Robert Holt


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Doctoral Student Supervision

Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.

The modification of chimeric antigen receptor T cells to overcome immune suppression mediated by indoleamine 2,3-dioxygenase (2024)

Chimeric antigen receptor (CAR) T cells are regarded as a major breakthrough for treating hematological malignancies. Through expression of a receptor comprising an extracellular antigen-binding domain and intracellular stimulatory domains, CAR T cells can be re-directed to target cells expressing the antigen of choice where they are activated to proliferate and kill their targets. Despite representing a major advancement in the treatment of “liquid” tumours, the success of CAR T cells in solid tumours has been hampered in part by the immunosuppressive tumour microenvironment. Tumours form a complex network of cells and signaling molecules that promote tumour growth while suppressing the immune response. One of these mechanisms of immunosuppression is indoleamine 2,3-dioxyganse (IDO). IDO degrades tryptophan into a set of immunosuppressive metabolites called kynurenines. Through these actions, IDO activates suppressive immune cells while inhibiting anti-tumour immunity. In CAR T-cell therapy, IDO expression can suppress T-cell mediated killing and compromise treatment efficacy.The aim of this thesis is to enhance the ability of CAR T cells to contend with IDO-mediated immunosuppression in the tumour microenvironment. This engineering strategy involves the enzymatic degradation of suppressive kynurenines, which first requires knowledge of the specific kynurenines involved. Herein I show that of all kynurenines, kynurenine itself most strongly suppresses CAR T cells through the aryl hydrocarbon receptor, and I demonstrate the ability of T cells expressing kynureninase (KYNase) to degrade kynurenine in vitro. I outline the optimization of a model system to assess IDO-mediated immunosuppression of human CAR T cells in vivo, and use this system to evaluate the therapeutic effect of KYNase modification. I show that, while KYNase expression is ineffective at rescuing human CAR T-cell suppression in a subcutaneous tumour model in vivo, the modification of the CAR itself may influence its ability to withstand the immunosuppressive milieu produced by IDO. Furthermore, I identify the solvents used in in vitro assays as a possible confounding factor that may influence the interpretation of decades of research into kynurenine. This thesis contributes towards a better appreciation of the variability in IDO-mediated immunosuppression in vitro and in vivo, between species, and across diverse model systems.

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Characterizing the granzyme-perforin pathway and its utility as a cell-to-cell delivery system for cellular therapeutics (2019)

Alongside small molecules and biologics, cell-based therapies are emerging as a third class of medical therapy. Additional sensors, actuators and control circuits would greatly expand the range of function and application of cellular therapeutics. To this end, a cell-to-cell delivery module has been developed by investigating and re-engineering the granzyme-perforin pathway of cytotoxic lymphocytes. A computational biophysical model of this process was developed and implemented using a spatial stochastic simulation algorithm, which indicated that hindered diffusion in the immune synapse is critical to ensure reliable granzyme internalization and that large amounts of granzyme escape the synapse, but should not have toxic effects due to rapid spatiotemporal dilution. Additionally, these results indicated that passive diffusion is sufficient for granzyme entry into the target cell, which motivated efforts to use granzyme as a molecular chaperone to transfer exogenous payloads from effector to target cells. Using a fluorescent protein payload, the subcellular localization of several granzyme B derived chaperones was characterized using fluorescence microscopy, and then their capacity to transfer the payload to target cells was evaluated in co-culture experiments. The results indicated that the motifs in granzyme B that are required for lytic granule loading are only functional and contiguous in the folded protein. Additionally, these experiments demonstrated that full length granzyme B is a suitable chaperone for delivering protein payloads to target cells via the granzyme-perforin pathway. Attempts were then made to use this system to deliver potent orthogonal toxins to apoptosis and lymphocyte resistant tumor cells. A range of granzyme B toxin fusion proteins were constructed, all of which retained toxic activity to varying degrees. To render target cells resistant to lymphocyte attack both small molecule and protein based inhibitors of apoptosis were tested in several cell lines, which delayed cell death, but did not stop it. Using effector target dose response curves, a moderate increase in target cell death was observed in cells targeted by lymphocytes expressing granzyme toxin fusion proteins, as compared to wild type lymphocytes, but the biological significance of this effect is uncertain. Approaches to improve this granzyme-perforin mediated delivery system and its therapeutic utility are discussed and explored.

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Interrogating the TCR-pMHC complex in health and disease using immunogenomics methods (2019)

The adaptive immune system is a complex network of cells working towards a common goal: detection and elimination of foreign cells that can harm the host. In cancer, malignant cells acquire mutations which can appear foreign to the adaptive immune system. The immune cells most directly involved in destruction of cancer cells are CD8+ T cells, using their T cell receptor (TCR) to recognize mutated peptides presented on cancer cells in the context of class I Major Histocompatibility Complex (MHC) molecules (pMHC). Immunogenomics methods offer ways to interrogate this TCR-pMHC interaction using genomics data.The aim of this thesis is to adapt and apply novel and existing immunoinformatic methods to cancer datasets to identify relationships between the immune system and cancer in a pan-cancer context. This involves prediction of cancer neoantigens derived from single nucleotide variants (SNVs) from tumours, and correlation of this neoantigen burden with outcomes and markers of immune inhibition. It involves extraction of TCR sequences from RNA-seq datasets to gain value-added information from these existing datasets, with demonstrated utility in solid tumours and lymphomas. Finally, it defines and explores the size of the self-immunopeptidome to classify individuals based on their ability to present peptides on class I MHC molecules. I show that T cell infiltration of solid tumours correlates with improved outcomes, neoantigen load, but also markers of T cell inhibition, suggesting that these individuals would benefit from checkpoint blockade therapy. In established tumours, the T cell repertoire is not clonal, and among the most abundant T cells in the tumour are viral-specific T cells also found in the normal repertoire. This information is obtained directly from existing RNA-seq datasets of tumours. When applied to RNA-seq of sorted T cell populations, clonally expanded T cells are detectable by their TCR, and alpha-beta pairing can be inferred. The self-immunopeptidome can be used to predict neoantigen load and is used to infer signatures of neoantigen immunogenicity. This thesis contributes towards a better understanding of the interaction between T cells and cancer cells, which can inform future strategies to improve immunotherapies in cancer.

