Cell surface engineering is an expanding field and whilst extensive research has been performed decorating cell surfaces with biomolecules, the engineering of cell surfaces with particles has been a largely unexploited area. This study reports on the assembly of cell-particle hybrids where irradiated tumor cells were surface engineered with adjuvant-loaded, biodegradable, biocompatible, polymeric particles, with the aim of generating a construct capable of functioning as a therapeutic cancer vaccine. Successfully assembled cell-particle hybrids presented here comprised either melanoma cells or prostate cancer cells stably adorned with Toll-like receptor-9 ligand-loaded particles using streptavidin-biotin cross-linking. Both cell-particle assemblies were tested in vivo for their potential as therapeutic cancer vaccines yielding promising therapeutic results for the prostate cancer model. The ramifications of results obtained for both tumor models are openly discussed. Graphical Abstract

Immune adjuvants are important components of current and prospective cancer vaccines. In this study we aimed at evaluating the use of a synthetic lipid A derivative, pentaerythritol lipid A (PET lipid A), loaded into poly(lactic-co-glycolic acid) (PLGA) particles, as a potential cancer vaccine adjuvant. PLGA particles (size range: 250 – 600 nm) were successfully formulated to include PET lipid A and/or the model tumor antigen, chicken ovalbumin (OVA). It was shown that particulated PET lipid A had a distinct advantage at promoting secretion of the immune potentiating cytokine, IL-12p70, and upregulating key costimulatory surface proteins, CD86 and CD40 in murine dendritic cells in vitro. In a murine tumor model, involving prophylactic vaccination with various permutations of soluble versus particulated formulations of OVA with or without PET lipid A, modest benefit was observed in terms of OVA-specific cell-mediated immune responses when PET lipid A was delivered in particles. These findings translated into a corresponding trend toward increased survival of mice challenged with OVA-expressing tumor cells (E.G7). In terms of translation of safe adjuvants into the clinic, these results promote the concept of delivering Toll-like receptor-4 (TLR-4) agonists in particles since doing so improves their adjuvant properties, whilst decreasing the chances of adverse effects due to off-target uptake by nonphagocytic cells.

Regulatory T (Treg) cells are one of the major immunosuppressive cell types in cancer and a potential target for immunotherapy, but targeting tumor-infiltrating (TI) Treg cells has been challenging. Here, using single-cell RNA sequencing of immune cells from renal clear cell carcinoma (ccRCC) patients, we identify two distinct transcriptional fates for TI Treg cells, Fate-1 and Fate-2. The Fate-1 signature is associated with a poorer prognosis in ccRCC and several other solid cancers. CD177, a cell surface protein normally expressed on neutrophil, is specifically expressed on Fate-1 TI Treg cells in several solid cancer types, but not on other TI or peripheral Treg cells. Mechanistically, blocking CD177 reduces the suppressive activity of Treg cells in vitro, while Treg-specific deletion of Cd177 leads to decreased tumor growth and reduced TI Treg frequency in mice. Our results thus uncover a functional CD177+ TI Treg population that may serve as a target for TI Treg-specific immunotherapy.

One of the primary goals of our group and our collaborators here at the University of Iowa is to develop therapeutic cancer vaccines using biodegradable and biocompatible polymer-based vectors. A major advantage of using discretely packaged immunogenic cargo over non-encapsulated vaccines is that they promote enhanced cellular immunity, a key requirement in achieving antitumor activity. We discuss the importance of co-encapsulation of tumor antigen and adjuvant, with specific focus on the synthetic oligonucleotide adjuvant, cytosine-phosphate-guanine oligodeoxynucleotides. We also discuss our research using a variety of polymers including poly(α-hydroxy acids) and polyanhydrides, with the aim of determining the effect that parameters, such as size and polymer type, can have on prophylactic and therapeutic tumor vaccine formulation efficacy. Aside from their role as vaccine vectors per se, we also address the research currently underway in our group that utilizes more novel applications of biodegradable polymer-based particles in facilitating other types of immune-based therapies.

Immune adjuvants, such as ligands for pathogen-associated molecular patterns (PAMPs), have been showing promise in boosting immune responses to tumor associated antigens, and delivering these adjuvants as discrete packages is considered advantageous over delivery in soluble form. Here we describe in detail, methods for independently loading a range of adjuvants into polymer-based biodegradable particles. We also describe the means by which to characterize these particles with respect to adjuvant loading and release kinetics as well as in terms of particle size, shape, and zeta-potential. These adjuvant-loaded particles have the potential to be used in dendritic cell-based uptake experiments performed in vitro or to be used in preclinical cancer vaccine research applications where they can be co-delivered with antigen-loaded particles or some other vaccine component comprising antigenic material.

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Cancer vaccines represent a promising treatment modality for a worldwide health problem. Whether as an adjuvant or as a stand-alone therapy, cancer vaccines represent a tumor-specific and systemic treatment potentially capable of eliminating metastatic lesions without the severe side-effects often associated with chemotherapy. Specifically, whole cell tumor vaccines have shown promise in preclinical and clinical settings and the studies presented here represent the beginnings of an approach to improve the antitumor potency of these vaccines. This project demonstrates as "proof of concept" the feasibility of manufacturing tumor cell-particle hybrids. The coupled use of these two components, whole tumor cells and cargo-carrying biodegradable particles, as one entity in a cancer vaccine system is a new line of research. Stable cell-particle hybrids were assembled using avidin-biotin chemistry where cargo-carrying PLGA particles (500 nm diameter) were coated with streptavidin and allowed to bind to tumor cells that had been indirectly labeled with biotin (using an integrin-specific biotinylated antibody). That successful cell-particle hybrids were assembled was determined by multiple means, including flow cytometry, laser scanning confocal microscopy and scanning electron microscopy. Two murine tumor cell lines (representing melanoma and prostate cancer) were investigated in this study and successfully demonstrated the general applicability of the assembly method. Particles appeared to be localized on the cell surface (rather than endocytosed) as determined by microscopic imaging. The cell-particle hybrid was shown to be stable to irradiation, an important consideration since whole tumor cells need to be treated with ionizing radiation prior to being used as vaccines in order to render them nonproliferative and immunogenic. We also characterized loading and release profiles of CpG, a prospective vaccine adjuvant, into PLGA particles. v We conclude that we have developed a method for manufacturing cell-particle hybrids comprising PLGA nanoparticles and irradiated tumor cells. The next step would be to use CpG-loaded particles in the assembled hybrid and test the anti-tumor immune efficiency of this cancer vaccine formulation in either a melanoma or prostate cancer model. vi TABLE OF CONTENTS LIST OF TABLES …………………………………………………………….

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