Sir Peter MacCallum Department of Oncology - Research Publications

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    IL-15 Preconditioning Augments CAR T Cell Responses to Checkpoint Blockade for Improved Treatment of Solid Tumors
    Giuffrida, L ; Sek, K ; Henderson, MA ; House, IG ; Lai, J ; Chen, AXY ; Todd, KL ; Petley, E ; Mardiana, S ; Todorovski, I ; Gruber, E ; Kelly, MJ ; Solomon, BJ ; Vervoort, SJ ; Johnstone, RW ; Parish, IA ; Neeson, PJ ; Kats, LM ; Darcy, PK ; Beavis, PA (CELL PRESS, 2020-11-04)
    Chimeric antigen receptor (CAR) T cell therapy has been highly successful in hematological malignancies leading to their US Food and Drug Administration (FDA) approval. However, the efficacy of CAR T cells in solid tumors is limited by tumor-induced immunosuppression, leading to the development of combination approaches, such as adjuvant programmed cell death 1 (PD-1) blockade. Current FDA-approved methods for generating CAR T cells utilize either anti-CD3 and interleukin (IL)-2 or anti-CD3/CD28 beads, which can generate a T cell product with an effector/exhausted phenotype. Whereas different cytokine preconditioning milieu, such as IL-7/IL-15, have been shown to promote T cell engraftment, the impact of this approach on CAR T cell responses to adjuvant immune-checkpoint blockade has not been assessed. In the current study, we reveal that the preconditioning of CAR T cells with IL-7/IL-15 increased CAR T cell responses to anti-PD-1 adjuvant therapy. This was associated with the emergence of an intratumoral CD8+CD62L+TCF7+IRF4- population that was highly responsive to anti-PD-1 therapy and mediated the vast majority of transcriptional and epigenetic changes in vivo following PD-1 blockade. Our data indicate that preservation of CAR T cells in a TCF7+ phenotype is crucial for their responsiveness to adjuvant immunotherapy approaches and should be a key consideration when designing clinical protocols.
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    Tissue-specific tumour microenvironments are an emerging determinant of immunotherapy responses
    Oliver, AJ ; Darcy, PK ; Kershaw, MH ; Slaney, CY (AME PUBL CO, 2020-08)
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    Chimeric Antigen Receptor T cell Therapy and the Immunosuppressive Tumor Microenvironment in Pediatric Sarcoma
    Terry, RL ; Meyran, D ; Fleuren, EDG ; Mayoh, C ; Zhu, J ; Omer, N ; Ziegler, DS ; Haber, M ; Darcy, PK ; Trapani, JA ; Neeson, PJ ; Ekert, PG (MDPI, 2021-09)
    Sarcomas are a diverse group of bone and soft tissue tumors that account for over 10% of childhood cancers. Outcomes are particularly poor for children with refractory, relapsed, or metastatic disease. Chimeric antigen receptor T (CAR T) cells are an exciting form of adoptive cell therapy that potentially offers new hope for these children. In early trials, promising outcomes have been achieved in some pediatric patients with sarcoma. However, many children do not derive benefit despite significant expression of the targeted tumor antigen. The success of CAR T cell therapy in sarcomas and other solid tumors is limited by the immunosuppressive tumor microenvironment (TME). In this review, we provide an update of the CAR T cell therapies that are currently being tested in pediatric sarcoma clinical trials, including those targeting tumors that express HER2, NY-ESO, GD2, EGFR, GPC3, B7-H3, and MAGE-A4. We also outline promising new CAR T cells that are in pre-clinical development. Finally, we discuss strategies that are being used to overcome tumor-mediated immunosuppression in solid tumors; these strategies have the potential to improve clinical outcomes of CAR T cell therapy for children with sarcoma.
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    Chimeric antigen receptor T cell therapies for thoracic cancers-challenges and opportunities
    Chan, JD ; Harrison, AJ ; Darcy, PK ; Kershaw, MH ; Slaney, CY (AME PUBL CO, 2020-08)
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    A New Safety Approach Allowing Reversible Control of CAR T Cell Responses
    Chen, AXY ; House, IG ; Beavis, PA ; Darcy, PK (CELL PRESS, 2020-07-08)
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    MAIT cells regulate NK cell-mediated tumor immunity
    Petley, E ; Koay, H-F ; Henderson, MA ; Sek, K ; Todd, KL ; Keam, SP ; Lai, J ; House, IG ; Li, J ; Zethoven, M ; Chen, AXY ; Oliver, AJ ; Michie, J ; Freeman, AJ ; Giuffrida, L ; Chan, JD ; Pizzolla, A ; Mak, JYW ; McCulloch, TR ; Souza-Fonseca-Guimaraes, F ; Kearney, CJ ; Millen, R ; Ramsay, RG ; Huntington, ND ; McCluskey, J ; Oliaro, J ; Fairlie, DP ; Neeson, PJ ; Godfrey, D ; Beavis, PA ; Darcy, PK (NATURE PORTFOLIO, 2021-08-06)
    The function of MR1-restricted mucosal-associated invariant T (MAIT) cells in tumor immunity is unclear. Here we show that MAIT cell-deficient mice have enhanced NK cell-dependent control of metastatic B16F10 tumor growth relative to control mice. Analyses of this interplay in human tumor samples reveal that high expression of a MAIT cell gene signature negatively impacts the prognostic significance of NK cells. Paradoxically, pre-pulsing tumors with MAIT cell antigens, or activating MAIT cells in vivo, enhances anti-tumor immunity in B16F10 and E0771 mouse tumor models, including in the context of established metastasis. These effects are associated with enhanced NK cell responses and increased expression of both IFN-γ-dependent and inflammatory genes in NK cells. Importantly, activated human MAIT cells also promote the function of NK cells isolated from patient tumor samples. Our results thus describe an activation-dependent, MAIT cell-mediated regulation of NK cells, and suggest a potential therapeutic avenue for cancer treatment.
