Cancer is one of the leading causes of death worldwide, with new cases expected to rise by 70 percent over the next two decades, making the search for an effective treatment more important now than ever before. Great strides have been made in recent years in the form of cancer immunotherapies. Inspired by discoveries about how the immune system works, immunotherapies are designed to reinforce the immune system’s ability to recognize and eliminate cancer cells.
New immunotherapeutics, such as cancer vaccines and checkpoint inhibitor drugs, have already shown great promise in improving the prognosis of patients suffering from various forms of cancer. However, these drugs can induce harmful side effects such as severe inflammatory responses and even autoimmune diseases. As such, the development of cancer immunotherapies has started to focus on improving efficiency, safety, and specificity by using genetic engineering approaches.
Genetic modification of T-lymphocytes (T-cells) and its application to cancer immunotherapy is currently under intense pre-clinical and clinical investigation, principally due to the important role that T-cells play in cell-mediated immune responses against cancer. The first stage of this response occurs when T-cells encounter B-cells or dendritic cells that have digested tumor-associated antigens (TAAs) and are displaying antigen peptide fragments bound to major histocompatibility complex (MHC) molecules (antigen-MHC). Receptors on the surface of T-cells (T-cell receptors or TCRs) then recognize and bind with the antigen-MHC complex, stimulating the T-cells to mature. The mature T-cells then either regulate the immune response (by releasing cytokines to promote further production of T-cells and generate helper T-cells) or become cytotoxic killer T-cells that recognize and directly attack cancerous cells.
In order to evade detection and attack by the immune system, cancerous cells not only create an immunologically-tolerant tumor microenvironment but they also employ numerous immunosuppressive mechanisms. This means that the natural immune response is not always effective against cancer for numerous reasons, including the observation that growing tumors down-regulate specific effector T-cell responses, such as interfering with T-cell trafficking and metabolism, and induce resistance to T-cell killing.
In order to combat cancer and its immunosuppressive effects, genetic modification of T-cells ex vivo can re-direct their specificity towards cancer cells once they are infused back into the patient. One such genetic modification is the transfer of chimeric antigen receptor (CAR) transgene cassettes into primary T-cells to produce so-called CAR T-cells (Figure 1). CARs are highly specific targeting molecules, typically combining the binding properties of a monoclonal antibody with signaling via the TCR complex. Therefore, the enforced expression of single-chain CARs on the T-cell surface allows T-cells to specifically target and bind with TAAs, providing the immune system with significant reinforcements and stimulating its emergence as a breakthrough cancer therapy.
CAR T-cells: An exciting opportunity for cancer immunotherapy
CAR T-cells have advantages over the way that normal T-cells work, since they can recognize and directly engage with TAAs independently of their expression of MHC (unlike TCRs), thus bypassing human leukocyte antigen (HLA) recognition. Consequently, CAR T-cells can avoid some of the main mechanisms that mediate tumor escape from TCR-mediated immunity. Furthermore, CARs can bind not only to proteins but also to carbohydrate, ganglioside, proteoglycan, and heavily glycosylated proteins to expand the range of potential targets that T-cells can attack and destroy (Table 1).
Table 1: CAR T-cell features and their associated benefits for targeting tumor cells.
|CAR T-cell feature||Benefit for targeting tumor cells|
|High specificity for TAAs||Reduces off-target effects|
|Monoclonal antibody-strength binding||Reinforces the immune system against tumor cells|
|MHC-independent binding||Avoids the main mechanisms of tumor resistance against TCR-mediated immunity|
|Can bind to proteins, carbohydrates, proteoglycans, and gangliosides||Expands the range of tumor cell targets T-cells can recognize|
CAR T-cells are currently undergoing considerable research, with over 100 clinical trials in progress. Early results have already revealed remarkable responses from patients with advanced refractory cancer, particularly those suffering from blood cancers, such as acute and chronic lymphoblastic leukemia. In these early studies, the most investigated target antigen to date has been CD19. Anti-CD19 CAR T-cells have experienced much clinical success, ushering in a new paradigm for evaluating CAR technology and increasingly becoming an attractive modality for cancer therapy, particularly given its high specificity and powerful elimination of target cells while avoiding the harmful side effects associated with other immunotherapies.
However, the methods by which T-cells are genetically modified for adoptive therapy are still being scrutinized, with some significant limitations and safety issues needing to be addressed before they can be routinely used in clinical applications. Therefore, to ensure the potential of CAR T-cell therapy can be fully leveraged, it is of utmost importance to understand how best to engineer them.
How are CAR T-cells generated?
In order to keep up with new CAR therapeutic approaches, suitable gene transfer technologies have had to be developed alongside them (Table 2). Approaches for introducing CAR transgene cassettes into primary T-cells have mainly used either viral-mediated transduction (e.g. using gamma-retroviral or lentiviral vectors, or adeno-associated virus) or non-viral gene transfer of DNA plasmids. Although viral methods have been widely used in early clinical studies and have experienced clinical success, they are typically limited in their application due to safety concerns of viral transfer (immunogenicity, insertional mutagenesis), production costs of clinical-grade viral vectors, and genetic payload restrictions.
