Date of Award

5-1-2023

Degree Type

Dissertation

Degree Name

PhD. Chemistry

Department

Chemistry and Biochemistry

Advisor

Gregory R Wiedman, Ph.D.

Advisor

Jessica Cottrell, Ph.D.

Committee Member

Joseph Badillo, Ph.D.

Committee Member

Constantine Bitsaktsis, Ph.D.

Committee Member

Yuri Kazakevich, Ph.D.

Keywords

Immunostimulatory Peptides, XBP1, CS1, Monomer, Dimer, Trimer

Abstract

This dissertation reports the design, synthesis, characterization, and biological studies of three uniquely structured peptides, derived from ubiquitinated multiple myeloma proteins and ubiquitinated B7H6 ligand. The peptides were made into linear or branched shapes, with the branched peptides containing both major histocompatibility complex (MHC) class two and NKp30 binding motifs properties in one peptide. This results in an immunocytotoxic response (via cytokines and chemokines) of both natural killer cells and cytotoxic T lymphocytes (CTLs). These shapes were determined for data comparison but also to increase cytokine proliferation, the shapes help with healthy cell aviation. Thus, the linear monomer (contains a CTL specific peptide, CS1), the V-shaped dimer (containing two CTL specific peptides, CS1 and XBP1 US), and the Y-shaped trimer (containing two CTL specific peptides, CS1 and XBP1 US, as well as NK cell specific TVPLN) that all contain structural differences. Since the CTL derived peptides have been previously found to contain great cancer-targeting immunostimulant via cytokine proliferation then it was determined that those same peptides be tested on NK-92MI cells. While cancer related B7H6 antigen that is often downregulated and even shed off from the surface of cancerous cells Thus, the B7H6 derived trimeric peptide tail is anticipated to improve NK cell binding and the unnatural peptide structure of the trimer in anticipated to increase the cytokine production. This dissertation presents a proof-of-principal breakthrough on the develop of bifunctional peptide antigens that can activate NK cell cytokine release.

The preclinical methodology initially delves into the physical binding of the peptides to the surface of NK-92MI cells, using flow cytometry. From this study, all peptides had higher than 70 percent binding capacity to NK-92MI cells at three different concentrations and at three different time points; and statistically the dimer was more significant at all concentration for two of the three timepoints. Similarly, all peptides triggered immunostimulatory cytokine secretion of interleukin 2 (IL-2), interferon gamma (IFNy), and tumor necrosis factor alpha (TNFa); but statistically only IFNy and TNFa showed significance at the lowest peptide concentration of both the monomer and dimer. Thus, the Luminex cell-based assays demonstrated the peptides capabilities of activating NK cell immunity using immuncytotoxicity. While cellular viability studies via MTT assay comparing the peptides and the concentrations established that the monomer was significantly more viable than that of the dimer and trimer at all concentrations. This pilot study therefore uncovers new bifunctional cancer targeting immunotherapeutic peptides for both NK cells and CTLs but also paves the way for additional analyses that may result clinical applications.

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