Sir Peter MacCallum Department of Oncology - Research Publications
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ItemNo Preview AvailableLow-dose carboplatin modifies the tumor microenvironment to augment CAR T cell efficacy in human prostate cancer models(NATURE PORTFOLIO, 2023-09-02)Chimeric antigen receptor (CAR) T cells have transformed the treatment landscape for hematological malignancies. However, CAR T cells are less efficient against solid tumors, largely due to poor infiltration resulting from the immunosuppressive nature of the tumor microenvironment (TME). Here, we assessed the efficacy of Lewis Y antigen (LeY)-specific CAR T cells in patient-derived xenograft (PDX) models of prostate cancer. In vitro, LeY CAR T cells directly killed organoids derived from androgen receptor (AR)-positive or AR-null PDXs. In vivo, although LeY CAR T cells alone did not reduce tumor growth, a single prior dose of carboplatin reduced tumor burden. Carboplatin had a pro-inflammatory effect on the TME that facilitated early and durable CAR T cell infiltration, including an altered cancer-associated fibroblast phenotype, enhanced extracellular matrix degradation and re-oriented M1 macrophage differentiation. In a PDX less sensitive to carboplatin, CAR T cell infiltration was dampened; however, a reduction in tumor burden was still observed with increased T cell activation. These findings indicate that carboplatin improves the efficacy of CAR T cell treatment, with the extent of the response dependent on changes induced within the TME.
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ItemNo Preview AvailableTSTEM-like CAR-T cells exhibit improved persistence and tumor control compared with conventional CAR-T cells in preclinical models(AMER ASSOC ADVANCEMENT SCIENCE, 2023-04-05)Patients who receive chimeric antigen receptor (CAR)-T cells that are enriched in memory T cells exhibit better disease control as a result of increased expansion and persistence of the CAR-T cells. Human memory T cells include stem-like CD8+ memory T cell progenitors that can become either functional stem-like T (TSTEM) cells or dysfunctional T progenitor exhausted (TPEX) cells. To that end, we demonstrated that TSTEM cells were less abundant in infused CAR-T cell products in a phase 1 clinical trial testing Lewis Y-CAR-T cells (NCT03851146), and the infused CAR-T cells displayed poor persistence in patients. To address this issue, we developed a production protocol to generate TSTEM-like CAR-T cells enriched for expression of genes in cell replication pathways. Compared with conventional CAR-T cells, TSTEM-like CAR-T cells had enhanced proliferative capacity and increased cytokine secretion after CAR stimulation, including after chronic CAR stimulation in vitro. These responses were dependent on the presence of CD4+ T cells during TSTEM-like CAR-T cell production. Adoptive transfer of TSTEM-like CAR-T cells induced better control of established tumors and resistance to tumor rechallenge in preclinical models. These more favorable outcomes were associated with increased persistence of TSTEM-like CAR-T cells and an increased memory T cell pool. Last, TSTEM-like CAR-T cells and anti-programmed cell death protein 1 (PD-1) treatment eradicated established tumors, and this was associated with increased tumor-infiltrating CD8+CAR+ T cells producing interferon-γ. In conclusion, our CAR-T cell protocol generated TSTEM-like CAR-T cells with enhanced therapeutic efficacy, resulting in increased proliferative capacity and persistence in vivo.
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ItemChimeric Antigen Receptor T cell Therapy and the Immunosuppressive Tumor Microenvironment in Pediatric Sarcoma(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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ItemCMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity(NATURE RESEARCH, 2017-09-07)Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell-mediated immunosurveillance. The success of therapies that disrupt PD-L1-mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression. Here we identify the uncharacterized protein CMTM6 as a critical regulator of PD-L1 in a broad range of cancer cells, by using a genome-wide CRISPR-Cas9 screen. CMTM6 is a ubiquitously expressed protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes, where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome, we find that CMTM6 displays specificity for PD-L1. Notably, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour-specific T cell activity in vitro and in vivo. These findings provide insights into the biology of PD-L1 regulation, identify a previously unrecognized master regulator of this critical immune checkpoint and highlight a potential therapeutic target to overcome immune evasion by tumour cells.
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ItemNo Preview AvailablePreclinical Evidence of the Efficacy of Lewis Y Car T Cells in Patient-Derived Models of Prostate Cancer(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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ItemNo Preview AvailableMyeloma natural killer cells are exhausted and have impaired regulation of activation(FERRATA STORTI FOUNDATION, 2021-09)Not available.
