Yi Wen

Defense Date

6-2-2014

Graduation Date

Summer 2014

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Pharmaceutics

School

School of Pharmacy

Committee Chair

Wilson Meng

Committee Member

Ellen Gawalt

Committee Member

Ira Buckner

Committee Member

Lauren O'Donnell

Committee Member

Jelena Janjic

Keywords

antibody delivery, antibody display system, beta-sheet, EAK16-II, immune modulation, self-assembling peptides

Abstract

Many diseases are associated with disruption of immune homeostasis. Monoclonal antibodies (mAb) targeting soluble antigens or immune cell surface molecules have been developed to modulate immunity. However, systemic administration can lead to severe immune related side effects. This dissertation investigated a versatile platform to display antibodies locally in diseased tissues to minimize unwanted systemic exposure. This system is a stimuli-responsive and injectable formulation utilizing in situ forming peptide co-assemblies and two linker proteins. The self-assembling peptides EAK16-II and EAKIIH6 co-assemble into fibrous structures with accessible His-tags when exposed to salts. Anti-His-tag antibodies (αH6-IgG) bind to these His-tags and provide fragment crystallizable region (Fc) for protein A/G (pAG). A therapeutic antibody can be displayed through non-covalent Fc-pAG interactions. Proof-of-concept studies demonstrate that antibodies were displayed via His-tags through the linker proteins, namely His-tag/αH6-IgG/pAG interactions. Displayed antibodies remained biologically active and were able to bind to cognate antigens. Upon administration to normal subcutaneous space, tumors, or transplant sites, a fluorescent dye labeled IgG was retained effectively at the site of injection. Antibodies targeting a cellular surface antigen were loaded onto the peptide co-assembly. Displayed antibodies were able to interact with cellular surface antigens and impede trafficking of target cells, resulting in potential therapeutic response in a murine skin transplantation model. Characterization of the peptide co-assembly indicates the His-tagged peptide (EAKIIH6) co-assembled with the parent peptide (EAK16-II) in a concentration dependent manner. Comparing the co-assembly efficiency of EAKIIH6 to other control peptides implies that EAKIIH6 might adopt a β-strand conformation and align with existing EAK16-II β-sheets during co-assembly. It was also found that the accessibility of His-tags could be tuned by changing the ratio of peptides. To conclude, these results demonstrate the peptide co-assembly based system could be used as a versatile antibody display system to modulate immunity. It may have the potential to improve therapeutical outcomes of mAbs as a local antibody delivery system, and it could also be utilized as a material strategy to immobilize target cells.

Format

PDF

Language

English

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