ORCID Profile
0000-0002-2331-5799
Current Organisation
University of South Australia
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Publisher: Springer Science and Business Media LLC
Date: 17-11-2020
DOI: 10.1186/S41181-020-00109-6
Abstract: The chimeric monoclonal antibody (mAb) chDAB4 (APOMAB®) targets the Lupus associated (La)/Sjögren Syndrome-B (SSB) antigen, which is over-expressed in tumors but only becomes available for antibody binding in dead tumor cells. Hence, chDAB4 may be used as a novel theranostic tool to distinguish between responders and nonresponders early after chemotherapy. Here, we aimed to ascertain which positron emitter, Zirconium-89 ([ 89 Zr]Zr IV ) or Iodine-124 ([ 124 I]I), was best suited to label chDAB4 for post-chemotherapy PET imaging of tumor-bearing mice and to determine which of two different bifunctional chelators provided optimal tumor imaging by PET using [ 89 Zr]Zr IV -labeled chDAB4. C57BL/6 J mice bearing subcutaneous syngeneic tumors of EL4 lymphoma were either untreated or given chemotherapy, then administered radiolabeled chDAB4 after 24 h with its biodistribution examined using PET and organ assay. We compared chDAB4 radiolabeled with [ 89 Zr] Zr IV or [ 124 I] I, or [ 89 Zr]Zr-chDAB4 using either DFO-NCS or DFOSq as a chelator. After chemotherapy, [ 89 Zr]Zr-chDAB4 showed higher and prolonged mean (± SD) tumor uptake of 29.5 ± 5.9 compared to 7.8 ± 1.2 for [ 124 I] I -chDAB4. In contrast, antibody uptake in healthy tissues was not affected. Compared to DFO-NCS, DFOSq did not result in significant differences in tumor uptake of [ 89 Zr]Zr-chDAB4 but did alter the tumor:liver ratio in treated mice 3 days after injection in favour of DFOSq (8.0 ± 1.1) compared to DFO-NCS (4.2 ± 0.7). ImmunoPET using chDAB4 radiolabeled with residualizing [ 89 Zr] Zr IV rather than [ 124 I] I optimized post-chemotherapy tumor uptake. Further, PET imaging characteristics were improved by DFOSq rather than DFO-NCS. Therefore, the radionuclide/chelator combination of [ 89 Zr] Zr IV and DFOSq is preferred for the imminent clinical evaluation of chDAB4 as a selective tumor cell death radioligand.
Publisher: American Society of Hematology
Date: 20-06-2017
DOI: 10.1182/BLOODADVANCES.2016002931
Abstract: High CD123 expression increases proliferation and results in enhanced survival in response to low concentration of IL-3 in vitro. High CD123-expressing LSCs downregulate chemokine receptor expression, affecting niche interactions.
Publisher: American Association for Cancer Research (AACR)
Date: 07-2018
DOI: 10.1158/1538-7445.AM2018-3910
Abstract: At the base of normal intestinal crypts, stem cells maintain the highly regenerative gut epithelium. These intestinal stem cells are well characterized for their high expression of the G coupled protein receptor LGR5 (also known as GPR49). Together with R-spondins (potent Wnt signaling modulators) and stem cell growth factors, LGR5 forms part of a signaling cascade responsible for the regulation of cellular proliferation. Key mutations in the APC or BRAF pathways of intestinal stem cells lead to lesions and metastatic colorectal cancer if not diagnosed and resected at an early stage. The majority of primary and metastatic tumors arising from these mutations overexpress LGR5. It has been reported that metastatic colorectal cancer (CRC) patients expressing higher levels of LGR5 in tumor biopsies had increased rates of relapse. BNC101 is a first-in-class anti-LGR5 humanized monoclonal antibody. Biacore analysis of BNC101 has demonstrated a high affinity to LGR5 (Kd=16nM) with no cross-reactivity to LGR4 or LGR6 receptors. Immunoprecipitation studies have also shown that BNC101 has no off-target binding. The murine equivalent of BNC101 has demonstrated antitumor activity in multiple CRC patient-derived xenografts. Flow cytometry studies on CRC cell lines have revealed that LGR5 is located intracellularly with a small fraction present on the cell surface. To better understand its mechanism of action, BNC101 was conjugated to Alexa Fluor 647 and incubated with human CRC cell lines. It was shown that BNC101 interacts with membrane-bound LGR5 and is internalized within 5 minutes. Incubation of the cells for 24 hours at clinically relevant concentrations led to accumulation of fluorophore-conjugated BNC101 within the cell. This intracellular accumulation of BNC101 was further demonstrated with receptor recycling kinetic studies whereby only partial receptor recycling occurred, which may be attributed to the large intracellular LGR5 pool. CHO cell lines overexpressing LGR5 with a GFP tag were used to determine co-localization of BNC101 with its LGR5 ligand. BNC101 is currently in a safety and dose escalation phase I clinical trial in patients with recurrent