Defense Date

2-28-2022

Graduation Date

Spring 5-21-2022

Availability

One-year Embargo

Submission Type

thesis

Degree Name

PhD

Department

Pharmaceutics

School

School of Pharmacy

Committee Chair

Wilson S Meng

Committee Member

James K Drennen III

Committee Member

Ellen S Gawalt

Committee Member

Devika S Manickam

Committee Member

Peter Wildfong

Keywords

hydrogel, self-assembling peptides, EAK, immune checkpoint inhibitor, Fc binding, localized delivery

Abstract

Renal cell carcinoma (RCC) is a type of cancer in which malignant cells develop in the kidney tubules. Immune checkpoint inhibitors (ICI), including anti-PD-1 antibodies (aPD1), as monotherapies or in combination settings have advanced to the front-line treatment for patients diagnosed with RCC. Systemic infusion of ICI may render subtherapeutic concentrations in the lesions and increase risks of immune-related adverse events. To overcome these limitations, locoregional drug delivery has gained substantial interests as a modality in immuno-oncology. However, conventional intratumoral injections may be ineffective because the high interstitial fluid pressure in tumors resists drug retention and results in rapid local elimination. In this dissertation work, we developed a unique injectable gel containing a self-assembling peptide AEAEAKAKAEAEAKAK (EAK) and FMNQAQRRFYEALHD, an immunoglobulin (Ig) constant fragment (Fc)-binding peptide to assist the locoregional delivery of ICI.

EAK is an amphiphilic peptide, which self-assembles into β-fibrils when ionic strength is raised by the addition of salt or in vivo injections. The helical peptide Z15 is miniaturized from the Fc-binding domain of Staphylococcus aureus protein A (SpA). The linkage of the two sequences using a spacer results in the bifunctional peptide, Z15_EAK, which exhibits self-assembling and Fc-binding functions. In physiological solutions, Z15_EAK forms characteristic β fibrils resembling those of EAK, which were also observed in transmission electron microscopy. The peptide also exhibits specific binding affinity for Ig, with Kd estimated in the low micromolar range. In a series of experiments in mice, Ig is retained in subcutaneous injection depots by the gel for extended durations, from 8 to 28 days. Pharmacokinetics (PK) modeling (Matlab Simbiology) was used to understand the impact of Z15_EAK on the local retention, and a bioinformatics method was developed to analyze antigenicity of the Z15_EAK sequence. Specifically, a PK model was constructed to predict the fraction of unbound Ig in the injection depot as a function of dosing regimen. The bioinformatics analysis reveals a relatively low potential of Z15_EAK to elicit immunogenicity compared to that of other protein A-derived Fc-binding domains.

The last phase of the project entailed using the Z15_EAK gel to deliver ICI in a mouse model of RCC. In addition to aPD1, adenosine deaminase (ADA), an endogenous enzyme that catabolizes adenosine was also included. The accumulation of extracellular adenosine in the tumor microenvironment is associated with resistance to PD1-targeted therapies in humans. The drug-loaded gels were injected near the tumor inoculating site ("peritumoral”) to render a sustained diffusion of aPD1 and ADA into the tumors and target T cells trafficking from the draining lymph nodes. The aPD1/ADA gel were shown shifted the local T cell population toward an effector phenotype while reducing the expansion of regulatory T cells (Tregs), which are immunosuppressive. The combined treatment delayed tumor growth and altered the expression of immune-related genes in the tumors. With all data considered, however, aPD1 appeared to be the main driver in the observed effects in mice received the combined treatment. Therefore, the contributions of ADA and the gel component need further investigation, with suggestions made in the last chapter of this document.

Language

English

Additional Citations

Pham, N.B., W. Liu, N.R. Schueller, E.S. Gawalt, Y. Fan, and W.S. Meng, Toward reducing biomaterial antigenic potential: a miniaturized Fc-binding domain for local deposition of antibodies. Biomaterials Science, 2019. 7(3): p. 760-772

NB Pham and WS Meng. Protein aggregation and immunogenicity of biotherapeutics. International Journal of Pharmaceutics (2020) 585: 119523

Download

Share

COinS