By Sreenidhi Venkatesh
Every year the incidence of cancer continues to increase, taking more than 150,000 lives each year in the UK (Cancer Research UK, n.d.). To combat this complex disease, treatments have evolved to include a plethora of options, including immunotherapy. Cancer immunotherapy involves harnessing the immune system to target and eradicate cancer. Immunotherapies include monoclonal antibodies, checkpoint inhibitors, vaccines, cytokines, and most recently Chimeric Antigen Receptor (CAR) T-Cell therapy (Cancer Research UK, n.d.).
Amongst the various ingenious mechanisms that cancer uses to hide from the body’s defences, down-regulating different parts of the antigen recognition, processing, and presentation machinery to avoid the immune system is one (Yang, 2015). CAR T-cell therapy, first developed in the 1960s, is one that compensates for the malfunctioning of this essential process. It involves the extraction of an individual’s T-cells through apheresis and genetically engineering the T-cells to be CAR T-cells which can recognise special proteins on the cancer cells. Following in-vitro growth and multiplication, the cells are injected into the blood stream where they target cancer cells (Cancer Research UK, n.d.).
In the first-generation of CAR T-cells, the genetic engineering led to them having the following structure: an intracellular signalling domain, a transmembrane domain of the T-cell receptor, and a single chain variable fragment (scFv) which comes from a monoclonal antibody (Zhao et al., 2018). The variable region was essential in the recognition of antigens specific to the cancer cells that were being targeted. The transduction of CAR genes allowed the T-cells to influence a range of responses that were not limited to those of the major histocompatibility complex. As the first-generation CAR design elicited a low proliferative capacity, the design underwent further modification. This produced the second generation of CAR T-cells, which had superior expansion of T-cells when repetitively exposed to antigens and better cytokine secretion (Zhao et al., 2018).
Following these improvements and modifications, CAR technology has been used to successfully treat B-cell malignancies and lymphocytic leukaemia. The main target in clinical trials has been CD19 which is exclusively expressed in B-cell leukaemia and lymphomas (Davila et al., 2012).
Despite the positive outcomes observed in clinical trials, CAR T-cells have a number of limitations that include side-effects and restricted usage. Side-effects such as severe cytokine release syndrome and neurotoxicity are significant (Brudno & Kochenderfer, 2019). They are characterised by a range of symptoms which include fever and headaches, and a reduction in B-cell count due to the destruction of B-cells in those targeting the CD19 protein. As the receptors on CAR T-cells are specific to a single molecule, they cannot be altered post-engineering. Aside from limiting the clinical use, there are high manufacturing costs associated too (Zhao et al., 2018).
Cho et al. (2018) have developed an interesting solution to this problem – split, universal, and programmable CAR T-cells (SUPRA CAR T-cells). Unlike the original CAR T-cells, SUPRA CARs are much more nuanced in their actions. SUPRA CARs are eponymous as ‘split’ is indicative of the CAR system carrying two components, a ‘zipCAR’ and ‘zipFv’ fragments. The ‘zipFv’ comprises the ligand binding scFv domain which is connected to a leucine zipper. The ‘zipCAR’ also has a leucine zipper attached to the extracellular facet of the T-cell. Genetic-engineering of the T-cells involves production and modification of ‘zipFvs’ and ‘zipCARs’ into pairs. These CAR T-cells are universal as various ‘zipFvs’ can be engineered to bind to the specific leucine zipper. Finally, they are programmable as they target specificity and adjust according to cell activity (Cho, Collins & Wong, 2018).
A great deal of control can be asserted over SUPRA CAR T-cells. If inhibition of CAR reconstitution is desired, a competing zipFV can be added which dimerises with the first. To determine the extent of the efficacy of the CAR T-cell, the affinity between the leucine zipper pairs or the affinity between the antigen and the variable region can be adjusted. Moreover, the concentration of ‘zipFv’ and expression level of ‘zpCARs’ can also be tuned. By manipulating these factors, the response induced by the T-cell can be modulated thereby reducing the various side-effects (Chen, 2018).
The studies conducted in the lab to assess the function of SUPRA CAR T-cells varied from the modular function studies described above to in-vivo studies. It was found that by engineering two orthogonal ‘zipFv’ fragments, independent control over different cell types could be established, thereby increasing the number of different immune responses. The researchers found that when cultured cells were treated with CAR T-cells and adaptor molecules for membrane localised proteins (Her2, Axl, or both), there was an increased killing efficiency. By developing the CAR so it would sense different antigens simultaneously, the escape of tumours could be prevented (Cho, Collins & Wong, 2018).
The in-vivo studies comprised of a xenograft model in which mice were injected with breast cancer cells which were Her2 positive. It was found that injecting primary human CD8+ T-cells with ‘zipCAR’ and ‘zipFv’ led to a “robust tumour clearance”. It was further found that the cytokine release seen was particular to when the ‘zipFv’ bound to the specific ‘zipCAR’ (Idrus, 2018). The study also tested a humanised SUPRA CAR system to prevent immunogenicity (foreign substances provoking an immune response). They created ‘zipCAR’ and ‘zipFv’ using zipper domains from human FOS and JUN transcription factors and found that the killing levels were similar to those that did not have a human origin. The study also showed that it could efficiently eliminate leukaemia in-vivo using a blood tumour model. Overall, these are indicative of possible positive outcomes that may be observed when used in humans to combat cancer (Cho, Collins & Wong, 2018).
This combinatorial targeting mechanism has greatly increased the versatility of the CAR T-cell treatment and will allow for fine modulations to be performed. The initial paper on SUPRA CAR T-cells was published in 2018, and the application of this therapy has not yet been expanded to include humans. As every cancer has varying levels of heterogeneity, the effectiveness of this therapy will differ. This suggests that some cancers may be more susceptible than others. Following further research which encompasses the efficacy of the therapy and application to humans, SUPRA CAR T-cells could pave a path for the next-generation of cancer treatments.
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