metastatic CRC. We were able to demonstrate BNC101 target engagement with LGR5 for the first time in tumor biopsies following treatment. Tumor biopsies were analyzed by mass spectrometry together with Matrix Assisted Laser Desorption/Ionization co-localization of BNC101 with LGR5 was observed, providing us with evidence for the first time that BNC101 infiltrates the patient tumor and engages with the overexpressed LGR5 receptor. Citation Format: Daniel J. Inglis, John Licari, Kristen R. Georgiou, Nicole L. Wittwer, Ross W. Hamilton, Donna M. Beaumont, Michaela A. Scherer, Tina C. Lavranos. Characterization of BNC101 a human specific monoclonal antibody targeting the GPCR LGR5: First-in-human evidence of target engagement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018 2018 Apr 14-18 Chicago, IL. Philadelphia (PA): AACR Cancer Res 2018 (13 Suppl):Abstract nr 3910.
Publisher: Springer Science and Business Media LLC
Date: 25-10-2023
Publisher: MDPI AG
Date: 24-04-2022
DOI: 10.3390/IJMS23094707
Abstract: Aluminium (Al) compounds are used as adjuvants in human and veterinary prophylactic vaccines due to their improved tolerability compared to other adjuvants. These Al-based adjuvants form microparticles (MPs) of heterogeneous sizes ranging from ~0.5 to 10 µm and generally induce type 2 (Th2)-biased immune responses. However, recent literature indicates that moving from micron dimension particles toward the nanoscale can modify the adjuvanticity of Al towards type 1 (Th1) responses, which can potentially be exploited for the development of vaccines for which Th1 immunity is crucial. Specifically, in the context of cancer treatments, Al nanoparticles (Al-NPs) can induce a more balanced (Th1/Th2), robust, and durable immune response associated with an increased number of cytotoxic T cells compared to Al-MPs, which are more favourable for stimulating an oncolytic response. In this review, we compare the adjuvant properties of Al-NPs to those of Al-MPs in the context of infectious disease vaccines and cancer immunotherapy and provide perspectives for future research.
Publisher: Elsevier BV
Date: 07-2022
DOI: 10.1016/J.BIOPHA.2022.113090
Abstract: The Fc region of a monoclonal antibody (mAb) can play a crucial role in its biodistribution and therapeutic activity. The chimeric mAb, chDAB4 (APOMAB®), which binds to dead tumor cells after DNA-damaging anticancer treatment, has been studied pre-clinically in both diagnostic and therapeutic applications in cancer. Given that macrophages contribute to the tumor accumulation of chDAB4 and its potency as an antibody drug conjugate in vivo, we next wanted to determine whether the Fc region of the chDAB4 mAb also contributed. We found that, regardless of prior labeling with chDAB4, dead EL4 lymphoma or Lewis Lung (LL2) tumor cells were phagocytosed equally by wild-type or Fcγ knock-down macrophage cell lines. A similar result was seen with bone marrow-derived macrophages from wild-type, Fcγ knock-out (KO) and NOTAM mice that express Fcγ but lack immunoreceptor tyrosine-based activation motif (ITAM) signaling. Among EL4 tumor-bearing wild-type, Fcγ KO or NOTAM mice, no differences were observed in post-chemotherapy uptake of
Publisher: Springer Science and Business Media LLC
Date: 06-07-2021
DOI: 10.1007/S11307-021-01620-1
Abstract: Early detection of tumor treatment responses represents an unmet clinical need with no approved noninvasive methods. DAB4, or its chimeric derivative, chDAB4 (APOMAB®) is an antibody that targets the Lupus associated antigen (La/SSB). La/SSB is over-expressed in malignancy and selectively targeted by chDAB4 in cancer cells dying from DNA-damaging treatment. Therefore, chDAB4 is a unique diagnostic tool that detects dead cancer cells and thus could distinguish between treatment responsive and nonresponsive patients. In clinically relevant tumor models, mice bearing subcutaneous xenografts of human ovarian or lung cancer cell lines or intraperitoneal ovarian cancer xenografts were untreated or given chemotherapy followed 24h later by chDAB4 radiolabeled with [ 89 Zr]Zr IV . Tumor responses were monitored using bioluminescence imaging and caliper measurements. [ 89 Zr]Zr-chDAB4 uptake in tumor and normal tissues was measured using an Albira SI Positron-Emission Tomography (PET) imager and its biodistribution was measured using a Hidex gamma-counter. Tumor uptake of [ 89 Zr]Zr-chDAB4 was detected in untreated mice, and uptake significantly increased in both human lung and ovarian tumors after chemotherapy, but not in normal tissues. Given that tumors, rather than normal tissues, were targeted after chemotherapy, these results support the clinical development of chDAB4 as a radiodiagnostic imaging agent and as a potential predictive marker of treatment response.