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ItemCross-talk between tumors at anatomically distinct sites(WILEY, 2021-01)Cancer tissue is not homogenous, and individual metastases at different anatomical locations can differ from the primary tumor and from one another in both their morphology and cellular composition, even within an individual patient. Tumors are composed of cancer cells and a range of other cell types, which, together with a variety of secreted molecules, collectively comprise the tumor microenvironment (TME). Cells of the TME can communicate with each other and with distant tissues in a form of molecular cross-talk to influence their growth and function. Cross-talk between cancer cells and local immune cells is well described and can lead to the induction of local immunosuppression. Recently, it has become apparent that tumors located remotely from each other, can engage in cross-talk that can influence their responsiveness to various therapies, including immunotherapy. In this article, we review studies that describe how tumors systemically communicate with distant tissues through motile cells, extracellular vesicles, and secreted molecules that can affect their function. In addition, we summarize evidence from mouse studies and the clinic that indicate an ability of some tumors to influence the progression and therapeutic responses of other tumors in different anatomical locations.
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ItemEARLY-PHENOTYPE LEWIS Y CAR-T CELLS PERSIST BETTER IN VIVO AND INDUCE SOLID TUMOR REGRESSION IN COMBINATION WITH ANTI-PD1(BMJ PUBLISHING GROUP, 2020-11)Background Chimeric antigen receptor (CAR-T) cells are a promising new therapy for patients with cancer. However, in contrast to their success in B cell malignancies, CAR-T cells targeting solid cancers have had limited success so far due to their poor proliferation and poor long-term persistence in vivo. To address this issue, we used naïve T cells to generate second-generation CAR-T cells recognizing the tumor antigen Lewis Y (LeY), termed ‘early’ CAR-T cells. Methods Purified naïve T cells were activated by CD3/CD28 soluble tetrameric antibody complex, retrovirally transduced (LeY scFv-CD3z-CD28 CAR) and expanded in IL-7/IL-15. The early LeY CAR-T cell function was tested in vitro for cytotoxicity (Cr-release and degranulation), proliferation, and cytokine secretion by CBA, either de novo or following chronic stimulation for 1 month. Finally, early CAR-T cell persistence and anti-tumor efficacy was assessed in the OVCAR3-NSG model, in the presence or absence of anti-PD-1. Results The early-CAR-T cells comprised stem cell memory-like (CD95+, CD62L+, CD45RA+) and central memory phenotype (CD95+, CD62L+, CD45RA-) T cells with increased expression of ICOS, Ki67, TCF7 and CD27 (Figure 1). The early-CAR-T cells retained potent antigen-specific cytotoxicity, and secreted significantly higher levels of cytokines (IFN-?, TNF-a and IL-2) and increased proliferation compared to conventional CAR-T cells. Importantly, early-CAR-T cells had a significantly higher proliferative capacity after long-term chronic stimulation compared to conventional CAR-T cells (figure 2), and CD4+ CAR-T cells were critical for effective early CD8+ CAR-T cell proliferation capacity in vitro (figure 3). Early CAR-T cells had significantly better in vivo tumor control compared to conventional CAR-T cells (Figure 4), this was associated with increased CAR-T cell persistence. Because chronically stimulated early-LeY-CAR-T cells expressed PD-1 (figure 2), and OVCAR-3 cells expressed PD-L1 when co-cultured with LeY-CAR-T cells (figure 5), we combined early LeY-CAR-T cells with anti-PD-1 therapy and observed complete tumour regression in these mice. Interestingly, early LeY-CAR-T cell plus anti-PD-1 treatment also enhanced the percentage of circulating stem-cell memory like CAR-T cells in vivo (figure 5). Abstract 126 Figure 1Early-CAR-T protocol, including Naïve-T cells purification and expansion in IL-7 and IL-15 promotes the maintenance of a TSCM and TCM phenotype. A) Scheme of the 7-day production protocol for Early-CAR-T cells. B) Phenotype by FACs of the conventional CAR-T cells and the Early-CAR-T cells. Pooled data in triplicate for 6 donors. C) Phenotype by Mass cytometry comparing the Conventional-CAR-T cells vs Early-CAR-T cells vs Early-CD8-CAR-T cells. Data for one