Sir Peter MacCallum Department of Oncology - Research Publications
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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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ItemGenerating CAR T cells from tumor-infiltrating lymphocytes.(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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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.
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ItemMultiplex immunohistochemistry accurately defines the immune context of metastatic melanoma(NATURE PORTFOLIO, 2018-07-24)A prospective study explored the heterogeneous nature of metastatic melanoma using Multiplex immunohistochemistry (IHC) and flow cytometry (FACS). Multiplex IHC data quantitated immune subset number present intra-tumoral (IT) vs the tumor stroma, plus distance of immune subsets from the tumor margin (TM). In addition, mIHC showed a close association between the presence of IT CD8+ T cells and PDL1 expression in melanoma, which was more prevalent on macrophages than on melanoma cells. In contrast, FACS provided more detailed information regarding the T cell subset differentiation, their activation status and expression of immune checkpoint molecules. Interestingly, mIHC detected significantly higher Treg numbers than FACS and showed preferential CD4+ T cell distribution in the tumor stroma. Based on the mIHC and FACS data, we provide a model which defines metastatic melanoma immune context into four categories using the presence or absence of PDL1+ melanoma cells and/or macrophages, and their location within the tumor or on the periphery, combined with the presence or absence of IT CD8+ T cells. This model interprets melanoma immune context as a spectrum of tumor escape from immune control, and provides a snapshot upon which interpretation of checkpoint blockade inhibitor (CBI) therapy responses can be built.
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ItemCAR-T Cells Inflict Sequential Killing of Multiple Tumor Target Cells(AMER ASSOC CANCER RESEARCH, 2015-05)Adoptive therapy with chimeric antigen receptor (CAR) T cells shows great promise clinically. However, there are important aspects of CAR-T-cell biology that have not been explored, particularly with respect to the kinetics of activation, immune synapse formation, and tumor cell killing. Moreover, the effects of signaling via the endogenous T-cell receptor (TCR) or CAR on killing kinetics are unclear. To address these issues, we developed a novel transgenic mouse (designated CAR.OT-I), in which CD8(+) T cells coexpressed the clonogenic OT-I TCR, recognizing the H-2K(b)-presented ovalbumin peptide SIINFEKL, and an scFv specific for human HER2. Primed CAR.OT-I T cells were mixed with SIINFEKL-pulsed or HER2-expressing tumor cells and visualized in real-time using time-lapse microscopy. We found that engagement via CAR or TCR did not affect cell death kinetics, except that the time from degranulation to CAR-T-cell detachment was faster when CAR was engaged. We showed, for the first time, that individual CAR.OT-I cells can kill multiple tumor cells ("serial killing"), irrespective of the mode of recognition. At low effector:target ratios, the tumor cell killing rate was similar via TCR or CAR ligation over the first 20 hours of coincubation. However, from 20 to 50 hours, tumor cell death mediated through CAR became attenuated due to CAR downregulation throughout the time course. Our study provides important insights into CAR-T-tumor cell interactions, with implications for single- or dual receptor-focused T-cell therapy.