Cancer is a heterogeneous disease as it can present itself in many different ways depending on the individual. So, we would need to continuously develop increasingly specific ways to ensure we can effectively combat this nasty medical condition.
On top of that, existing treatment methods such as radiotherapy and chemotherapy have been shown to induce toxicity as well as cellular damages. This, in turn, hampers the efficacy of cancer therapy and calls for alternative strategies to overcome the problems faced by the aforementioned methods.
Interestingly, a very promising field within the cancer therapy landscape has been on the rise over recent years. It has even been dubbed by the scientific community as the “fifth pillar” of cancer treatment. That field, in short, is called immunotherapy.
What is immunotherapy?
Cancer immunotherapy is all about enlisting help from the patient’s own immune system to get rid of the cancer cells from their body. In other words, we are basically attempting to strengthen the patient’s immune system so that it is capable of fighting off tumours.
The earliest versions of cancer immunotherapy date back to 1891 where William Coley, the father of immunotherapy, was the first guy who successfully utilised the immune system to combat cancer. He got that idea after observing that adding mixtures of live and inactivated microorganisms called Streptococcus pyogenes and Serratia marcescens led to the regression of tumours within sarcoma patients.
As we move forward in time, cancer immunotherapy was soon equipped with a variety of other treatment methods. One of them includes checkpoint inhibitor blockade therapy, which is basically all about blocking (a.k.a. inhibiting) proteins that play a big role in regulating the activation of immune cells. Examples of such proteins would be the programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4).
Meanwhile, other treatment strategies in the field of immunotherapy would involve using antibodies, known as bispecific antibodies, or even our own immune cells, known as the adoptive transfer of immune cells, to fend off cancer cells.
In this article, however, we are going to extend our discussion on the adoptive cell transfer technique, placing a particular focus on a variant of engineered immune cells known as the chimeric antigen receptor T/NK cells (CAR-T/NK cells) and their applications in immunotherapy.
CAR-T cells
The basic stuff
Let’s start with the basics first.
T cells - also known as T lymphocytes - are white blood cells with a central role in adaptive immunity. Their job description consists of eliminating infected host cells, secreting cytokines, initiating an attack by activating other cells of the immune system, as well as controlling the degree of our immune response towards pathogens.
It should also be noted, however, that not all T cells have the ability to destroy infected cells. In fact, we can divide T cells into three categories - cytotoxic T lymphocytes (CD8+ T lymphocytes), helper T lymphocytes (CD4+ T lymphocytes), and memory T lymphocytes. Of these, only cytotoxic T lymphocytes will be granted the license to kill.
To do this job, cytotoxic T lymphocytes have a receptor that is… creatively termed the T-cell receptor (TCR) which allows them to recognise specific pathogens through their unique proteins (called antigens). Once these T cells recognise these antigens, they can then start to initiate an attack against these foreign invaders.
Redesigning the T cell
On that note, chimeric antigen receptor T cells (CAR-T cells) are basically cytotoxic T lymphocytes that have been genetically-engineered to express genes encoding for antigen receptors on their surface. These antigen receptors, in turn, would be able to identify cancer antigens in a very specific manner.
The chimeric antigen receptor is structured in a way that it consists of an external region which recognises tumour antigens. This external region is connected to another important region inside the cell which is necessary for the activation of the entire CAR-T cell (check out Figure 1).
Once the CAR-T cell recognises a tumour antigen, it will get activated and start producing toxic proteins called cytokines. These proteins are important mediators of cell signalling and cell-to-cell interactions, and they in turn assist the clearance of those cancer cells.
Figure 1: How a CAR-T cell works. CAR-T cell recognizes tumour antigens present on the surface of the tumour cell using the chimeric antigen receptor and targets them for killing. This figure was adapted from Ramos et al.’s (2016) CAR-T Cell Therapy for Lymphoma.
At the point of writing, we have already developed three generations of CAR-T cells (see Figure 2 as a guide). To begin with, the first generation is relatively simple as it only contains one intracellular region known as CD3ζ. Meanwhile, the second generation consists of the CD3ζ region together with a helper molecule called 4-1BB. Finally, the third generation is made of the CD3ζ region, 4-1BB region, and another helper molecule called CD28.