Table 2: Comparison of viral and non-viral electroporation delivery modes for CAR T-cell generation.
|Delivery mode||Viral||Non-viral using advanced electroporation techniques|
|Tool||Gamma RV, LV, AAV||Transposon/transposase systems||CRISPR, TALEN, ZFN||mRNA|
|Efficient CAR T-cell generation||√||√||√||√|
|Stable integration of CAR||+
|Allow co-delivery of multiple/different substrates||–||+||+||+|
|Low risk of insertional mutagenesis||–||+||++||+++|
Alternatively, non-viral transfection of DNA plasmids via advanced electroporation techniques, as well as liposomal formulation, nanoparticles, and cell-penetrating peptides, are increasingly being adopted due to their low immunogenicity and low risk of insertional mutagenesis. In particular, non-viral electroporation methods offer further advantages over viral methods, such as being more cost-effective and allowing the delivery of larger gene inserts. Early results suggest CAR T-cells generated by non-viral electroporation methods are effective in treating certain types of cancer (e.g. Philadelphia chromosome-positive acute lymphoblastic leukemia resistant to tyrosine kinase inhibitors). Therefore, all this indicates that non-viral transfection via electroporation is likely to become one of the preferred methods over viral-mediated transduction for engineering CAR T-cells in the future.
Although non-viral bioprocessing methods have great potential over viral methods in terms of their clinical application in human cancer therapy, they present some limitations that need to be overcome before they can be adopted for routine clinical use. Specifically, it has been difficult to validate non-viral methods in human applications, mainly because of the low efficiency of gene transfer they provide and subsequent insufficient integration into the immune system. Yet, enhanced electroporation techniques combined with DNA transposition methods have started to show great promise in resolving these challenges, thus providing fresh impetus for their application in human cancer therapy.
Emerging methods enhance non-viral CAR gene transfer
DNA transposition has emerged as a non-viral gene insertion method to generate CAR T-cells. In DNA transposition, transposons (defined segments of DNA) move from one genomic location to another facilitated by one or more proteins, called the transposase. Transposons have been unveiled as a simple, yet powerful, genetic editing tool for mutagenesis (that is, to remove and/or integrate genetic sequences ex vivo) in vertebrates. Transposon-transposase systems have been shown to successfully transfer CAR transgene cassettes into T-cells to produce CAR T-cells for safe, inexpensive, and effective therapeutic purposes, such as the fish-derived Sleeping Beauty (SB) and insect-derived piggyBac human-adapted transposition systems.
Advanced electroporation technologies have transformed the capability of these transposition systems in the non-viral generation of CAR T-cells. Such systems provide highly effective methods for genetically modifying T-cells, amongst other cell types, and can be ideal for more complex transfection scenarios where multiple or even different substrate types need to be co-delivered (for example, CAR T-cell generation). Systems are available that even enable closed transfection to be performed for up to 1×109 cells, allowing for large-scale generation of CAR T-cells for immunotherapy development. These technologies can improve the efficiency of non-viral gene transfer, and so enhance the safe and effective integration of CAR T-cells into a patient’s immune system.
Given that non-viral transposition is still at an early stage in its clinical application, enhanced approaches to using it are still being investigated to potentially heighten the availability of safe, low cost, and efficient CAR T-cell cancer therapy in routine clinical use. For example, SB transposition of CAR genes from minimalistic DNA vectors called minicircles (MCs) have been found to produce a higher proportion of non-toxic MC-derived CAR transposons compared to those produced by viral methods.
As well as non-viral transposition systems, other new methods have shown promise in reducing the unwanted off-tissue toxicity that can be produced by genetically modified T-cells. By using transiently expressed mRNAs, CAR expression can be switched on or off to limit on-target, off-tissue toxicity to normal tissue. Yet, this technique cannot provide the long-term expression needed for maximal CAR T-cell function and sustained defense against cancer, so requires further investigation.
More recently, CRISPR/Cas9 has been used to introduce CAR sequences into T-cells. Targeted integration of CAR sequences into the TCR locus has allowed for endogenous control of CAR expression with parallel knockout of the TCR, which may generate a more effective and safer CAR T-cell population. CRISPR/Cas9 has also been used to knockout the inhibitory checkpoint PD-1 receptor in T-cells to potentially improve the efficiency of T-cell based therapeutics. These studies highlight the potential of CRISPR/Cas9 genome editing to advance the efficiency, safety, and effectiveness of immunotherapies.
CAR T-cell immunotherapies are becoming increasingly important in treating cancer, especially as non-viral gene modification technologies become more advanced and our understanding of immunology improves. The next challenge is to address how best to ensure robust and sustained CAR T-cell activity, and obtain the required anti-tumor effects without producing off-target toxicity, to improve patient outcomes.
The future of T-cell cancer therapy is likely to involve precision treatments that target the specific molecular mechanisms underpinning cancer in individual patients or groups of patients. This may also combine CAR T-cell therapy with other treatments (e.g. vaccines, checkpoint blockade drugs) to complement each of their respective limitations. Ultimately, manufacturing processes might be able to consistently produce T-cell therapies to the desired specification through automated engineering, or they might become available from a scalable allogeneic “off the shelf” universal source.
As cancer incidence worldwide continues to rise at an alarming rate, the race to find an effective treatment has never been so important. Yet, the remarkable progress that cancer immunotherapies have made in recent years has given us fresh hope. Advancing genetic engineering approaches and new technologies are enabling us to reinforce our natural immune defenses against cancer, generating ever stronger CAR T-cell therapies to provide cancer patients with the best care possible.