Browsing by Subject "Nanorings"

Now showing 1 - 3 of 3
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    Chemically self-assembled antibody nanorings (CSANS): Design and characterization of an anti-CD3 IGM biomimetic.
    (2010-10) Li, Qing
    Based on our development of a highly efficient protocol for the chemically controlled self-assembling of protein nanorings, we have sought to exploit our methodology for engineering multivalent chemically self-assembled antibody-nanorings (CSANs) for tissue imaging and drug delivery. Two novel DHFR-DHFR-anti-CD3 scFv fusion proteins were constructed (13DDantiCD3 and 1DDantiCD3). In addition, the two DHFR cysteines were mutated to either alanine or serine to enhance correct folding. The protein was expressed in BL21 (DE3) cells, renatured with the SLS-based refolding protocol and purified by methotrexate affinity chromatography. Incubation of 13DDantiCD3 with the chemical dimerizer, bisMTX, resulted in almost exclusive formation of the bivalent CSANs, while incubation with 1DDantiCD3 resulted in formation of octavalent CSANs. Both antibody nanorings selectively blocked the killing of the CD3+ human T-leukemia HPB-MLT by a diphtheria-anti-CD3 immunotoxin. FACS analysis revealed nearly identical dissociation constants for both the selfassembled and parental monoclonal antibody and a 3-fold lower K d for the octavalent species. The chemically dimerized scFv's were shown to be stable in cell culture at 37°C and the dimerization was shown to be reversible by the addition of excess amounts of the non-toxic FDA approved DHFR antagonist trimethoprim. We also demonstrate that, similar to the parental bivalent anti-CD3 monoclonal antibody (MAB), anti-CD3 CSANs selectively bind to CD3+ leukemia cells, and undergo rapid internalization through a caveolin-independent pathway that requires cholesterol, actin polymerization and protein tyrosine kinase activation. While treatment with the monoclonal antibody leads to T-cell activation and nearly complete loss (i.e. 90%) of surface displayed T-cell receptor (TCR), only 25-30% of the TCR down regulate and no significant T-cell proliferation is observed after treatment of peripheral blood mononuclear cells (PBMCs) with anti-CD3 CSANs. Consistent with the proliferation findings, 15-25% less CD25 (IL-2 receptor) was found on the surface of PBMCs treated with either the polyvalent or bivalent anti-CD3 CSANs, respectively, than on PBMCs treated with the parental MAB. Comparative experiments with F(ab')2 derived from the MAB confirm that the activation of the T-cells by the MAB is dependent on the Fc domain, and thus interactions of the PBMC T-cells with accessory cells, such as macrophages. Taken together, our results demonstrate that anti-CD3 CSANs with valencies ranging from 2 to 8 could be employed for radionuclide, drug or potentially oligonucleotide delivery to T-cells without, as has been observed for other antibody conjugated nanoparticles, the deleterious affects of activation observed for MAB. Further the CSAN construct may be adapted for the preparation of other multivalent scFvs.
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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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    Toward therapeutic nanoassemblies: the design and modeling of protein-protein interactions.
    (2009-11) White, Brian Richard
    Unraveling the nanoscale processes of biological pathways via the testing, replication, and visualization of the underlying mechanisms remains a persistent challenge in the study of these critical life-governing systems. Recent advances in the field of chemically induced dimerization have unlocked multiple tools for the exploration of these facets of biology, including the development of switchable signaling systems, assertion of control over protein localization in the cell, and regulation of gene expression. An additional revelation through protein complexation by chemical induction is the construction of multivalent protein-based nanostructures, capable of bearing multiple targeting agents. However, stochastic assembly of these proteins has proven unsatisfactory in generating homogeneous populations. Herein, we have taken the initial steps toward developing a protein-based biomolecular language for nanostructural assembly. Through gel filtration analysis, we have characterized the ability of interfacial point mutations to modulate the stability of a bis-methotrexate (bis-MTX) induced E. coli dihydrofolate reductase (DHFR) dimer over a dynamic range of 1.5 kcal/mol. Furthermore, we have employed single-molecule fluorescence assays to demonstrate the stabilization of a heterodimeric DHFR dimer, yielding 4-fold selectivity for the heterodimer over either corresponding homodimer. In addition to our experimental characterization of the chemically induced DHFR dimer, we have also taken steps toward the construction of a tripartite computational model of dimerization in an effort to predict the effects of further mutations. We have tested a number of molecular mechanics force fields against quantum mechanical benchmarks and discovered that the MMFF94, OPLS2005, and AMBER force fields yield the most accurate electrostatic and configurational treatment of the complex bis-MTX dimerizer. While initial attempts at calculating the binding free energy of the macromolecular complex have been unsuccessful, we have gleaned important insights into the complexities of modeling this three-body system. The advances described within the following work delineate important aspects of protein interface remodeling in a chemically induced system and provide an avenue toward the further development of both a computational model of protein interactions and the future directed assembly of protein based materials and therapeutic nanostructures.