Tiara Byrd

Medulloblastoma-Biology and microenvironment: A review

Medulloblastoma (MB) is a cancer of the cerebellum and the most common primary pediatric malignancy of the central nervous system. Classified as a primitive neural ectoderm tumor; it is thought to arise from granule cell precursors in the cerebellum. The standard of care consists of surgery, chemotherapy and age-dependent radiation therapy. Despite aggressive multimodality therapy; approximately 30% of MB patients remain incurable. Moreover, for long-term survivors, the treatment related sequelae are often debilitating. Side effects include cerebellar mutism, sterility, neurocognitive deficits, and a substantial risk of developing secondary cancers. In a quest for more effective and targeted therapies, scientists have begun to investigate the biological events that not only initiate but also sustain the malignant phenotype in MB. Of particular interest is, the role of the tumor microenvironment in tumor pathogenesis. This review seeks to highlight several key processes observed in cancer biology, particularly the involvement of the tumor microenvironment, with relevant examples from MB.

A homing system targets therapeutic T cells to brain cancer

Successful T cell immunotherapy for brain cancer requires that the T cells can access tumour tissues, but this has been difficult to achieve. Here we show that, in contrast to inflammatory brain diseases such as multiple sclerosis, where endothelial cells upregulate ICAM1 and VCAM1 to guide the extravasation of pro-inflammatory cells, cancer endothelium downregulates these molecules to evade immune recognition. By contrast, we found that cancer endothelium upregulates activated leukocyte cell adhesion molecule (ALCAM), which allowed us to overcome this immune-evasion mechanism by creating an ALCAM-restricted homing system (HS). We re-engineered the natural ligand of ALCAM, CD6, in a manner that triggers initial anchorage of T cells to ALCAM and conditionally mediates a secondary wave of adhesion by sensitizing T cells to low-level ICAM1 on the cancer endothelium, thereby creating the adhesion forces necessary to capture T cells from the bloodstream. Cytotoxic HS T cells robustly infiltrated brain cancers after intravenous injection and exhibited potent antitumour activity. We have therefore developed a molecule that targets the delivery of T cells to brain cancer.Therapeutic T cells bearing ligands engineered to optimize adhesion and transmigration through the blood–brain barrier can be targeted to brain tumours.

Abstract A25: TEM8 specific CAR T cells induce regression of patient-derived xenograft and metastatic models of triple-negative breast cancer

Lacking marked expression of human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR), triple-negative breast cancer (TNBC) is a breast cancer subtype in desperate need of targeted therapy options. Tumor endothelial marker 8 (TEM8), initially identified as a tumor endothelium marker in colon cancer, has been shown to be upregulated in TNBC. To confirm this, we stained primary TNBC tissues for TEM8; in all cases TEM8 was expressed with no expression in normal breast tissue. TEM8 is expressed by TNBC cell lines as indicated by flow cytometry and Western blot. We thus engineered chimeric antigen receptor (CAR) T cells to specifically target TEM8 in TNBC. TEM8 CAR T cells distinctly recognized TEM8, secreted immunostimulatory cytokines, and killed TEM8-positive TNBC cells in vitro. In vivo, the adoptive transfer of TEM8 CAR T cells induced regression against orthotopic patient-derived xenograft (PDX) models, including the aggressive claudin-low TNBC PDX, WHIM12. Systemic administration of TEM8 CAR T cells also induced regression against a lung metastasis TNBC model. In all models, treatment with TEM8 CAR T cells resulted in a survival advantage in mice compared to controls. Hence, TEM8 may serve as an attractive targeted immunotherapy of TNBC. Citation Format: Tiara Byrd, Kristen Fousek, Antonella Pignata, Christopher Szot, Heba Samaha, Lacey Dobrolecki, Htoo Zarni Oo, Poul Sorensen, Matthew Ellis, Michael Lewis, Meenakshi Hegde, Bradley Fletcher, Brad St. Croix, Nabil Ahmed. TEM8 specific CAR T cells induce regression of patient-derived xenograft and metastatic models of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A25.

392. Super-Resolution (STED) Imaging Reveals Simultaneous Co-Docking of Tandem Chimeric Antigen Receptors to Two Target Antigens Enhancing T Cell Functionality and Mitigating Antigen Escape

Background: Tandem chimeric antigen receptors (CARs) are bispecific CAR molecules with two different target recognition exodomains joined in tandem. Tandem CARs mediate distinct T cell activation to either target antigen and super-additive functionality upon encounter of both targets simultaneously. Tandem CAR T cells successfully mitigate antigen escape and exhibit enhanced antitumor activity compared to their unispecific counterparts. Stimulated emission depletion (STED) microscopy can achieve a sub-diffractive lateral resolution of ~ 50 nm enabling imaging of the immunological synapse at near single molecule levels.Hypothesis: Super resolution STED Imaging can differentiate heterodimers created by Tandem CAR molecules simultaneously co-docking to both target antigens at the immunological synapse (IS).Methods: Human T cells were retrovirally transduced to express a Tandem CAR molecule specific for HER2(ErbB2) and IL13Rα2. Conjugates of Tandem CAR T cells and the human glioblastoma cell line U373 were fixed after 30 minutes of incubation then labelled with primary unconjugated monoclonal antibodies against HER2 and IL13Rα2 tumor ligands. The IS was examined at the T cell/ U373 interface using confocal and STED microscopy then confirmed with in situ proximity ligation assay (PLA). Unispecific CAR T cells, T cells co-expressing HER2 and IL13Rα2 CARs and non-transduced T cells were used as experimental controls.Results: Confocal microscopy showed co-clustering of HER2 and IL13Rα2 at the IS in contrast to only HER2 or IL13Rα2 localized to the IS for the IL13Rα2 CAR and HER2 CAR T cell/GBM conjugates, respectively. Using a fixed intensity threshold for all conjugates analyzed, quantification of receptor accumulation at the IS revealed that there was increased collective clustering of target molecules at the IS (p= 0.0002). Super-resolution STED microscopy was used to interrogate the quality and quantity of co-localized Tan CAR target ligands at the immune synapse. Measurement of co-localized HER2 and IL13Rα2 aggregate diameters showed a distribution of 80-200 nm aggregate size. The aggregate size has high correlation to the predicted size of CAR-tumor ligand-antibody-antigen complex, indicating increased probability of co-docking of the target ligands at the Tan CAR synapse. Co-localization of the two tumor antigens was present with a very low frequency in the bi-specific T cell - tumor synapse with their average aggregate size ranging from 300-500 nm, suggesting that these ligands formed independent and not “co-docked” conjugates. Co-localization was absent in the single CAR -tumor synapse. In situ PLA with a maximum distance of 30-40 nm showed accumulation of HER2/IL13Rα2 heterodimer signals at the immunological synapse of Tandem CAR Tcell/ GBM IS.Conclusion: Tandem CAR molecules simultaneously engage two target antigens mediating significantly enhances T cell activation through a bifunctional immunological synapse.