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  • br ACCEPTED MANUSCRIPT br Another interesting approach to en


    Another interesting approach to enhancing anti-PD-1 therapies has been the use of PD-1 receptor-expressing nanovesicle decoys88. Cellular membranes can be engineered into nanoscale vesicles using a membrane extrusion method. One group generated nanovesicles with high levels of PD-1 receptor from HEK193T cells stably overexpressing PD-1. The nanovesicles remained in circulation longer than PDL-1 9004-65-3 and induced significantly greater CD8+ tumor infiltration and tumor regression in melanoma tumor models. They could further enhance antitumor effects by co-delivering encapsulated 1 methyltrypophan (a potent inhibitor of IDO) to offset the actions of co-localized immunosuppressive cell populations.
    It is also possible to enhance the effects of PD-1 blockade by increasing activation of professional APCs. Activated APCs release proinflammatory cytokines including type-1 interferons 9004-65-3 that promote the formation of antigen-specific T-cells. Cytosolic cyclic dinucleotides are used as signaling molecules by bacteria and potent immune-stimulating danger signals. Endogenous cytosolic DNA can also be converted to cyclic dinucleotide cGMP-AMP synthase (cGAS)89. Cytosolic dinucleotides potently stimulate type-1 INF responses in a TLR-independent mechanism by activating the endoplasmic reticulum protein STING90. STING agonists appear very promising in preclinical tumor models and are being investigated in clinical trials to enhance responses to checkpoint inhibitors. However, the intracellular localization of STING may be problematic even for direct tumoral injections of cyclic dinucleotides as cytosolic accumulation is poor. Wilson and colleagues generated cationic poly(beta-amino ester) (PBAE) NPs for the intracellular delivery of cyclic dinucleotide STING agonists91. NP formulation increased STING activation 10-fold compared to direct tumoral injection of cyclic dinucleotides and improved tumor growth delay alone or in combination with anti-PD-1 antibodies in B16F10 melanoma tumor models.
    3.2.2 NP Delivery of T-cell Activators
    Enhancing cytotoxic T-cells by activating stimulatory surface receptors has also been explored as a way to overcome resistance to checkpoint inhibitors and T-cell modulation by immunosuppressive cell-types in the TME. Agonists to OX40 have been tested in clinical trials but early results have shown modest efficacy with conventional systemic delivery92,93. Enrichment of these drugs in tumors using NP delivery appears to improve their efficacy. One study compared in vivo cytokine modulation and tumor growth delay using free and PLGA NP-delivered OX40 stimulatory antibodies94. NP formulation significantly enhanced CD8+ and NK tumor infiltration, proinflammatory cytokine signaling, and anti-tumor cytotoxicity compared to free antibody. Another study attempted to enhance the activation of antigen-specific clonal T-cells at the point of engagement with tumors using tumor-targeted OX40-stimulating NPs. They generated specific AFP targeted CD8+ T-cells (AFP 158-166) and PLGA polymeric NPs co-decorated with AFP binding antibodies and stimulating OX40 antibodies95. Dual-targeted NPs efficiently enhanced T-cell activation and cytotoxic killing of AFP-expressing HepB2 hepatocellular tumor cells but had no AFP negative SMMC-7721 cells indicating that the efficacy of OX40 stimulation is enhanced by temporal co-localization with target cells.
    Simultaneous OX40 activation and PD-1 disinhibition has also been investigated to maximize CD8+ T-cell activation. Systemic co-administration of activating OX40 and inhibitory PD-1 antibodies is inefficient because only a small fraction of T-cells will be engaged by both antibodies. Mi et al. developed polymeric NPs co-decorated with OX40 and PDL-1 antibodies to enable spatialtemporal co-delivery of both signals to T-cells96. The dual-targeted NPs stimulated potent systemic immune responses in B16 melanoma and 4T1 breast cancer models. They observed complete tumor regression in almost 40% of animals treated with co-decorated NPs compared to only 5% in animals treated with both
    antibodies as systemic agents or singly decorated NPs. Durable immunity was achieved as 83% of cured mice were resistant to tumor re-challenge. In a slightly different paradigm, another group generated NPs co-decorated with inhibitory PDL-1 antibodies and stimulatory 41BB antibodies to simultaneously engage tumor and T-cells97. Dual-targeted NPs improved tumor growth delay and resulted in expansion of tumor-specific T-cell clonal populations. These studies provide strong evidence for the importance of simultaneous (or near simultaneous) delivery of complimentary signals to maximize the efficacy of cancer immunotherapies and nicely highlight advances not possible with conventional drug delivery.