Publisher: BMJ
Date: 03-2023
Abstract: Emerging evidence suggests that the mechanism of chemotherapy-induced cell death may influence the antitumor immune response in patients with cancer. Unlike immunologically silent apoptosis, pyroptosis is a lytic and inflammatory form of programmed cell death characterized by pore formation in the cell membrane and release of proinflammatory factors. Gasdermin E (GSDME) has recently gained attention after cleavage of GSDME by certain chemotherapeutics has been shown to elicit pyroptosis. This study investigated the immunomodulatory effects of a mesothelin-targeting antibody drug conjugate (ADC) in mouse models of breast and colon cancer. The antitumor effects of the ADC were studied in EMT6 breast cancer and CT26 colon cancer syngeneic mouse models. The immunomodulatory effects of the ADC were assessed by analysis of tumor-infiltrating immune cells using flow cytometry. ADC mechanism of action was evaluated by morphology, biological assays, ADC-mediated cleavage of key effector proteins, and CRISPR/Cas9-mediated knockout (KO). Finally, the antitumor effect of ADC and Fms-like tyrosine kinase-3 ligand (Flt3L) combination therapy was evaluated in tumors expressing GSDME as well as in GSDME-silenced tumors. The data demonstrated that the ADC controlled tumor growth and stimulated anticancer immune responses. Investigation of the mechanism of action revealed that tubulysin, the cytotoxic payload of the ADC, induced cleavage of GSDME and elicited pyroptotic cell death in GSDME-expressing cells. Using GSDME KO, we showed that GSDME expression is critical for the effectiveness of the ADC as a monotherapy. Combining the ADC with Flt3L, a cytokine that expands dendritic cells in both lymphoid and non-lymphoid tissues, restored control of GSDME KO tumors. Together, these results show for the first time that tubulysin and a tubulysin containing ADC can elicit pyroptosis, and that this fiery cell death is critical for antitumor immunity and therapeutic response.
Publisher: Springer Science and Business Media LLC
Date: 16-08-2021
Publisher: Elsevier BV
Date: 11-2020
Publisher: Cold Spring Harbor Laboratory
Date: 02-05-2022
DOI: 10.1101/2022.05.01.490250
Abstract: Aggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of CAR-T cell therapy have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T cell therapy also remain limited. We profiled an extensive biobank of patients’ biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR-T cells from peripheral blood of glioblastoma and diffuse midline glioma patients and characterized their phenotype and function in vitro . Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control. Here we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite glioblastoma patients having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an IL-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors. Markers associated with tumor control included those related to T-cell homing, infiltration, and cytotoxicity. Targeting GD2 using a clinically deployed CAR-T therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors. GD2 is a tumor antigen of significant interest for targeting immunotherapy. A single preclinical study has shown the effectiveness of GD2-CAR-T cell therapy in an orthotopic xenograft model of diffuse midline glioma. Similarly, there is one previous preclinical study of GD2-CAR-T therapy in a orthotopic glioblastoma xenograft model but tumor control was achieved only following intracranial injection of CAR-T cells. Given that GD2-CAR-T therapy is already being evaluated clinically for other tumor indications, it is important to establish whether there is an acceptable rationale for its use in brain tumors. This is the first description of a GD2-targeted CAR-T cell therapy that shows antitumor effectiveness in a preclinical model of human glioblastoma following intravenous administration. It is also the first study to investigate the potential effects that the immune profile of glioblastoma patients may have on the feasibility of CAR-T cell manufacturing. The results of this study have led to the initiation of an Australian phase 1 clinical trial program aiming to test GD2-specific CAR-T cells for the treatment of childhood and adult primary brain tumors. The study provides valuable insights into the microenvironmental factors that influence the effectiveness of CAR-T cell therapy for this type of tumor, paving the way for further optimization of CAR-T cell technology for treatment of aggressive primary brain tumors such as glioblastoma.