Figure 2: The possible structures of chimeric antigen receptor (CAR) on CAR-T and CAR-NK cells. CAR-T cells contain external regions that are derived from monoclonal antibodies. This external region is connected to another region that spans the cell membrane and interacts with the TCR-associated region in the cell (shown in red). The first-generation CAR-T cells possess an internal region called the CD3ζ. Its job is basically to mediate the activation of the CAR-T cell. In contrast, the second-generation CAR-T cells possess an additional helper molecule (4-1BB), whereas the third-generation CAR-T cells are composed of multiple internal helper molecules - such as 4-1BB and CD28 - in addition to CD3ζ. On the other hand, CAR-NK cells possess a different internal molecule called DNAX activating protein 10 (DAP10) or 12 (DAP12) instead of CD3ζ. This figure was adapted from Petty et al’s Chimeric Antigen Receptor Cell Therapy: Overcoming Obstacles to Battle Cancer (2020).
How are CAR-T cells developed?
The development of CAR-T cells involves the isolation of T cells from the patient through a process known as leukapheresis. Once that’s done, the scientist will then introduce viral vectors that express the chimeric antigen receptor into the isolated T cells. After that, these T cells will be cultured in the lab.
Moving on, lymphodepleting chemotherapy, a process by which lymphocytes are being destroyed to prepare the body for the introduction of CAR-T cells as well as limit the competition between normal lymphocytes and CAR-T cells, takes place after the expansion of CAR-T cells. After that, the infusion of CAR-T cells into the patient follows suit.
Figure 3: A schematic of the CAR-T cell therapy procedure. T cells are first isolated through leukapheresis. The gene expressing the chimeric antigen receptor is then incorporated into the T cells via a viral vector. Moving forward, these newly formed CAR-T cells are expanded outside the human body (ex vivo) and delivered back to the patient once the lymphodepleting chemotherapy is completed. This figure was adapted from McGuirk et al’s (2017) Building blocks for institutional preparation of CTL019 delivery.
Interestingly, in addition to deriving CAR-T cells from the patients themselves (thereby generating autologous CAR-T cells), we can also develop CAR-T cells using the cells obtained from healthy individuals by following a similar procedure as discussed earlier. This, in turn, leads to the production of allogeneic CAR-T cells.
What are the limitations of their power?
Researchers have been very optimistic about these engineered cells as they have presented positive results regarding their efficacy during preclinical studies of their applications in colorectal cancer, cervical cancer, and B-cell acute lymphoblastic leukaemia treatments.
However, even though CAR-T cells may appear to be the panacea for cancer treatment, clinical applications have also demonstrated multiple health complications associated with this treatment strategy.
First of all, patients with low numbers of T cells cannot undergo CAR-T cell therapy as the expansion of those is challenging. One study, in particular, revealed that the cell population density affects their ability to expand after noticing how underpopulated cells are more prone to dying because they get exposed to reactive oxygen species, which are highly toxic compounds produced by cells under oxidative stress as a result of being in low-density areas.
Furthermore, clinical research has also observed the development of cytokine release syndrome (CRS) after the infusion of CAR-T cells into patients, which thereby introduces further barriers to their utilisation. In essence, cytokine release syndrome is an inflammatory response involving the excessive production of specific cytokines in the human body by white blood cells. If left untreated, this can lead to feverish symptoms as well as organ failure. Meanwhile, CAR-T cell therapy can also impose the danger of graft-versus-host disease (GVHD) onto patients. This may lead to inflammation in multiple regions of the body, and in the worst possible case, death.
In addition, cancer cells have the annoying ability to minimise the expression of specific antigens on their surface, which could further hamper the efficacy of CAR-T cells. They are essentially capable of doing this because they can undergo a process called immunoediting and consequently avoid getting detected by our immune system.
All in all, these problems are clear evidence that further research should be conducted in order to devise potential solutions to the limitations of CAR-T cells.
CAR-NK cells
Over recent years, alternative approaches to CAR-T cells are emerging in the field of oncology, with one great example of which being the utilisation of natural killer (NK) cells instead.
Again, the basics first
Natural killer (NK) cells are a component of innate immunity. These cells do not actually recognise antigens with the same level of specificity as T cells. Instead, they possess activating and inhibitory receptors on their surface, where the balance of which triggers their activation.
