We have previously demonstrated that in the presence of the chemical dimerizer bisMTX, DHFR-DHFR (DHFR2) and DHFR-DHFR-antiCD3 (DHFR2-antiCD3) fusion proteins can spontaneously assemble into a range of chemically self-assembled nanorings (CSANs) whose size varies depending on the length and composition of the linker peptide between the DHFRs. If the linker is a single glycine, we observed that rings containing 7 to 10 DHFR2 fusion proteins with an average ring size were composed of 8 monomers. The research presented in the thesis focused on exploring our methodology for engineering multivalent CSANs for delivery of vaccine adjuvants and for redirecting immune cells specificity for cancer immunotherapy. In the first part, we have synthesized a MTX analog for the purpose of driving the equilibrium towards formation of heterodimers of CSANs. The binding affinities of this MTX analog with either wild type DHFR or several DHFR mutants were determined. In the second part of the thesis, we assembled bispecific antiCD3/CD22 CSANs by mixing of an equal proportion of two DHFR2 linked by a single glycine and fused to two different antibodies: antiCD3 scFv that binds to the CD3 receptor on immune T-cells and antiCD22 scFv targeting CD22, an antigen widely expressed on B-leukemias or lymphomas. We firstly determined the binding affinity of engineered antiCD22 CSANs to CD22+ B lymphoma cells by comparison with those for the parental monoclonal antibody followed by investigations of their internalization by these cells using confocal microscopy. In addition, we studied the activation of T cells by cytokine profiling, immunophenotyping and T cell functional assays in vitro after treatment with bispecific antiCD3/CD22 CSANs. In the presence of target B lymphoma cells, cytolytic efficacy of redirected T cells was also determined. Finally, we assessed the ability of trimethoprim, a non-toxic FDA approved competitive inhibitor of DHFR to carry out disassembly of the bispecific antibody nanorings. Finally, to explore the potential of DHFR2 based CSANs to be used as vaccines by the multivalent display of an antigen and adjuvant, we prepared CSANs assembled with the bisMTX dimerizer and CSANs assembled with the bisMTX-CpG, bisMTX dimerizer linked to CpG oligonucleotides, and compared their immune responses in mice. We studied the immunogenicity of CpG CSANs as well as CSANs by cytokine profiling, immunophenotyping using mouse immune cells in vitro. And we determined titers of neutralization antibodies in the sera of immunized mice.
University of Minnesota Ph.D. dissertation. May 2016. Major: Medicinal Chemistry. Advisor: Carston Wagner. 1 computer file (PDF); xii, 153 pages.
Self-Assembled Antibody Nanorings as Prosthetic Antigen Receptors for Redirecting T Cells against Tumor Cells and as Platform for Delivery of Vaccine Adjuvants for Cancer Immunotherapy.
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