Surgery (St Vincent's) - Theses
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ItemCombining TIL and CAR for adoptive cell therapy in metastatic melanoma( 2019)Background Metastatic melanoma is a highly lethal disease, and until recently patients had limited therapeutic options. Knowledge and understanding of the role the immune system plays in tumour development and its therapeutic potential has recently gained momentum and immunotherapeutic agents have emerged as the gold standard of therapy in treating this cancer. Adoptive cell therapy (ACT) has been shown to have high rates of tumour regression with durable, complete responses and potential 'cure'. Tumour-infiltrating Lymphocytes (TIL) and Chimeric Antigen Receptor (CAR) therapies are examples of ACT. Each has their own advantages, limitations and toxicities. As the complexity of the immune system and its targets is increasingly appreciated, combining immunotherapies is emerging as a promising avenue for improving patient oncological outcomes. This project explores the efficacy of dual specific T cells by combining TIL and CAR therapies. Aim To establish a model system transducing TIL with anti-Her2 CAR (TIL-CAR) and assessing function against autologous melanoma tumour cells that express Her2 antigen. Method TIL were generated from patient derived metastatic melanoma tumours and tumour cell lines were established in a biobank. TIL were thawed and activated using CD3/28 beads and transduced with second generation anti-Her2 CAR (scFv-erbB2-CD28-zeta) using a retronectin protocol. Patient matched PBMCs were transduced for functional comparison. Melanoma tumour lines in the biobank were found to innately express Her2 antigen to varying degrees. Some melanoma tumour lines were transduced and sorted to create higher expressing Her2 antigen lines for functional comparison. Flow cytometry was used to confirm cell phenotype and antigen/CAR expression. Functional testing was performed using ELISA and chromium release assays. An in vivo ACT model in NSG mice was performed comparing TIL and TIL-CAR. Results TIL were successfully cultured from metastatic melanoma tumour pieces. Despite TIL proliferating at lower rates than PBMCs, both were successfully transduced to express anti-Her2 CAR. When TIL were transduced to express anti-Her2 CAR they were functionally active through both TCR and CAR and produced greater amounts of interferon gamma against Her2 expressing tumour lines. TIL-CAR had greater cytotoxic activity when cultured against autologous melanoma tumour lines, but the benefit transduced TIL over PBMCs varied in response between tested patients. The advantage of TIL-CAR over PBMC-CAR did not demonstrate consistent trends across this limited group of patients. The functional activity may be influenced by the level of Her2 expression in the co-cultured tumour cells as well as by the phenotype of T cell populations. Results of an in vivo pilot study in mice demonstrated reduction in tumour size when TIL-CAR were used in an ACT protocol. The primary limitation of this study was the low proliferation rate of TIL following transduction which required extended periods in culture. Conclusions Combination of TIL-CAR is a novel concept. TIL can be transduced to express anti-Her2 CAR. Metastatic melanoma cells in our biobank constitutively express Her2 antigen. TIL-CAR tend to show greater activity in interferon gamma and cytotoxic functions compared to parental (non-transduced) TIL when cultured against Her2 expressing tumour lines. When compared to the activity of PBMCs transduced with the same CAR the additional benefit conferred by TIL-CAR is inconsistent. Protocols would benefit from further optimisation to generate a 'younger' phenotype capable of more rapid and sustainable proliferative potential and facilitate earlier delivery of therapy if used in a clinical setting.
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ItemStrategies for engineering skeletal muscle: an important link in the neuroprosthetic interface of bionic limbs( 2019)Limb amputation is a major cause of disability in our community, for which motorised prosthetic devices offer a return to function and independence. Advanced robotic limb technology utilises a range of mind-prosthetic interfacing strategies to intuitively drive the limb. These approaches include direct recording of peripheral nerves, brain-recording interfaces, or the transposition of transected nerves to remaining muscle groups for myoelectric recording. All of the current methods are hampered by delicate neural biology, either leading to premature device failure or introducing unnecessary surgical risk. As an alternative, this thesis proposes a new solution: to develop a bioelectrode using tissue engineered skeletal muscle as a signal amplifier of activity from residual nerves for intuitive prosthetic control. Conceptually, the fabrication of such a device would begin with a tissue biopsy from the patient from which a pool of myogenic stem cells would be derived and expanded. These autologous cells would be used for the tissue engineering of skeletal muscle fibres, primed with neurotrophic biofactors to optimise the tissue for innervation. Flexible recording wires could be incorporated into this fabrication step, thus eliminating the trauma of electrode insertion and also optimising its biocompatibility. The bioelectrode device could also be designed to patient or prosthetic anatomy as required. This thesis developed key elements of the above proposal. Firstly, a bioprinting technique was established to tissue engineer functional skeletal muscle using a gelatin methacryloyl (GelMA) bioink. Bioprinting enabled the rapid deposition of muscle progenitor cells (primary mouse myoblasts) in layered fibres, reminiscent of native muscle architecture. Fabrication parameters were optimised to produce fibres with high cell viability and print resolution. These bioprinted constructs were then able to support advanced maturation of cells into multinucleated muscle fibres, as evidenced by molecular analysis and functional testing. There was a significantly greater upregulation of genetic markers of myogenesis when compared to monolayer myotube cultures, and this result was complemented with functional testing that demonstrated mature patterns of calcium handling and electrical activity. The bioprinted muscle was then implanted in the nude rat to assess its capacity for innervation and vascularisation. The tissue construct was implanted in an in vivo chamber, which was supplied by a surgically formed arteriovenous loop and transected nerve. After only two weeks, independent bundles of mature muscle fibres had developed, with histological evidence of neural integration and vascularisation. In vivo electrophysiological studies confirmed the presence of innervation by demonstrating muscle activity in response to neural stimulation. Lastly, the triad of bioprinted muscle, vasculature and nerve was housed in a customised 3D printed chamber as a prototype for a bioelectrode that could be surgically grafted onto transected peripheral nerves after limb amputation. To conclude, this thesis developed the principle elements of designing a bioelectrode for neuroprosthetic interfacing and provides the foundation for further tissue optimisation and integration of electrodes. Although the work presented is in the frontier stages of development, it offers an exciting glimpse into the future of modern medicine and brings the dream of mind-controlled motorised prosthetic limbs closer to everyday reality.