Publisher: American Association for Cancer Research (AACR)
Date: 07-2018
DOI: 10.1158/1538-7445.AM2018-1881
Abstract: BNC105 is a phase II potent and highly selective disruptor of tumor microvasculature that causes the rapid onset of hypoxia and necrosis in solid tumors. BNC105 targets the colchicine-binding site on tubulin, causing chronic disruption of adhesion molecules, and was developed to be best-in-class with high specificity to actively iding cells. It has one of the largest therapeutic windows of its class and has been shown to have direct cytotoxic activity on tumor cells. It is this highly tumor-specific mechanism of action that has positioned BNC105 as a therapeutic with high potential in the hematologic cancer setting. Previous studies of BNC105 have shown that treatment with BNC105 results in the activation of c-Jun N-terminal kinase (JNK), phosphorylation of ATF2, and the induction of ATF3 and Noxa, leading to acute apoptosis in chronic lymphocytic leukemia (CLL) cells. These findings led to the commencement of a phase 1/2 trial of BNC105 in patients with CLL. The present study was designed to investigate the effect of BNC105 treatment on acute myeloid leukemia (AML), a disease that currently has limited treatment options. To assess the utility of BNC105 therapy in this setting, six AML cell lines representing different subtypes, including the high-risk FLT3-ITD subtype, were initially used in proliferation and cytotoxicity assays. The production of reactive oxygen species (ROS), cell cycle distribution and cell signaling by Western blot were all assessed after treatment. All tested AML cell lines were highly sensitive to treatment with BNC105 with an IC50=0.2nM to 1.3nM after 48 hours treatment. Analysis of apoptosis induction revealed cell line-specific effects however, a consistent dose-dependent increase in phosphorylation of JNK was observed across all cell lines. AML patient s les obtained from the South Australian Cancer Research Biobank (SACRB) were exposed to BNC105 at clinically relevant doses for up to 72 hours and cellular viability and apoptosis induction were assessed by Annexin V/ 7AAD staining and caspase 3 and 7 activation measured. BNC105 induced caspase activity and significantly decreased viability in a dose- and time-dependent manner, including the FLT3 mutant subtype patient s les. In comparison, bone marrow mononuclear cells from healthy controls were much less affected by BNC105. Effects of BNC105 on the leukemic stem cell (LSC) phenotype population were also investigated. The LSC-containing population, measured by CD34/CD38 and GPR56 or CD93 staining, was targeted by BNC105 in all AML patient s les tested. These results suggest that AML cells can be directly targeted by BNC105 at clinically relevant concentrations and hence further clinical investigation of BNC105 is warranted for AML treatment in a patient population with high unmet need. Citation Format: Daniel J. Inglis, Debora A. Casolari, Tran Nguyen, Donna M. Beaumont, Nicole L. Wittwer, David Ross, Richard D'Andrea, Tina C. Lavranos. The microtubule-disrupting drug BNC105 is a potent inducer of apoptosis in AML patient s les [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018 2018 Apr 14-18 Chicago, IL. Philadelphia (PA): AACR Cancer Res 2018 (13 Suppl):Abstract nr 1881.
Publisher: BMJ
Date: 09-2022
Abstract: Aggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of chimeric antigen receptor (CAR) T-cell therapy in patients with glioblastoma have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T-cell therapy also remain limited. We profiled an extensive biobank of patients’ biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR- T cells from patients with peripheral blood of glioblastoma and diffuse midline glioma and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control. Here we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite patients with glioblastoma having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an interleukin-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors. Targeting GD2 using a clinically deployed CAR-T-cell therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors.
No related grants have been discovered for Nicole Wittwer.