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    Preclinical Evidence of the Efficacy of Lewis Y Car T Cells in Patient-Derived Models of Prostate Cancer
    Risbridger, GP ; Porter, LH ; Zhu, J ; Byrne, D ; Lister, N ; Azad, A ; Hofman, M ; Vela, I ; Taylor, RA ; Neeson, P ; Darcy, P ; Trapani, J (The Endocrine Society, 2021-05-03)
    Abstract Chimeric antigen receptor T (CAR T) cell therapy is an adoptive immunotherapy that has led to new treatments for lymphoma, leukemia, and other blood cancers; however, its efficacy for prostate cancer remains unproven. Here we report pre-clinical evidence of the efficacy of CAR T cell therapy against the Lewis Y antigen (LeY) using patient-derived models of prostate cancer. To assess the expression of LeY on prostate tumours, we performed immunohistochemistry on a cohort of 41 patient-derived xenografts (PDXs). Cytoplasmic and membrane expression were separately assessed and quantified, for each patient. Overall, 61% (25/41) of PDXs were positive for membrane LeY expression, of which 18 PDXs had greater than 50% membrane-positive cells, and considered most suitable to detection and stable binding by anti-LeY CAR T’s. To determine the in vitro sensitivity to CAR T cytotoxicity, we selected 4 PDXs with high and 2 PDXs with low LeY expression using 3 androgen receptor (AR)-positive adenocarcinomas and 3 AR-negative tumors expressing neuroendocrine markers. Next we established organoids for in vitro co-culture assays where organoids were co-incubated with an equal number of anti-LeY+ CAR T cells or Empty vector control CAR T cells (Ev CAR T). Using time-lapse microscopy we reported destruction of organoids by LeY+ CAR T cells as indicated by their morphological collapse and uptake of propidium iodide from the culture medium; control Ev CAR T cells produced no cytotoxicity. Over the 48h assay, the level of target cell death of the LeY+ organoids was correlated to the intensity LeY surface expression. Target cell death mediated by the CAR T cells required perforin and granzyme B, as potent and highly specific small molecule inhibitors of perforin (SN34960) and granzyme B (C20) applied alone or in combination greatly decreased PI uptake, indicating organoid survival. Neither inhibitor adversely affected CAR T cell viability as measured by PI and Annexin V staining. This demonstrated canonical activation of granule exocytosis pathway by the CAR T cells, leading to organoid cell death. To assess CAR T cell efficacy in vivo, we selected one PDX with high LeY expression. Monotherapy with CAR T cells failed to decrease tumour volume compared to vehicle control. However, CAR T cells given after a single dose of the chemotherapeutic agent carboplatin greatly and durably reduced tumour burden, with residual tumour mass being less than 1% of their original size (0.56 ± 0.23% of tumour volume at the start of treatment). Overall, these data provide preclinical evidence that: i) high membrane expression of LeY correlates with in vitro and in vivo CAR T cell-induced tumour cell death via the canonical perforin/granzyme B mechanism; and, ii) membrane LeY can be used as a biomarker for patient selection.
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    Myeloma natural killer cells are exhausted and have impaired regulation of activation
    D'Souza, C ; Keam, SP ; Yeang, HXA ; Neeson, M ; Richardson, K ; Hsu, AK ; Canfield, R ; Bezman, N ; Robbins, M ; Quach, H ; Ritchie, DS ; Harrison, SJ ; Trapani, JA ; Prince, HM ; Beavis, PA ; Darcy, PK ; Neeson, PJ (FERRATA STORTI FOUNDATION, 2021-09)
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    Generating CAR T cells from tumor-infiltrating lymphocytes.
