Abstract
By equipping T cells with chimeric antigen receptors (CARs), their targeting can be rerouted to selectively destroy blood-based cancer cells. The recent development of CAR T cell therapies has revolutionised the treatment of relapsed/refractory cancers and provides new hope to patients that would otherwise face terminal diagnoses.
Unfortunately, CAR T cells involve complex manufacturing processes that drive high prices and limit patient access. Though there are 7 FDA-approved CAR T cell therapies, none are available in New Zealand due to our small economy and market size. New Zealand-based patients must either enrol for limited clinical trial positions, or travel overseas for treatments, often costing upwards of $500,000. The high price of CAR T cell therapy is largely due to the per-patient basis of manufacturing. The CAR T cells must derive from the patient’s blood to avoid the onset of graft reactivity and erroneous toxicities. Likewise, CAR T cell therapies are limited by relying on the T cells of patients that have experienced multiple rounds of chemotherapy.
In contrast, novel therapies are engineering natural killer (NK) cells with CAR for enhanced cancer recognition. Like CAR T cells, CAR NK cells are similarly manufactured ex vivo prior to infusion into the patient. However, unlike T cells, NK cells do not react to the same host-specific residues that restrict T cells to their donors. This means NK cells can be administered in an off-the-shelf setting. In essence, large quantities of CAR NK cells can be manufactured from one donor for treating multiple patients, therefore diluting the manufacturing costs. CAR NK cell therapies are still in their infancy and restricted to clinical trials. However, the results are promising and yielding similar response rates to CAR T cells for some indications. Implementing a CAR NK cell platform in New Zealand would enable lower costs for treatment and achieve more equitable access to these revolutionary cell therapies.
Within this thesis, we established New Zealand’s first CAR NK cell pipeline for preclinical manufacturing and analysis. By combining cost-effective, commercially available reagents with in-house techniques, we optimised an accessible protocol for expanding and engineering vast quantities of NK cells. The expanded NK cells express a range of activating receptors for cancer recognition and display anti-cancer properties in vitro.
With an expansion platform established, we sought to further integrate it into CAR T cell manufacturing. During the production of CAR T cells, the T cells are isolated from patient blood, while the remaining cells are discarded. By applying our protocol to the waste product, we were able to simultaneously expand a pure population of NK cells in parallel with the CAR T cells. The NK cells were applied as a pretreatment before CAR T cells and significantly enhanced tumour clearance and survival in mouse models of breast cancer, acute lymphoblastic leukaemia and non-Hodgkin’s lymphoma.
Following the success of NK and CAR T cell combination therapies, we further engineered the CAR T cells to secrete beneficial cytokines that enhance both NK and T cell fitness. Different molecular techniques and orientations were trialled and an optimal setting in which T cells can express CAR and IL-21 was attained. Successful expression of IL-21 resulted in a more youthful population of CAR T cells and warrants further investigation in the future.
While endogenous NK cells can directly recognise malignant cells, tumour-targeting can be enhanced with anti-tumour antibodies. When antibodies bind to tumour cells, NK cells recognise the constant (Fc) region of the antibody and are encouraged to destroy the respective tumour. Avelumab is an antibody that binds specifically to PD-L1; a molecule that is highly expressed on tumour cells to suppress NK and T cell attack. Avelumab therefore functions to block the interaction with PD-L1, while encouraging NK cells to lyse the bound tumour cells. By introducing mutations into the Avelumab Fc region, we enhanced the subsequent activation of NK cells. However, it was revealed that activated NK cells upregulate their own PD-L1 molecules, and the enhanced Avelumab led to NK cell fratricide. Therefore, the use of anti-PD-L1 anti-bodies should be reconsidered when anti-cancer activity is expected to be largely mediated by endogenous or adoptively transferred NK cells. The findings of this thesis provide a baseline for developing NK cell therapies in New Zealand. A CAR NK cell protocol has been established, enabling further research to improve the treatment of blood-borne and solid malignancies. By converting the protocol to comply with good manufacturing practices, CAR NK cells can be generated in a cost-effective manner for clinical applications in New Zealand.