Adjuvant-Loaded Subcellular Vesicles Derived From Disrupted Cancer Cells for Cancer Vaccination.

Targeted subunit vaccines for cancer immunotherapy do not capture tumor antigenic complexity, and approaches employing tumor lysate are often limited by inefficient antigen uptake and presentation, and low immunogenicity. Here, whole cancer cells are processed to generate antigen-rich, membrane-enclosed subcellular particles, termed "reduced cancer cells", that reflect the diversity and breadth of the parent cancer cell antigen repertoire, and can be loaded with disparate adjuvant payloads. These vesicular particles enhance the uptake of the adjuvant payload, and potentiate the activation of primary dendritic cells in vitro. Similarly, reduced cancer cell-associated antigens are more efficiently presented by primary dendritic cells in vitro than their soluble counterparts or lysate control. In mice, vaccination using adjuvant-loaded reduced cancer cells facilitates the induction of antigen-specific cellular and humoral immune responses. Taken together, these observations demonstrate that adjuvant-loaded reduced cancer cells could be utilized in cancer vaccines as an alternative to lysate.

Activation and expansion of human T cells using artificial antigen-presenting cell scaffolds.

Synthetic antigen-presenting cells (APCs) are used to mediate scalable ex vivo T-cell expansion for adoptive cell therapy. Recently, we developed APC-mimetic scaffolds (APC-ms), which present signals to T cells in a physiological manner to mediate rapid and controlled T-cell expansion. APC-ms are composed of individual high-aspect-ratio silica microrods loaded with soluble mitogenic cues and coated with liposomes of defined compositions, to form supported lipid bilayers. Membrane-bound ligands for stimulation and co-stimulation of T-cell receptors are presented via the fluid, synthetic membranes, while mitogenic cues are released slowly from the microrods. In culture, interacting T cells assemble the individual APC-ms microrods into a biodegradable 3D matrix. Compared to conventional methods, APC-ms facilitates several-fold greater polyclonal T-cell expansion and improved antigen-specific enrichment of rare T-cell subpopulations. Here we provide a detailed protocol for APC-ms synthesis and use for human T-cell activation, and discuss important considerations for material design and T-cell co-culture. This protocol describes the facile assembly of APC-ms in ~4 h and rapid expansion or enrichment of relevant T-cell clones in <2 weeks, and is applicable for T-cell manufacturing and assay development.

Scaffolds that mimic antigen-presenting cells enable ex vivo expansion of primary T cells.

Therapeutic ex vivo T-cell expansion is limited by low rates and T-cell products of limited functionality. Here we describe a system that mimics natural antigen-presenting cells (APCs) and consists of a fluid lipid bilayer supported by mesoporous silica micro-rods. The lipid bilayer presents membrane-bound cues for T-cell receptor stimulation and costimulation, while the micro-rods enable sustained release of soluble paracrine cues. Using anti-CD3, anti-CD28, and interleukin-2, we show that the APC-mimetic scaffolds (APC-ms) promote two- to tenfold greater polyclonal expansion of primary mouse and human T cells compared with commercial expansion beads (Dynabeads). The efficiency of expansion depends on the density of stimulatory cues and the amount of material in the starting culture. Following a single stimulation, APC-ms enables antigen-specific expansion of rare cytotoxic T-cell subpopulations at a greater magnitude than autologous monocyte-derived dendritic cells after 2 weeks. APC-ms support over fivefold greater expansion of restimulated CD19 CAR-T cells than Dynabeads, with similar efficacy in a xenograft lymphoma model.

Liposomal Delivery Enhances Immune Activation by STING Agonists for Cancer Immunotherapy.

Overcoming the immunosuppressive tumor microenvironment (TME) is critical to realizing the potential of cancer immunotherapy strategies. Agonists of stimulator of interferon genes (STING), a cytosolic immune adaptor protein, have been shown to induce potent anti-tumor activity when delivered into the TME. However, the anionic properties of STING agonists make them poorly membrane permeable, and limit their ability to engage STING in the cytosol of responding cells. In this study, cationic liposomes with varying surface polyethylene glycol (PEG) levels were used to encapsulate cGAMP to facilitate its cytosolic delivery. In vitro studies with antigen-presenting cells (APCs) revealed that liposomal formulations substantially improved the cellular uptake of cGAMP and pro-inflammatory gene induction relative to free drug. Liposomal encapsulation allowed cGAMP delivery to metastatic melanoma tumors in the lung, leading to anti-tumor activity, whereas free drug produced no effect at the same dose. Injection of liposomal cGAMP into orthotopic melanoma tumors showed retention of cGAMP at the tumor site and co-localization with tumor-associated APCs. Liposomal delivery induced regression of injected tumors and produced immunological memory that protected previously treated mice from rechallenge with tumor cells. These results show that liposomal delivery improves STING agonist activity, and could improve their utility in clinical oncology.

