Browsing by Subject "Immunoengineering"

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (hMSC) chondrogenesis. We combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in hMSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce hMSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

Following this, we modified this anti-inflammatory engineered cartilage to incorporate rabbit MSCs and evaluated this therapeutic strategy in a pilot study in vivo in rabbit osteochondral defects. Rabbits were fed a custom doxycycline diet to induce gene expression in engineered cartilage implanted in the joint. Serum and synovial fluid were collected and the levels of doxycycline and inflammatory mediators were measured. Rabbits were euthanized 3 weeks following surgery and tissues were harvested for analysis. We found that doxycycline levels in serum and synovial fluid were too low to induce strong overexpression of hIL-1Ra in the joint and hIL-1Ra was undetectable in synovial fluid via ELISA. Although hIL-1Ra expression in the first few days local to the site of injury may have had a beneficial effect, overall a higher doxycycline dose and more readily transduced cell population would improve application of this therapy.

In addition to the 3D woven PCL scaffold, cartilage-derived matrix scaffolds have recently emerged as a promising option for cartilage tissue engineering. Spatially-defined, biomaterial-mediated lentiviral gene delivery of tunable and inducible morphogenetic transgenes may enable guided differentiation of hMSCs into both cartilage and bone within CDM scaffolds, enhancing the ability of the CDM scaffold to provide chondrogenic cues to hMSCs. In addition to controlled production of anti-inflammatory proteins within the joint, in situ production of chondro- and osteo-inductive factors within tissue-engineered cartilage, bone, or osteochondral tissue may be highly advantageous as it could eliminate the need for extensive in vitro differentiation involving supplementation of culture media with exogenous growth factors. To this end, we have utilized controlled overexpression of transforming growth factor-beta 3 (TGF-β3), bone morphogenetic protein-2 (BMP-2) or a combination of both factors, to induce chondrogenesis, osteogenesis, or both, within CDM hemispheres. We found that TGF-β3 overexpression led to robust chondrogenesis in vitro and BMP-2 overexpression led to mineralization but not accumulation of type I collagen. We also showed the development of a single osteochondral construct by combining tissues overexpressing BMP-2 (hemisphere insert) and TGF-β3 (hollow hemisphere shell) and culturing them together in the same media. Chondrogenic ECM was localized in the TGF-β3-expressing portion and osteogenic ECM was localized in the BMP-2-expressing region. Tissue also formed in the interface between the two pieces, integrating them into a single construct.

Since CDM scaffolds can be enzymatically degraded just like native cartilage, we hypothesized that IL-1 may have an even larger influence on CDM than PCL tissue-engineered constructs. Additionally, anti-inflammatory engineered cartilage implanted in vivo will likely affect cartilage and the underlying bone. There is some evidence that osteogenesis may be enhanced by IL-1 treatment rather than inhibited. To investigate the effects of an inflammatory environment on osteogenesis and chondrogenesis within CDM hemispheres, we evaluated the ability of IL-1Ra-expressing or control constructs to undergo chondrogenesis and osteogenesis in the prescence of IL-1. We found that IL-1 prevented chondrogenesis in CDM hemispheres but did not did not produce discernable effects on osteogenesis in CDM hemispheres. IL-1Ra-expressing CDM hemispheres produced robust cartilage-like ECM and did not upregulate inflammatory mediators during chondrogenic culture in the presence of IL-1.

