Date of Award

Spring 5-15-2017

Degree Type

Dissertation

Degree Name

PhD. Chemistry

Department

Chemistry and Biochemistry

Advisor

David Sabatino, Ph.D.

Committee Member

Constantine Bitsaktsis, Ph.D.

Committee Member

Monika Raj, Ph.D.

Committee Member

Cecilia Marzabadi, Ph.D.

Keywords

B7H6, immunotherapy, NK cells, NKp30, peptide vaccines, novel therapy, cancer, cancer biomarker

Abstract

Cancer-based immunotherapy has led the evolution of biologics that can stimulate immune responses towards tumor eradication. The synthesis of small to intermediate size molecules with the targeting and effector functions of mAb may represent a novel class of immunotherapeutics that may overcome the limitations of their biological counterparts.Towards this objective, B7H6 has been identified as a protein ligand localized on the cell surface of transformed tumor cells. B7H6 binds specifically to the activating receptor NKp30, constitutively expressed on all resting and active NK cells. Upon ligand:receptor binding, B7H6 triggers NK cell activation and release of chemokines and pro-inflammatory cytokines such as TNF-a and IFN-g, leading to tumor cell lysis and death. B7H6 was found to be expressed on the surface of tumors but not on healthy cells making it an interesting bio-marker for NK cell mediated cytotoxicity. Moreover, it was recently discovered that tumor cells escape immunosurveillance by the downregulation or shedding of B7H6. In an effort to overcome this resistance mechanism, the development of new B7H6 derived ligands that may effectively target NKp30 and activate NK cells towards B7H6 deficient tumors may result in a more effective NK-cell-based cancer therapy approach.

The rational design of a novel class of immunostimulatory peptides (IPs) derived from the binding interface of B7H6:NKp30 was developed. The IPs were composed of the B7H6 active site sequence for NKp30 binding and immunostimulatory activity. The secondary structures of the peptides were evaluated by CD spectroscopy in H2O, PBS and a H2O:TFE mixture which demonstrated flexible peptide structures which adapted to the solvent conditions. The evaluation of their biological properties showed an occupancy of the synthetic peptides to a human NK cell line comparable to the natural NKp30 ligand, B7H6, and the human anti-NKp30 monoclonal antibody (mAb). Moreover, the immunostimulatory activity of B7H6 was demonstrated by the secretion of the pro-inflammatory cytokines, tumor necrosis factor-alfa (TNF-a) and interferon gamma (IFN-g) by the human NK cell line. The IPs were found to be non-toxic, without any observable evidence of early or late stage apoptosis within the NK92-MI cells. Taken altogether, this novel class of synthetic peptides may prove to be a promising lead in the development of a new peptide-based immunotherapy approach. Alternatively, Pep42, a cyclic peptide selected from phage display bio-panning experiments, CTVALPGGYVRVC, has been shown to bind specifically to the heat shock family member Glucose-Regulated Protein 78 (GRP78) receptor, while internalizing poorly within tumor cells by receptor mediated endocytosis. GRP78 has been found to be exclusively overexpressed and cell surface localized on a variety tumor cells, where it promotes cancer initiation, proliferation and metastatic spread, but not on healthy tissue. This thesis also describes the application of Pep42, its FITC-labeled derivatives, and a series of poly(ethylene glycol)-linked Pep42 sequences in tumor immunotherapy applications, and their incorporation within bioactive proteins such as B7H6. This thesis describes progress towards the design, development and application of novel semi-synthetic antibody mimics for NK-dependent treatment of tumors that overexpress GRP78 at the cell surface.

 
 

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