Defense Date

7-9-2024

Graduation Date

Summer 8-10-2024

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Pharmaceutics

School

School of Pharmacy

Committee Chair

Wilson S. Meng

Committee Member

Devika S. Manickam

Committee Member

Ellen S. Gawalt

Committee Member

Peter Wildfong

Committee Member

Patrick Flaherty

Keywords

Injectable scaffold, localization, immunotherapy, lymph nodes, cell therapy, 3D cell culture, spheroids, biomaterials, T cell tolerance

Abstract

Dysregulation of immune homeostasis is fundamental to the development of autoimmune disorders. Fibroblastic reticular cells (FRCs) within lymph nodes play crucial roles in forming a microenvironment conducive to immune cell interactions and regulating autoreactive T cells driving autoimmune diseases. Studies have shown the potential of FRCs as a form of personalized cell therapy to correct dysregulated immune responses. Successful translational efforts, however, require the cells to be formulated as injectable suspensions while retaining their native architecture, which is essential for their regulatory functions. The main challenge resides in retaining the native reticular organization of FRCs lost in conventional monolayer cultures used for expansion and/or functional manipulation. Organizing FRCs into three-dimensional (3D) multicellular clusters would recapitulate their critical phenotypic attributes.

In this dissertation, I describe a scaffolding method based on the self-assembling peptide EAK in which the amphiphilic sequence [AEAEAKAK]2 is biotinylated at the N-terminus. The modified peptide, referred to as EAKbt, was designed to transform human FRCs harvested from conventional monolayer cultures into injectable units. Above a certain concentration (> 1 mg/mL) and ionic strength (>20 mM), EAKbt self-assembled into loosely associated fibrils. Cross-linking the peptide with avidin transformed the fibrils into a dense network of coacervates. The key finding is that avidin cross-linked EAKbt drove the assembly of human FRCs into porous 3D cell clusters (clFRCs). The clFRC formulation is a robust process for guiding FRCs into an injectable formulation in which metabolic activities and biomarkers are preserved. The functional capacity of clFRCs was demonstrated in clFRCs cocultured with a human T cell line (Jurkat) or peripheral blood mononuclear cells (PBMCs). clFRCs attract and elicit infiltration of T cells. Importantly, clFRCs rendered an immunosuppressive milieu in the cocultures which is indicative of skewing the immune cells into anti-inflammatory, tolerogenic phenotypes. These results demonstrate the potential of clFRCs as a modality for stromal cell delivery for immune modulation.

Studies in humans and rodents have shown the complexities and limitations of cell-based interventions in immunological diseases. Repeatedly, cells that were exogenously manipulated in vivo were observed to undergo phenotypic drifts in vivo. I attempted to address this challenge by formulating Adenosine (Ado), a small molecule with a plasma half-life of less than 10 seconds, into a physical state which could be incorporated into clFRCs. The concept is to repurpose Ado, an FDA-approved nucleoside currently indicated for restoring cardiac rhythm but has been shown extensively in rodents to have immune regulatory functions, into an injectable crystalline suspension. It enables the delivery of Ado more than twice the dose in the same volume of the conventional formulation. The crystals were found to be stable in a protein-rich environment and, therefore, conducive for local administration into small anatomical spaces. The crystalline Ado formulation was found to exhibit localized and systemic anti-inflammatory effects.

In addition to clFRCs and crystalline Ado, I also present in this dissertation a peptide that binds to tumor necrosis factor receptor II (TNFRII) as an “adjuvant” for clFRCs. Additionally, a chapter is devoted to capture the current perspectives of the impact of interfacial phenomena on protein aggregation and immunogenicity. In conclusion, this dissertation provides original findings and opens new research avenues for advancing localized immunotherapies.

Language

English

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