Date of Award

Summer 8-17-2020

Degree Type

Dissertation

Degree Name

PhD Molecular Bioscience

Department

Biology

Advisor

Constantine Bitsaktsis, Ph.D.

Committee Member

David Sabatino, Ph.D.

Committee Member

Robert Korngold, Ph.D.

Committee Member

Allan Blake, Ph.D.

Committee Member

Jessica Cottrell, Ph.D.

Keywords

Cancer, immunotherapy, drug development, cancer-targeting immunostimulatory peptides

Abstract

This dissertation reports the synthesis and biological applications of bifunctional trimeric peptides with B7H6-derived NKp30 binding motifs that serve to activate an immunocytotoxic response in natural killer cells and a GRP78-binding motif that can target tumors that express surface GRP78. In this manner the cancer-targeting immunostimulatory peptides are anticipated to directly bind and activate effector NK92-MI cells while also recognizing and binding to target A549 tumor cells to facilitate NK cell-dependent immunocytotoxicity of the targeted tumors. The NKp30 binding peptide motifs are derived from the tumor associated B7H6 antigen that is often downregulated or shed from the surface of tumors resulting in evasion of NK-dependent immunity and tumor resistance. Moreover, surface GRP78 is expressed on tumors but not healthy cells thereby enabling GRP78-targeting peptides to selectively bind to tumors. Therefore, the bifunctional peptides are designed to overcome this limitation and restore NK cell immunity while providing targeted cancer immunotherapy in tumors that express surface GRP78. This thesis provides a proof-of-concept discovery on the potential to develop bifunctional peptide ligands that can restore and/or activate NK cell activity against these tumor types.

Our preclinical approach initially studied direct and/or competition binding assays of the trimeric peptides to both surface NKp30 and GRP78 cell lines, NK92-MI and A549 cells respectively, using flow cytometry. From this analysis, a lead synthetic bifunctional trimeric peptide 13 displayed binding to the NK and A549 cells with some degree of receptor binding specificity. This trimeric peptide also triggered NK-dependent immunostimulatory activity with secretion of IFN-g and IL-8. In co-culture with the surface GRP78 expressing A549 lung tumor cells, the lead trimeric peptide 13 pre-activated NK92-MI cells resulting in observable association and cytotoxicity of the target A549 cancer cells, presumably due to the detectable release of proinflammatory cytokines, TNF-a, IFN-g and IL-8 from the NK cells. These cell-based assays confirmed the capabilities of the lead trimeric peptide 13 to activate NK cell immunity and effect immuncytotoxicity of the target A549 tumor cells. The pre-activated NK92-MI cells with the lead trimeric peptide 13 also sustained a reduced tumor growth rate when compared to the untreated control, thereby also supporting the in vivo immunotherapeutic effects of the lead peptide in mice models with the A549 tumor. This pilot study therefore reveals the cancer immunotherapeutic potential of a lead cancer-targeting immunostimulatory peptide and paves the way for additional investigations that may facilitate its translation from pre-clinical to clinical applications.

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