donor representative of 3 different donors Abstract 126 Figure 2Early-CAR-T cells are comparable in vitro to conventional CAR-T cells in terms of killing but have a better proliferation capacity that persists after chronic stimulation. The long-term stimulated early- CAR-T cells maintain their memory phenotype and upregulated PD-1. A) Chromium release assay against the LeY+ cell line (OVCAR3), data for one donor representative of 3 other donors. B) Cytokine secretion evaluated by CBA after coculture with the LeY+ cell line (OVCAR3) or with the LeY- cell line (MDA-MB435). C) Division index of CAR-T cells quantified with CTV. D) Evaluation of the differentiation, proliferation and cytotoxicity of the CAR-T cells after chronic stimulation Abstract 126 Figure 3Early-CD4+- CAR-T cells are critical for the proliferation capacity of the Early-CD8+-CAR-T cells. A) Scheme of the CD4-depletion protocol to compare Early-CD8-CAR-T proliferation with or without CD4-T cells. B) Division index of CD4-depleted Early-CAR-T cells, CD8-T cells from bulk Early-CAR-T cells, and from CD4+ T cells from bulk Early-CAR-T-cells quantified with CTV Abstract 126 Figure 4Early-CAR-T cells show in vivo a better persistence and a better proliferation capacity associated with a better tumoral control. A) Design of the in vivo experiment (n=7 mice per group) B) T-cell persistence in peripheral blood was measured by FACS. C) Speakman correlation (Day 13) between Tumor size and% CAR-T- cells. D) Tumor kinetic and Kaplan-Meier analysis of survival of OVCAR-bearing NSG mice treated with Conventional CAR-T cells, or Early-CAR-T cells or Low-dose of Early-CAR-T cells Abstract 126 Figure 5Anti-PD1 treatment enhance the efficacy of the Early-CAR-T cells. A) Upregulation of PD-L1 on OVCAR3 when expanded in the supernatant from co-culture of OVCAR3 with LeY-CAR-T cells. B) Design of the in vivo experiment (n=7 mice per group). C) T-cell persistence, phenotype and anti-human IgG4 in peripheral blood were measured by FACS. D) Tumor kinetic of OVCAR-bearing NSG mice treated with Early-CAR-T cells or Early-CAR-T cells + Nivolumab Conclusions Our early CAR-T cells have better cytokine secretion and proliferation than conventional CAR-T cells. Early CAR-T cells also have superior anti-tumor efficacy in vivo, they have better persistence and maintain the circulating T cell memory pool. Importantly, low dose early-LeY-CAR-T cells combined with anti-PD1-treatment leads to complete clearance of LeY+ solid tumors in vivo. The early CAR-T cell production protocol is directly translatable for improving CAR-T cell efficacy in clinical trials for patients with solid tumors.
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ItemAgonist immunotherapy restores T cell function following MEK inhibition improving efficacy in breast cancer(NATURE PUBLISHING GROUP, 2017-09-19)The presence of tumor-infiltrating lymphocytes in triple-negative breast cancers is correlated with improved outcomes. Ras/MAPK pathway activation is associated with significantly lower levels of tumor-infiltrating lymphocytes in triple-negative breast cancers and while MEK inhibition can promote recruitment of tumor-infiltrating lymphocytes to the tumor, here we show that MEK inhibition adversely affects early onset T-cell effector function. We show that α-4-1BB and α-OX-40 T-cell agonist antibodies can rescue the adverse effects of MEK inhibition on T cells in both mouse and human T cells, which results in augmented anti-tumor effects in vivo. This effect is dependent upon increased downstream p38/JNK pathway activation. Taken together, our data suggest that although Ras/MAPK pathway inhibition can increase tumor immunogenicity, the negative impact on T-cell activity is functionally important. This undesirable impact is effectively prevented by combination with T-cell immune agonist immunotherapies resulting in superior therapeutic efficacy.MEK inhibition in breast cancer is associated with increased tumour infiltrating lymphocytes (TILs), however, MAPK activity is required for T cells function. Here the authors show that TILs activity following MEK inhibition can be enhanced by agonist immunotherapy resulting in synergic therapeutic effects.
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ItemChimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity(NATL ACAD SCIENCES, 2018-02-27)Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell.