Supramolecular Peptide and Protein Assemblies for Applications in Immunotherapy and 3D Cell Culture

Peptide-based self-assembling biomaterials are a promising platform for biomedical applications such as immunomodulation, drug delivery, tissue repair and regeneration, cell delivery, and combinations thereof. However, many of these applications would benefit from the incorporation of folded proteins which have several advantages over their peptide counterparts. Despite their essential contribution to research progress over the years, bioactive peptides often fail to recapitulate the dynamic, high-affinity, and multifunctional nature of whole proteins. The ability to integrate and control the incorporation of protein components into self-assembling peptide materials would greatly broaden their applicability in biological contexts, particularly immune engineering and tissue engineering. In previous work, a strategy was established for inducing desired sets of expressed functional proteins to assemble directly into nanofibers or hydrogels through the use of a novel assembly tag known as the “βtail”. βtail proteins can be expressed and purified in a monomeric state, but they assemble in a modular fashion into compositionally defined nanofibers or gels when mixed with additional fibrillizing peptides. In this dissertation, we leveraged the novel βtail technology to elevate the function of self-assembling peptide materials for active immunotherapy and 3D cell culture applications. In concentrated form, peptide assemblies are well-studied matrices for the culture of many different cell types, but in more dilute formulations, they make nanofibers that are usefully immunostimulatory. The βtail system is useful for incorporating proteins in both contexts. The first half of this thesis (Chapters 3 and 4) describes the development of protein-bearing nanofibers for immunomodulation. We chose to focus on the protein C3dg, a late product of the complement cascade and key interface between innate and adaptive immunity. This protein has received considerable interest as a molecular adjuvant, but its utility in immunotherapies was yet to be fully realized. This was, in large part, due to an inability to assemble multiple copies of C3dg without utilizing chemistries that denature the protein or occlude its binding site. We overcame this issue with the βtail platform: by expressing a βtail-tagged version of C3dg and assembling multivalently into peptide nanofibers, we were able to enhance the humoral and cell-mediated immunogenic effects of C3dg. We initially investigated βtail-C3dg as a component in an active immunotherapy to mitigate TNF-mediated inflammation (Chapter 3). Active immunotherapies offer important advantages over existing biologics such as monoclonal antibodies (mAb), particularly towards chronic inflammatory diseases. Supramolecular assemblies based on a peptide system (Q11) containing βtail-tagged C3dg, B-cell epitopes from TNF, and the universal T-cell epitope PADRE raised strong antibody responses against both TNF and C3dg, and prophylactic immunization with these materials significantly improved protection in a lethal TNF-mediated inflammation model. Additionally, in a murine model of psoriasis induced by imiquimod, the C3dg-adjuvanted nanofiber vaccine performed as well as anti-TNF monoclonal antibodies. Nanofibers containing only βtail-C3dg and lacking the TNF B-cell epitope also showed improvements in both models, suggesting that supramolecular C3dg, by itself, played an important therapeutic role. We observed that immunization with βtail-C3dg caused the expansion of an autoreactive C3dg-specific T-cell population, which we believe acted to dampen the immune response, preventing excessive inflammation. These findings led us to believe that molecular assemblies displaying C3dg warrant further development as active immunotherapies. Due to its apparent anti-inflammatory characteristics, we sought to investigate the broad use of βtail-C3dg as a component in an active immunotherapy against another inflammatory molecule, complement component C5a (Chapter 4). There are no reported C5a B cell epitopes, so the epitopes investigated herein were predicted using the Kolaskar Tongaonkar Antigenicity Test and assembled with βtail-tagged C3dg and PADRE. Two out of the three selected epitopes raised IgG antibodies against C5a, and mice immunized with these formulations exhibited significantly reduced serum C5a concentrations. Interestingly, mice receiving prophylactic immunization with nanofiber formulations containing βtail-C3dg, C5a B-cell epitope, and PADRE exhibited reduced protection in a lethal sepsis model compared to formulations containing only the C5a B-cell epitope and PADRE. When we investigated the T-cell populations, we found that the combined C3dg/C5a immunizations elicited TH1-polarized autoreactive C3dg-specific T-cells. Because C5a plays a role in effector T-cell responses, we hypothesized that its combination with C3dg may have induced an inflammatory T-cell population against the C3dg component. Because formulations containing only the C5a B cell epitope and PADRE demonstrated efficacy, we proceeded to investigate formulations lacking the C3dg component. In a model of collagen antibody-induced arthritis, prophylactic immunization significantly improved the clinical severity of the disease. Despite the unexpectedly adverse contribution made by the βtail-C3dg component, this work represents a promising application of an active immunotherapy targeting complement C5a. The second half of this thesis (Chapters 5 and 6) focuses on the development of a tailored hydrogel matrix for prostate cancer cell growth in vitro using self-assembling peptides and proteins. One of the most significant challenges in establishing phenotypically accurate cultures of prostate cancer cells is constructing appropriate 3D culture environments. The utilization of matrices such as Matrigel has enabled the field to establish some prostate cancer organoid cultures, but Matrigel’s poor batch-to-batch consistency and “one size fits all” nature makes it difficult to customize for different cell types or different contexts. In response to these shortcomings, we designed a chemically defined nanofiber hydrogel (bQ13) that exhibits improved short- and long-term cytocompatibility for human prostate cancer cells compared to alternative commercially available 3D culture matrices. Building upon the success of bQ13, we sought to elevate the platform’s versatility by incorporating functional, structurally intricate proteins that interact directly with ligands and receptors to provoke a specific cellular response. Utilizing the βtail technology, we were able to assemble a range of concentrations of both green fluorescent protein (GFP) and the cell-binding domains of fibronectin (FN) into bQ13 nanofibers without altering the nanofiber structure or the mechanical properties of the hydrogel. The βtail did not interfere with the function of FN, allowing for the adhesion and spreading of cells in 2D and enhancing cell survival and proliferation in 3D. Additionally, the incorporation of an enzymatically cleavable sequence allowed for the controlled release of GFP from the bQ13 matrix, allowing for possible cell-monitoring applications. These results highlight the expanded adaptability of the bQ13 platform as a defined 3D matrix that can be tailored with folded proteins for various applications. This thesis outlines the first use of the βtail in specific biological applications and demonstrates its promising utility as a versatile biomaterial. The works discussed herein also propose significant contributions in the arenas of active immunotherapy and tissue engineering.