Inflammatory bowel disease (IBD) is a chronic disorder characterized by persistent inflammation in the gastrointestinal tract. Current therapies for IBD, such as anti-inflammatory or immunosuppressant drugs and anti-cytokine biologics, only temporarily alleviate symptoms and vary widely in effectiveness among patients. Consequently, there exists a critical unmet need for a long-lasting and broadly effective IBD treatment. Natural antibodies against the small molecule epitope phosphorylcholine (PC) are an important component of innate immunity with diverse functions including the clearance of bacterial and autologous targets in a non-inflammatory manner. The cells that produce these antibodies, B1a cells, however, have been shown to be reduced in patients with IBD, with this decrease being associated with a more advanced disease state. It has also been demonstrated that the adoptive transfer of B1a cells in a murine model of IBD results in the increased production of anti-PC antibodies and lessens disease severity. Furthermore, active immunotherapies are an alternative to monoclonal antibody biologics and a promising approach for generating a long-lasting IBD therapy because they exploit the ability of a patient’s own immune system to produce antibodies against a therapeutic target. Building on these concepts, we strove to develop an active immunotherapy consisting of PC as an epitope displayed on self-assembling peptide nanofibers to produce a therapeutic anti-PC antibody response for the treatment of IBD. The first part of this dissertation (Chapter 3) describes the process of designing, determining the therapeutic efficacy of, and gaining mechanistic insights into a nanofiber-based anti-PC active immunotherapy. We began by developing conjugation strategies for attaching PC to Q11 self-assembling peptides to render PC immunogenic. In an effort to find a balance between immunogenicity, stability, and ease of synthesis, we compared phosphoramidite and phosphodiester linkages for PC and concluded that the phosphodiester linkage was critical for PC epitope integrity. We then investigated how altering the multivalency of PC on Q11 nanofibers could further augment the anti-PC antibody response by synthesizing two nanofiber and PC conjugates with either 1 or 1-4 PC copies per Q11 peptide termed PC-Q11 and PCM-Q11, respectively. Intraperitoneal (i.p.) immunization with PCM-Q11 was found to induce a significantly greater anti-PC antibody response than i.p. immunization with PC-Q11. Additionally, PCM-Q11 was more selectively taken up by and able to activate natural antibody-producing B1a cells compared to all other B cells than PC-Q11 or Q11 alone. Further, control over the immune phenotype elicited was achieved via the inclusion of a T-cell epitope and/or CpG adjuvant, with the addition of both greatly augmenting the immune response elicited. We then evaluated the efficacy of immunizations with PCM-Q11/T-cell epitope with or without CpG in several different dextran sodium sulfate (DSS)-induced murine colitis models. We first investigated the ability of these immunizations to prevent severe disease when administered prior to the induction of a 30-day chronic DSS colitis model. Interestingly, immunization with both formulations was protective, significantly improving weight loss, disease severity indices, and colon lengths over unimmunized controls. This efficacy was repeated in a 1 cycle (10-day) DSS colitis model in both male and female mice and not attributed to CpG administration alone. Immunizations against PC also lowered bacterial spread to the spleen due to colon damage, with this effect being more pronounced in female mice. Additionally, we determined the efficacy of PCM-Q11 immunizations in a therapeutic setting where mice received one cycle of DSS colitis followed by three immunizations and then one more cycle of DSS colitis. This showed that immunizations with PCM-Q11 have therapeutic efficacy by significantly improving weight loss, disease activity indices, colon lengths, and bacterial spread to the spleen in this model. Furthermore, we have conducted several other studies to gain mechanistic insight into the observed efficacy of PCM-Q11 immunizations. We found that anti-PC immunization decreases microbiome diversity. We were also able to use flow cytometry to detect IgG and IgM antibodies in serum from mice immunized with PCM-Q11 that bind apoptotic colon epithelial cells in vitro. Additionally, we have shown through passive transfer of PCM-Q11 immunized sera that induced anti-PC antibodies offer some protection against severe DSS-induced colitis. Moreover, through both histological examination of colon damage and immunofluorescence imaging of tight junction proteins, we determined that PCM-Q11 immunizations were not acting by improving barrier function in the colon. Finally, we observed reduced efficacy of PCM-Q11 immunizations in an Il10-/- murine model of colitis. Collectively, this data demonstrates that immunization with PCM-Q11 was both preventative and therapeutic in multiple DSS-induced models of colitis in mice, with considerable efficacy attributed to the induced anti-PC antibody response. In the second part of this dissertation (Chapter 4), peptide nanofibers were modified for use as oral vaccines. While oral delivery offers direct access for eliciting immune responses within the gastrointestinal tract, it poses substantial obstacles for vaccines to overcome including acidic and proteolytic environments, thick mucus barriers, and a limited window for absorption. We therefore focused on two main design improvements to peptide nanofibers: synthesis with protease-resistant D-amino acids and incorporation of muco-penetrative peptide sequences rich in proline, alanine, and serine (PAS). We showed that D-amino acid Q11 was not degraded in simulated gastrointestinal environments in contrast to its L-amino acid counterpart. Additionally, we determined that PASylation enhanced muco-penetration in vitro and accelerated nanofiber transport through the GI tract in vivo. Ultimately, however, we found that oral immunization with PASylated L-amino acid nanofibers with cholera toxin B subunit mucosal adjuvant was the optimal formulation for the generation of both local and systemic immune responses. The primary areas affected in IBD are the distal small intestines and the colon, so the induction of therapeutic antibodies in these tissues is paramount. Thus, we sought to translate the above design principles to the small molecule epitope, PC, to enable oral administration. Oral immunization with PASylated anti-PC formulations was able to generate both local and systemic anti-PC immune responses. We then illustrated that oral vaccination with PASylated PC-bearing nanofibers was effective in both therapeutic and prophylactic models of DSS-induced colitis, observing comparable reductions in disease severity to i.p. anti-PC immunizations, thus, increasing the translatability of our therapy by offering a needle-free formulation. Overall, this data indicates that PASylated supramolecular peptide nanofibers are a promising platform for oral immunization. This dissertation outlines an encouraging first example of an active immunotherapy engaging natural antibody responses against phosphorylcholine as a durable therapy for IBD. More broadly, the strategies developed offer a potentially versatile approach for engaging natural antibody therapies and oral nanofiber peptide vaccines towards a variety of inflammatory and infectious diseases.

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.