Browsing by Subject "EGFR"

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    Directing T Cells with Chemically Self-Assembled Nanorings as an Immunotherapy for Targeting Hematological Malignancies and Solid Tumors
    (2021-12) Mews, Ellie
    Engineering cell-to-cell interactions has proven to be quite valuable due to the vast number of therapeutic applications that benefit from this technology. Although genetically engineering artificial receptors onto a patient’s cells has shown some success, preparation is costly, current applications are limited, and modifications are permanent. To address some of these concerns our lab has developed a non-genetic approach to facilitate selective cell-to-cell interactions with chemically self-assembling nanorings (CSANs). We have shown that functionalizing the CSAN construct with cancer antigen targeted protein scaffolds and a T-cell targeted single chain antibody fragment (?CD3 scFv) forms a mixture of bispecific nanorings that facilitate T cell interactions with the tumor. In this dissertation, the concept of directing T cell activity with bispecific CSANs is initially validated against CD19+ B cell lymphoma following the production and characterization of an ?CD19-DHFR2 fusion protein monomer. Previous work has demonstrated that the CSAN platform can also be used to target overexpressed tumor associated antigens on solid tumors; therefore, the remainder of this dissertation details the application of bispecific CSAN directed T cells against established brain tumors.Few therapeutic options are available for treating central nervous system (CNS) solid tumors, especially upon recurrence. Recent preclinical studies have shown promising results for eradicating various solid tumors by targeting the overexpressed immune checkpoint molecule, B7-H3. However, due to several therapy-related toxicities and reports of tumor escape, the full potential of targeting this pan-cancer antigen has yet to be realized. Here, we designed and characterized bispecific CSANs that target the T cell receptor, CD3ε, and tumor associated antigen, B7-H3. Two different B7-H3 targeted proteins were incorporated into the CSAN scaffold, a single chain variable fragment (scFv) derived from the 8H9 monoclonal antibody and an affibody that was affinity matured via yeast display technology. We show that both B7-H3 targeted protein scaffolds form bispecific nanorings with the ?CD3 monomer to increase T cell infiltration and facilitate selective cytotoxicity of B7-H3+ medulloblastoma cells. Additionally, ?B7-H3-?CD3 CSANs directed robust T cell responses against preclinical models of established medulloblastoma and glioblastoma tumors. Furthermore, the combination of ?EGFR-?CD3 CSANs and ?B7-H3-?CD3 CSANs further improved the anti-tumor immune response in these models, suggesting therapeutic synergism between EGFR and B7-H3. Intraperitoneal (IP) injections of ?B7-H3-?CD3 bispecific CSANs were found to effectively cross the blood-tumor barrier into the brain and elicit significant anti-tumor T cell activity intracranially as well as systemically in an orthotopic medulloblastoma model. Moreover, following treatment with ?B7-H3-?CD3 CSANs, intratumoral CD4+ and CD8+ T cells were found to primarily have a central memory phenotype that displayed significant levels of characteristic activation markers. Importantly, we demonstrate that the bispecific CSAN directed T cell cytotoxicity is not dependent on MHC class I interactions with target cells, suggesting that downregulation of MHCI expression as an immune evasion mechanism would not affect CSAN directed anti-tumor activity. Furthermore, due to the modularity of the nanorings, non-specific T cell activation against the ONS 2303 medulloblastoma cell line can be reduced by tuning the valency of the ?CD3 targeted monomer in the oligomerized CSAN. Collectively, these results demonstrate the ability of our multi-valent, bispecific CSANs to direct potent anti-tumor T cell responses and indicate its potential utility as an alternative or complementary therapy for immune cell targeting of B7-H3+ brain tumors.
