnCvLjbWOE/edit?usp=sharing)
(If figure 3 does not appear above, please use the following link to view: https://docs.google.com/document/d/1ASvLFO1RGOxCYJxm6G9_UsgUTYtS8u2gOznBPh1A-04/edit?usp=sharing)
(If table 1 does not appear above, please use the following link to view: https://docs.google.com/document/d/1PnCScHcXvwOG123U9cWky2aDIIIApmKUvGMQv1v1cH8/edit?usp=sharing)
(If table 2 does not appear above, please use the following link to view: https://docs.google.com/document/d/1m0ZvLyBbvZi4vpxwCDuTwB9toffz8H4fNA33Xp7zX_c/edit?usp=sharing)
(If table 3 does not appear above, please use the following link to view: https://docs.google.com/document/d/15jpaMLzh6Ux-cbnK3dvRs6H5hzzDgjOKBB5Tot-tQJs/edit?usp=sharing)
This research study addressed the problem: how can CAR T cell therapy be adapted and altered to be effective and viable in the challenging TME that exists within solid tumors? The hypothesis was proven correct that it will take a combination of many different solutions to solve this very large problem including not only CARs but TRUCKS, armored CARs and future immunotherapy vehicles. Although there are many solutions being investigated, most of them are still in the preclinical phase which means that there is a long way to go before CAR T cell therapy can become the standard of care for solid tumors.
This research study has talked about two classification systems in order to design strategies to treat solid tumors with immunotherapy: 1) Summarization of categorization of barriers that need to be overcome to allow for effective CAR T cell therapy in solid tumors: i) the antigen dilemma, ii) TME, iii) T cell exhaustion, and iv) tumor penetration; 2) A novel categorization of strategies being used to overcome the TME barrier: i) addition/deletion of signal pathways, ii) metabolic alteration, iii) targeting the structure and stroma of the tumor, and iv) the use of cytokines.
Ultimately, CAR T cell therapy has the possibility to revolutionize future cancer treatment, preventing many cancer related fatalities.
*Citations were done in MLA style with endnotes used for in text citations.
I would like to thank the following people for their help and contributions to my project:
- Dr. Nicole Propokishyn, University of Calgary, for supervising my research.
- Ms. Amanda Millette, my science fair coordinator, for taking me through the steps of a science fair project and helping me edit and reference.
- Dr. Persis Amrolia, University College London, for virtually collaborating with me.
- Dr. Taku Kambayashi, University of Pennsylvania, for virtually collaborating with me.
- Dr. Dave Siddhu, University of Calgary, for answering my questions when I first learnt about CAR T cell therapy and for introducing me to Dr. Nicole Propokishyn.
- Mrs. Alana Hayman, my biology teacher, for reviewing my presentation.
- Mr. Irfaan Sorathia, my physics teacher, for helping me scan my logbook and assisting me with any technological issues.
- Mrs. Valerie McClements, my English teacher, for helping me with my referencing.
- Dr. Jehangir Appoo for helping me edit.