    Mills, JK ; Henderson, MA ; Giuffrida, L ; Petrone, P ; Westwood, JA ; Darcy, PK ; Neeson, PJ ; Kershaw, MH ; Gyorki, DE (SAGE Publications, 2021)
    Background: Tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T-cell therapies have demonstrated promising, though limited, efficacy against melanoma. Methods: We designed a model system to explore the efficacy of dual specific T cells derived from melanoma patient TILs by transduction with a Her2-specific CAR. Results: Metastatic melanoma cells in our biobank constitutively expressed Her2 antigen. CAR-TIL produced greater amounts of IFN compared with parental TIL, when co-cultured with Her2 expressing tumor lines, including autologous melanoma tumor lines, although no consistent increase in cytotoxicity by TIL was afforded by expression of a CAR. Results of an in vivo study in NSG mice demonstrated tumor shrinkage when CAR-TILs were used in an adoptive cell therapy protocol. Conclusion: Potential limitations of transduced TIL in our study included limited proliferative potential and a terminally differentiated phenotype, which would need addressing in further work before consideration of clinical translation.
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    HIGH DOSE-RATE BRACHYTHERAPY OF LOCALIZED PROSTATE CANCER CONVERTS TUMORS FROM COLD TO HOT
    Keam, S ; Halse, H ; ThuNgoc, N ; Wang, M ; Losio, NVK ; Mitchell, C ; Caramia, F ; Byrne, D ; Haupt, S ; Ryland, G ; Darcy, P ; Sandhu, S ; Blombery, P ; Haupt, Y ; Williams, S ; Neeson, P (BMJ PUBLISHING GROUP, 2020-11)
    Background Prostate cancer is frequently cured with high dose-rate brachytherapy (HDRBT) radiation as a front-line treatment. Although considered to be an immune-excluded tissue, immune responses to radiation are implicated in driving tumour-eradication in prostate cancer.1 This has not been proven, and yet is used as the rationale for clinical trials combining radiation and immunotherapies.2 We hypothesise that there is a predictable relationship between radiation and the immune responses in prostate cancer that could be used to provide sound rationale for specific immune interventions in solid tumours that are made possible by radiation therapy. Methods We present here new results stemming from our recently published immunoprofiling study of world-unique pre- and post-radiation tissues from 24 prostate cancer patients (figure 1A), RadBank cohort).3 These samples were assessed using immune cell multiplex IHC, gene expression profiling, digital spatial profiling (DSP) and computational analysis of cell distribution. Results This study unequivocally revealed that high dose-rate radiation converts predominately ‘cold’ prostate tumour tissue to a more activated ‘hot’ state comprised of two sub-types (high and a less activated intermediate state). These changes were evident in increased tumour inflammation gene signatures and immune checkpoint expression, immune cell composition changes, and alterations in spatial interactions. However, as 20% of the patients did not respond, we also explored pre-treatment gene signatures of patient responses to radiation – identifying potential mechanisms that prime tissues to respond more favourably. Most recently, we have explored three other important facets of the immune response to HDRBT: (i) putative differential drivers of high and intermediate responses (figure 1B), (ii) TCR clonality changes (figure 1C), and (iii) the influence of clinical features (e.g. Gleason grade) and treatment (e.g. androgen deprivation) (figure 1D). Differential expression analysis has identified key molecules (e.g. CD40LG and Lck expression) which are associated with higher activation responses. TCR sequencing of pre- and post-HDRBT tissue and peripheral circulating cells is also suggestive of engagement of the adaptive immune system and the emergence of tumor-specific T cells. Finally, multivariate analysis has also revealed that higher grade tumours exhibit higher basal levels of activation and IC expression – making them less sensitive to immune activation by HDRBT. Abstract 580 Figure 1The effect of prostate brachytherapy on immune contexts(A) Study of immune response in 24 patients treated with HDRBT at Peter MacCallum Cancer Center ((DOI:10.1136/jitc2020-000792). Examples of new insights including (B) molecules associated with higher activation levels (e.g. Lck and CD40LG/CD154), (C) changes in T cell receptor dominance and diversity in tissue and peripheral circulation, and (D) effects of clinical attributes on immune modulators (e.g. TGFbeta) and TIS activation states. Conclusions We have begun to resolve clear patient and clinical classifiers based on immune responses to radiation, and identified patient groups likely to benefit from immune therapy alongside radiation. Importantly, these classifications are associated with baseline gene expression profiles that may be used for pre-clinical stratification and more sophisticated treatment paradigms. Ethics Approval All participants provided consent covering tissue research as part of a prospective tissue collection study for prostate radiobiology research, approved by the Human Research Ethics Committee at the Peter MacCallum Cancer Centre (PMCC; HREC approvals 10/68, 13/167, 18/204). Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal. References Dudzinski SO, et al., Combination immunotherapy and radiotherapy causes an abscopal treatment response in a mouse model of castration resistant prostate cancer. J Immunother Cancer 2019. 7(1): p. 218. Kwon E.D., et al., Ipilimumab versus placebo after radiotherapy in patients with metastatic castration-resistant prostate cancer that had progressed after docetaxel chemotherapy (CA184-043): a multicentre, randomised, double-blind, phase 3 trial. Lancet Oncol 2014;15(7): p. 700–12. Keam SP, et al., High dose-rate brachytherapy of localized prostate cancer converts tumors from cold to hot. J Immunother Cancer 2020;8(1).