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    FGFR1-induced soluble factors promote mammary tumorigenesis and chemoresistance
    (2013-08) Bade, Lindsey Kay
    The fibroblast growth factor receptor (FGFR) family consists of four receptor tyrosine kinases that are known regulators of cellular processes such as proliferation, migration, survival, and angiogenesis. Anomalous expression or uncontrolled activation of these receptors or their ligands has been correlated with progression of various types of cancer, including breast cancer. Specifically, the chromosomal locus of FGFR1, 8p11-12, is found to be aberrantly amplified in approximately 10% of patients diagnosed with breast cancer. Patients who harbor the FGFR1 amplification do not respond well to current therapies and develop resistance to hormone-based therapies. Therefore, understanding the molecular mechanisms of how FGFR1 overexpression promotes tumorigenesis may provide insights into better targets for novel, more effective therapies. The work presented here shows that FGFR1 activation significantly upregulates expression of the ligands AREG and EREG at the transcript and protein levels both in vitro and in vivo, which then activate EGFR signaling. AREG is critical for normal ductal morphogenesis in the mammary gland and has also been linked to breast cancer progression. Studies examining AREG expression in human breast cancers have found AREG expression to significantly correlate with regional lymph node metastases, large tumor size, and high-grade tumors. While EREG promotes proliferation of several normal and cancerous cell types, the role of EREG has not been extensively characterized in the mammary gland. However, recent studies have demonstrated that EREG is a potent mediator of metastasis of breast cancer cells to the lung and that overexpression of EREG is an indicator of poor prognosis for inflammatory breast cancer patients. EGFR, a member of the ErbB receptor tyrosine kinase family, has been well studied in the mammary gland, and it is known that EGFR is required for normal mammary gland ductal morphogenesis. Alternatively, overexpression or constitutive activation of EGFR in the mammary gland has been linked to mammary tumorigenesis. Additionally, overexpression of EGFR in the breast is associated with recurrence of earlier stage breast cancers and decreased disease-free and overall survival in later stage breast cancer patients. Notably, we demonstrate that EGFR activation is at least in part required for FGFR1-induced proliferation and migration and ERK1/2 activation, as inhibition of EGFR with the small molecule kinase inhibitor erlotinib significantly blocks these processes. Moreover, we show that FGFR1 and EGFR are co-expressed in TNBC cell lines and that both FGFR1 and EGFR can mediate Doxorubicin chemoresistance. We further show that FGFR1 upregulates expression of the cytokine LIF, which then signals through gp130/JAK to activate STAT3 in vitro. Directly inhibiting either FGFR1 or STAT3 significantly reduces chemoresistance and increases apoptosis in vitro. Furthermore, inhibition of FGFR1 with the small molecule inhibitor PD173074 results in increased chemosensitivity and apoptosis in a mouse model of mammary tumorigenesis. These results are significant because they are the first to show that FGFR1 signals through EGFR and that FGFR1 mediates chemoresistance through activation of STAT3. This study furthers our understanding of FGFR1-amplified mammary tumorigenesis and presents alternative factors for targeted therapies for patients with FGFR1-amplified breast cancers.
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    The Multifaceted Role of Calprotectin (S100A8/A9) in Head and Neck Squamous Cell Carcinoma
    (2017-11) Argyris, Prokopios
    Calprotectin (S100A8/A9) is formed as a heterodimeric protein complex of calcium regulating S100A8 and S100A9 encoded by genes mapped to the chromosomal locus 1q21.3 of the epidermal differentiation complex. Whereas extracellular calprotectin presents proinflammatory and antimicrobial properties, intracytoplasmic S100A8/A9 appears to play important roles in cell differentiation, cell cycle progression and proliferation, cell migration and survival. While highly upregulated in a variety of cancers, calprotectin is downregulated in head and neck squamous cell carcinoma (HNSCC); loss of S100A8/A9 is associated with increased DNA methylation and poor overall survival rates in HNSCC patients. Using immunohistochemical analysis for S100A8 and S100A9 we found that S100A8/A9 levels gradually decreased during progression of head and neck tumorigenesis from intra-oral premalignant (precancerous) epithelial dysplasia to invasive HNSCC. Furthermore, S100A8/A9 expression positively correlated with the level of squamous differentiation of the primary tumor. To investigate the localization of the calprotectin complex during cell cycle progression, S100A8/A9-expressing human HNSCC cells and immortalized oral keratinocytes were synchronized at G1/S and G2 phases of the cell cycle. During cell division, S100A8/A9 appeared to translocate from the cytoplasm to the microtubule-organizing centers, decorated the mitotic spindles and co-localized with casein kinase II (CK2). Calprotectin nuclear migration is consistent with a role of S100A8/A9 in the control of the G2/M checkpoint. To probe the role of calprotectin in DNA damage responses (DDR), we exposed S100A8/A9-expressing and S100A8/A9-negative carcinoma cells to genotoxic agents. Interestingly, following low doses of X-radiation and incubation with camptothecin, recruitment of the DNA repair regulatory molecules 53BP1 and γH2AX increased significantly in all calprotectin-positive carcinoma cells but failed to increase in calprotectin-negative cancer cells, suggesting impaired DDR in the absence of S100A8/A9. Furthermore, post-radiation DNA fragmentation was more prominent in calprotectin-positive cells as assessed by comet assays. S100A8/A9-negative HNSCC cells also appeared more resistant to cisplatin, while S100A8/A9-expressing carcinoma cells were more sensitive even at lower cisplatin doses. TCGA data indicated that more than 363 apoptosis–related genes were significantly upregulated by S100A8/A9–high HNSCCs compared to S100A8/A9–low neoplasms, including CASP1, -3, -4, -5, -7, -8, -9, -10 and -14. Intracellular calprotectin appeared to promote caspase-mediated DNA fragmentation following radio- and chemotherapy, contributing to S100A8/A9-dependent apoptotic death of carcinomatous cells. In addition, in vitro and ex vivo experiments showed that S100A8/A9 levels were inversely correlated to membranous and cytoplasmic EGFR expression, a negative prognosticator for HNSCC. Calprotectin-associated control of DNA damage responses, post-radiation sensitivity and cisplatin cytotoxicity, and EGFR expression could contribute to the increased overall survival rates of patients with S100A8/A9-high HNSCCs. Our current data further supports the tumor-suppressive role of calprotectin in HNSCC and points to new molecular targets for therapy.