Novel phosphatidylserine-binding molecule enhances antitumor T-cell responses by targeting immunosuppressive exosomes in human tumor microenvironments

Abstract

BackgroundThe human tumor microenvironment (TME) is a complex and dynamic milieu of diverse acellular and cellular components, creating an immunosuppressive environment, which contributes to tumor progression. We have previously shown that phosphatidylserine (PS) expressed on the surface of exosomes isolated from human TMEs is causally linked to T-cell immunosuppression, representing a potential immunotherapeutic target. In this study, we investigated the effect of ExoBlock, a novel PS-binding molecule, on T-cell responses in the TME.MethodsWe designed and synthesized a new compound, (ZnDPA)6-DP-15K, a multivalent PS binder named ExoBlock. The PS-binding avidity of ExoBlock was tested using an in vitro competition assay. The ability of this molecule to reverse exosome-mediated immunosuppression in vitro was tested using human T-cell activation assays. The in vivo therapeutic efficacy of ExoBlock was then tested in two different human tumor xenograft models, the melanoma-based xenomimetic (X-)mouse model, and the ovarian tumor-based omental tumor xenograft (OTX) model.ResultsExoBlock was able to bind PS with high avidity and was found to consistently and significantly block the immunosuppressive activity of human ovarian tumor and melanoma-associated exosomes in vitro. ExoBlock was also able to significantly enhance T cell-mediated tumor suppression in vivo in both the X-mouse and the OTX model. In the X-mouse model, ExoBlock suppressed tumor recurrence in a T cell-dependent manner. In the OTX model, ExoBlock treatment resulted in an increase in the number as well as function of CD4 and CD8 T cells in the TME, which was associated with a reduction in tumor burden and metastasis, as well as in the number of circulating PS+ exosomes in tumor-bearing mice.ConclusionOur results establish that targeting exosomal PS in TMEs with ExoBlock represents a promising strategy to enhance antitumor T-cell responses.

Tumor-Associated Exosomes: A Potential Therapeutic Target for Restoring Anti-Tumor T Cell Responses in Human Tumor Microenvironments

“…Exosomes can be isolated using a variety of techniques that are based on utilizing one or more of their characteristic features such as density, shape, size, and surface molecules. The most popular method of isolation based on published literature is ultracentrifugation (UC) [8][9][10]21,22]. UC protocols for exosome isolation often include prior low-and mediumspeed centrifugation steps to exclude cells and cell debris respectively, as well as membrane filtration to exclude larger vesicles and organelles [8][9][10]21,23].…”

“…The most popular method of isolation based on published literature is ultracentrifugation (UC) [8][9][10]21,22]. UC protocols for exosome isolation often include prior low-and mediumspeed centrifugation steps to exclude cells and cell debris respectively, as well as membrane filtration to exclude larger vesicles and organelles [8][9][10]21,23]. Another popular technique used to isolate exosomes is called size exclusion chromatography (SEC) [10,22], which is a size-based isolation technique.…”

“…UC protocols for exosome isolation often include prior low-and mediumspeed centrifugation steps to exclude cells and cell debris respectively, as well as membrane filtration to exclude larger vesicles and organelles [8][9][10]21,23]. Another popular technique used to isolate exosomes is called size exclusion chromatography (SEC) [10,22], which is a size-based isolation technique. When size exclusion columns are used, different fractions are analyzed for their protein content as well as vesicle number, and the fractions with the highest vesicle to protein ratio are pooled to constitute the final exosome preparation [10].…”

“…In the first part of this review, we summarize these in vitro data, while also emphasizing the role of two exosomal lipids, phosphatidylserine (PS) and ganglioside GD3, that were causally linked with exosome-mediated suppression of human T cell function [8,9]. In this section, we also shed light on some recent results supporting the viability of targeting PS+ immunosuppressive exosomes in human TME using a novel PS-binding molecule called ExoBlock [10].…”

“…In this context, we focus upon the design of two novel human tumor xenograft platforms that has now made it possible to evaluate the pre-clinical efficacies of multiple different immune-based therapies [11,12] and to begin to monitor the presence and function of tumor-associated exosomes in vivo. In this segment, we also discuss the in vivo studies demonstrating the efficacy of blocking exosomes with ExoBlock using these two tumor xenograft models [10].…”