To go into the finer details, the level of NK cell activity is dependent on whether a specific molecule called the major histocompatibility complex class I (MHC class I) is present on its neighbouring cells. To put it simply, normal cells would have the MHC class I molecules on their surface. Consequently, NK cells would recognise them and thereby avoid a “friendly fire”. Conversely, cancer cells would be attacked by NK cells as they won’t have these molecules present on their surface. Once these NK cells are activated, they basically trigger the production of highly toxic proteins like perforin and granzyme to defend our body.
All in all, the ability of NK cells to recognise changes in the expression of certain molecules on cells was eventually exploited to develop CAR-NK cells.
Figure 4: How NK cells recognise cancer cells. When NK cells encounter cells that present tumour antigens but don’t have the MHC class I molecule expressed on their surface, they will activate and start producing cytotoxic products against their target cell. On the other hand, if NK cells detect the normal expression of the MHC class I molecule on a cell but nothing that signals a cancer cell, their very own inhibitory receptors will suppress activation. This figure was adapted from Shah et al. (2009)’s NK antibody therapy: KIR-ative intent.
Upgrading the NK cell
Just like CAR-T cells, CAR-NK cells are also made out of a chimeric antigen receptor, though the main difference between them is that CAR-NK cells have an NK cell-specific domain called the DNAX activating protein 10 (DAP10) and 12 (DAP12). The good thing about these two domains is that they can activate the NK cell more efficiently as opposed to the CD3ζ domain as seen in CAR-T cells. Hence, as it is relatively easier to activate NK cells as opposed to T cells because T cells require the presence of specific antigens, CAR-NK cells are being considered a very promising form of immunotherapy.
In addition, studies have also demonstrated that unlike patients who were treated with CAR-T cells, those who were treated using CAR-NK cells do not suffer from graft-versus-host disease, and therefore CAR-NK cell therapy may be the solution to overcome some of the issues associated with CAR-T cell therapy. Moreover, patients treated with CAR-NK cells have been observed to have a lowered risk of developing cytokine release syndrome, and part of the reason for that is because NK cells have a relatively shorter lifespan (approximately one to two weeks if there aren’t many cytokines in the environment) in contrast to CAR-T cells.
Not to mention, although cancer cells are able to downregulate their MHC class I molecules in order to escape detection by CAR-T cells. This is the precise signal for CAR-NK cells to attack these cells and so, the cancer cells would be eliminated in no time!
The limitations of CAR-NK cells
Based on the previous section, it does seem like CAR-NK cells offer the exact solution we need to tackle the problems associated with CAR-T cells. But even though CAR-NK cells have shown very promising clinical results as of now, certain limitations may still hinder its widespread application.
For example, CAR-NK cell’s relatively short lifespan of one to two weeks may affect their overall efficacy. Plus, the hostile nature of the tumour environment could suppress their trafficking and effector functions.
How can we improve the effectiveness of CAR-T and CAR-NK cells?
Although CAR-T and CAR-NK cells have both presented great potential for cancer treatment, further investments into its research and development initiatives remains a necessity in order to overcome their unique set of drawbacks.
For instance, we could potentially use the CRISPR-Cas9 technology to enhance the overall abilities of these cells at eliminating tumours as CRISPR-Cas9 offers a much more precise way for editing genes.
Meanwhile, recent clinical studies have also demonstrated that combinational therapies – a form of therapy that uses a combination of agents and drugs in order to treat cancer patients – could enhance the anti-tumoural effect of CAR cells as well. Intriguingly, a Phase 1 clinical study used an anti-cancer drug (the PD-1 inhibitor) in conjunction with CAR-T cells. And it turns out that this combination aided the expansion of CAR-T cells and helped B-cell lymphoma patients to recover sooner, all of which whilst experiencing negligible side effects! So, moving forward, this could be a plausible treatment strategy that maximises the best of both worlds.
Why should you care?
In 2020 alone, the number of new cancer-related cases and deaths worldwide were estimated to be approximately 19 million and 10 million, respectively. Besides that, the healthcare cost of cancer is projected to reach $245 billion by 2030 according to the American Association for Cancer Research.
It is probably pretty clear at this point that immunotherapy is one powerful weapon that we are equipped with during our constant battle against cancer. Therefore, it is imperative for us to continue pushing forward with an innovative mindset for our future ideas may be the key to unlocking a cheap yet effective treatment against this unfortunate condition.
Author
Filippos Maniatis
BSc Biological Sciences, Imperial College London
MSc Bioscience Entrepreneurship, UCL