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Novel in vitro methods for the discovery of functional T-cell receptor epitopes from large peptide-coding libraries (2019)

Though it is understood that T-cells are a critical component of the immune system’s ability to destroy foreign invaders and attack cancerous cells, very little is known regarding the specific epitopes that are recognized by T-cells to carry out these functions. Generally, the epitopes mediating this immunity are short, contiguous peptides derived from antigenic proteins presented on major histocompatibility complex (MHC) molecules for inspection by T-cells. The ability to rapidly and deeply search peptide space to determine specific peptide epitopes that are naturally processed, presented, and capable of eliciting functional T-cell responses is a critical unmet need in the study of adaptive immunity. Here, I describe a novel method for deep T-cell epitope profiling that enables simultaneous in vitro interrogation of target cell populations encoding high-diversity minigene libraries with T-cell populations-of-interest. Targets eliciting T-cell reactivity are selectively isolated by fluorescence-activated cell sorting (FACS) and identified by deep amplicon sequencing. The approach was extensively validated using known murine T-cell receptor (TCR)/peptide-MHC pairs and it was shown that this method can unambiguously identify canonical minigenes from libraries of vastly more candidate antigens in parallel than would be feasibly tractable using conventional methods.The capability of this strategy was extended by applying a synthetic biology approach. Using pairs of immortalized natural killer (NK)-like effector cell lines and naturally tolerated target cell lines, I showed that fully reconstituting the TCR/CD3 complex in effectors and expressing relevant MHC-/minigene-coding sequences in targets is sufficient to re-direct the cytotoxicity of NK-like cell lines towards antigenic targets of recombinant TCR. These results provide indication that it should be possible to use an entirely synthetic framework for functionally screening recombinant TCR-of-interest against minigene libraries without the requirement to first isolate and expand primary T-cell clones or donor-derived antigen-presenting cells.The high-throughput T-cell antigen profiling methods described and validated in this thesis could allow investigators to generate TCR epitope data broader in scope than previously possible to better understand basic T-cell biology, develop better predictive models of T-cell reactivity, and rationally design T-cell-based immunotherapeutics for the treatment of cancer, infectious disease, and autoimmunity.

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Identfying and targeting immunogenic mutations in ovarian cancer (2016)

A hallmark of cancer is the accumulation of mutations, and a small proportion of these give rise to mutant neoantigens – mutated peptides bound to Major Histocompatibility Complex (MHC) and recognized by T cells. Accumulating evidence suggests that mutant neoantigens (hereafter referred to as “neoantigens”) underlie successful immune therapies in cancers with high mutation loads, such as melanoma. Moreover, neoantigen-specific vaccines have successfully targeted highly mutated murine tumor models. However, less is known about neoantigen-specific T cell responses in cancers with moderate mutation loads, such as ovarian cancer. I hypothesized that (1) modified peptide-based vaccination schedules can lead to enhanced antigen-specific T cell responses; (2) neoantigen-specific vaccines can elicit T cell responses that eradicate murine ovarian tumors; and (3) neoantigen-reactive T cells are detectable in human ovarian tumors and peripheral blood. To activate high frequencies of antigen-specific T cells, I developed a vaccination method involving repeated, daily immunizations with long peptides and adjuvant. This method elicited robust T cell responses that eliminated established murine tumors. I used these enhanced vaccination methods to target tumor-specific mutations identified by exome- and RNA-sequencing of the ovarian tumor model ID8-G7. Prophylactic and therapeutic vaccinations were performed targeting all expressed mutations that had a predicted MHCI binding affinity
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Investigation of Novel Schizophrenia Candidate Genes through Biochemical and Computational Methods (2010)

No abstract available.

Master's Student Supervision

Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.

Gene synthesis by assembly of short oligonucleotides (2010)

In principle, a pre-constructed library of all possible short oligonucleotides could be used to construct many distinct gene sequences. This approach requires computational methods to accurately determine the assembly procedure, but relieves the current technological constraints of custom oligonucleotide synthesis. In order to assess the feasibility of such an approach, I examined T4 DNA Ligase activity on short oligonucleotides and found that ligation is dependent on the formation of a double-stranded DNA duplex of at least five base pairs flanking the site of ligation. However, ligations could be performed with overhangs smaller than five nucleotides and oligonucleotides as small as octamers, in the presence of a second, complementary oligonucleotide. As a proof of principle for DNA synthesis through the assembly of short oligonucleotides, I performed a hierarchical ligation procedure whereby octamers were combined to construct a target 128 bp segment of the human beta–actin gene coding sequence. Thus, the construction of synthetic genes, without the need for custom oligonucleotide synthesis, is feasible. Algorithmic methods were then developed to extend this approach to DNA on the order of thousands of base pairs.

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