Mews, Ellie2022-02-152022-02-152021-12https://hdl.handle.net/11299/226383University of Minnesota Ph.D. dissertation. 2021. Major: Medicinal Chemistry. Advisors: Carston Wagner, Barry Finzel. 1 computer file (PDF); 206 pages.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.enB7-H3CD19EGFRGlioblastomaMedulloblastomaNanoringsThesis or Dissertation