Biophysical induction of cell release for minimally manipulative cell enrichment strategies.

The use of autologous cells harvested and subsequently transplanted in an intraoperative environment constitutes a new approach to promote regeneration. Usually cells are isolated by selection methods such as fluorescence- or magnetic- activated cell sorting with residual binding of the antibodies or beads. Thus, cell-based therapies would benefit from the development of new devices for cell isolation that minimally manipulate the target cell population. In the clinic, 5 to 10 percent of fractures do not heal properly and CD31+ cells have been identified as promising candidates to support bone regeneration. The aim of this project was to develop and prototype a simple system to facilitate the enrichment of CD31+ cells from whole blood. After validating the specificity of a commercially available aptamer for CD31, we combined this aptamer with traditional magnetic bead strategies, which led to enrichment of CD31+ cells with a purity of 91±10%. Subsequently, the aptamer was attached to agarose beads (Ø = 100-165 um) that were incorporated into a column-based system to enable capture and subsequent release of the CD31+ enriched cells. Different parameters were investigated to allow a biophysical-based cell release from beads, and a simple mixing was found sufficient to release initially bound cells from the optimized column without the need for any chemicals that promote disassociation. The system led to a significant enrichment of CD31+ cells (initial population: 63±9%, released: 87±3%) with excellent cell viability (released: 97±1%). The composition of the released CD31+ fraction indicated an enrichment of the monocyte population. The angiogenic and osteogenic potential of the released cell population were confirmed in vitro. These results and the simplicity of this system highlight the potential of such approach to enable cell enrichment strategies in intraoperative settings.

Engineered Materials for Cancer Immunotherapy.

Immunotherapy is a promising treatment modality for cancer as it can promote specific and durable anti-cancer responses. However, limitations to current approaches remain. Therapeutics administered as soluble injections often require high doses and frequent re-dosing, which can result in systemic toxicities. Soluble bolus-based vaccine formulations typically elicit weak cellular immune responses, limiting their use for cancer. Current methods for ex vivo T cell expansion for adoptive T cell therapies are suboptimal, and achieving high T cell persistence and sustained functionality with limited systemic toxicity following transfer remains challenging. Biomaterials can play important roles in addressing some of these limitations. For example, nanomaterials can be employed as vehicles to deliver immune modulating payloads to specific tissues, cells, and cellular compartments with minimal off-target toxicity, or to co-deliver antigen and danger signal in therapeutic vaccine formulations. Alternatively, micro-to macroscale materials can be employed as devices for controlled molecular and cellular delivery, or as engineered microenvironments for recruiting and programming immune cells in situ. Recent work has demonstrated the potential for combining cancer immunotherapy and biomaterials, and the application of biomaterials to cancer immunotherapy is likely to enable the development of effective next-generation platforms. This review discusses the application of engineered materials for the delivery of immune modulating agents to the tumor microenvironment, therapeutic cancer vaccination, and adoptive T cell therapy.

Solvation effects in calculated electrostatic association free energies for the C3d-CR2 complex and comparison with experimental data.

The complement system is an integral part of the innate immune system that participates in the clearance of pathogens from the body. The association between complement protein fragment C3d and B or T cell-receptor complement receptor (CR) 2 represents a crucial link between innate and adaptive immunities. The goal of this study is to predict association abilities of C3d and CR2 mutants by theoretically calculating electrostatic free energies of association and to assess the importance of solvation effects in the calculations. We demonstrate that calculated solvation free energy differences and Coulombic free energies of association are more sensitive than electrostatic free energies of association in solution and, thus, more accurate in predicting previously published experimental data for the association abilities (relative to the parent proteins) of specific C3d and CR2 mutants. We show that a proportional relationship exists between the predicted solvation free energy differences and the experimental data, while an inversely proportional relationship exists between the predicted Coulombic free energies of association and the experimental data. Our results yield new insights into the physicochemical properties underlying C3d-CR2 association. We discuss the predictive validity of Coulombic, solvation, and solution electrostatic free energies of association and the generalization of our method for theoretical mutagenesis studies of other systems. This is a basic study, aimed toward improving our understanding of the theoretical basis of immune system regulation at the molecular level. Such insight can serve as the groundwork for the design of regulators with tailored properties, vaccines, and other biotechnology products.