ST3GAL1 and ßII-spectrin pathways control CAR T cell migration to target tumors.

Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T cell immunotherapies have mostly failed in individuals with solid tumors. Here, with a CRISPR-Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 ß-galactoside α-2,3-sialyltransferase 1 (ST3GAL1) as a negative regulator of the cancer-specific migration of CAR T cells. Analysis of glycosylated proteins revealed that CD18 is a major effector of ST3GAL1 in activated CD8+ T cells. ST3GAL1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR T cells with enhanced expression of ßII-spectrin, a central LFA-1-associated cytoskeleton molecule, reversed ST3GAL1-mediated nonspecific T cell migration and reduced tumor growth in mice by improving tumor-specific homing of CAR T cells. These findings identify the ST3GAL1-ßII-spectrin axis as a major cell-intrinsic program for cancer-targeting CAR T cell migration and as a promising strategy for effective T cell immunotherapy.

In Situ One-Step Fluorescence Labeling Strategy of Exosomes via Bioorthogonal Click Chemistry for Real-Time Exosome Tracking In Vitro and In Vivo.

Exosomes are cellular components with promising uses in cancer diagnostics and therapeutics, and their imaging and tracking are essential to study their biological properties. Herein, we report on an in situ one-step fluorescence labeling strategy for exosomes via bioorthogonal click chemistry. First, exosome donor cancer cells were treated with tetraacetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) to generate unnatural azide groups (-N3) on their surface via metabolic glycoengineering. Then, the azide groups were labeled with near-infrared fluorescent dye-conjugated dibenzylcyclooctyne (DBCO-Cy5) via bioorthogonal click chemistry. After 2 days of incubation, the DBCO-Cy5-labeled exosomes (Cy5-Exo) were successfully secreted from the donor cancer cells and were isolated via classical ultracentrifugation, providing a high-yield of fluorescent dye-labeled exosomes. This in situ one-step bioorthogonal click chemistry offers improved labeling efficiency, biocompatibility, and imaging sensitivy compared to standard exosomes (ST-Exo), purified with classical ultracentrifugation or carbocyanine lipophilic dye (DiD)-labeled exosomes (DiD-Exo) in vitro. In particular, the Cy5-Exo were successfully taken up by A549 cells in a time-dependent manner, and they could escape from lysosome confinement, showing their possible use as a delivery carrier of therapeutic drugs or imaging agents. Finally, intraveneously injected Cy5-Exo were noninvasively tracked and imaged via near-infrared fluorescence (NIRF) imaging in tumor-bearing mice. This new fluorescence labeling strategy for natural exosomes may be useful to provide better understanding of their theranostic effects in many biomedical applications.

PD-1Hi CAR-T cells provide superior protection against solid tumors.

Chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising treatment option for several hematologic cancers. However, efforts to achieve the same level of therapeutic success in solid tumors have largely failed mainly due to CAR-T cell exhaustion and poor persistence at the tumor site. Although immunosuppression mediated by augmented programmed cell death protein-1 (PD-1) expression has been proposed to cause CAR-T cell hypofunction and limited clinical efficacy, little is known about the underlying mechanisms and immunological consequences of PD-1 expression on CAR-T cells. With flow cytometry analyses and in vitro and in vivo anti-cancer T cell function assays, we found that both manufactured murine and human CAR-T cell products displayed phenotypic signs of T cell exhaustion and heterogeneous expression levels of PD-1. Unexpectedly, PD-1high CAR-T cells outperformed PD-1low CAR-T cells in multiple T cell functions both in vitro and in vivo. Despite the achievement of superior persistence at the tumor site in vivo, adoptive transfer of PD-1high CAR-T cells alone failed to control tumor growth. Instead, a PD-1 blockade combination therapy significantly delayed tumor progression in mice infused with PD-1high CAR-T cells. Therefore, our data demonstrate that robust T cell activation during the ex vivo CAR-T cell manufacturing process generates a PD-1high CAR-T cell subset with improved persistence and enhanced anti-cancer functions. However, these cells may be vulnerable to the immunosuppressive microenvironment and require combination with PD-1 inhibition to maximize therapeutic functions in solid tumors.

Exosome-guided direct reprogramming of tumor-associated macrophages from protumorigenic to antitumorigenic to fight cancer.

Highly immunosuppressive tumor microenvironment containing various protumoral immune cells accelerates malignant transformation and treatment resistance. In particular, tumor-associated macrophages (TAMs), as the predominant infiltrated immune cells in a tumor, play a pivotal role in regulating the immunosuppressive tumor microenvironment. As a potential therapeutic strategy to counteract TAMs, here we explore an exosome-guided in situ direct reprogramming of tumor-supportive M2-polarized TAMs into tumor-attacking M1-type macrophages. Exosomes derived from M1-type macrophages (M1-Exo) promote a phenotypic switch from anti-inflammatory M2-like TAMs toward pro-inflammatory M1-type macrophages with high conversion efficiency. Reprogrammed M1 macrophages possessing protein-expression profiles similar to those of classically activated M1 macrophages display significantly increased phagocytic function and robust cross-presentation ability, potentiating antitumor immunity surrounding the tumor. Strikingly, these M1-Exo also lead to the conversion of human patient-derived TAMs into M1-like macrophages that highly express MHC class II, offering the clinical potential of autologous and allogeneic exosome-guided direct TAM reprogramming for arming macrophages to join the fight against cancer.

Investigation of d-Amino Acid-Based Surfactants and Nanocomposites with Gold and Silica Nanoparticles as against Multidrug-Resistant Bacteria Agents.

d-amino acid-based surfactants (d-AASs) were synthesized and their antimicrobial activity was evaluated. N-α-lauroyl-d-arginine ethyl ester hydrochloride (d-LAE), d-proline dodecyl ester (d-PD), and d-alanine dodecyl ester (d-AD) were found to have antibacterial activity against both Gram-positive and -negative bacteria, but less efficacy against Gram-negative bacteria. For these reasons, combining antimicrobial agents with nanoparticles is a promising technique for improving their antibacterial properties to eliminate drug-resistant pathogens. d-LAE coated on gold (AuNP) and silica (SiNP) nanoparticles has more efficient antibacterial activity than that of d-LAE alone. However, unlike d-LAE, d-PD has enhanced antibacterial activity upon being coated on AuNP. The antibacterial d-AASs and their nanocomposites with nanoparticles were synthesized in an environmentally friendly manner and are expected to be valuable new antimicrobial agents against multidrug-resistant (MDR) pathogens.

Advantage of extracellular vesicles in hindering the CD47 signal for cancer immunotherapy.

The cluster of differentiation 47 (CD47) protein is abundantly expressed on various malignant cells and suppresses the phagocytic function of macrophages and dendritic cells. High CD47 expression levels are correlated with poor cancer survival. Antagonizing CD47 antibodies with potent antitumor effects have been developed in clinical trials, but have critical side effects, inducing anemia and thrombocytopenia. To develop a safe and potent CD47 blockade, we designed extracellular vesicles (EVs) harboring signal regulatory protein alpha (SIPRα)-EV-SIRPα (EVs that express SIPRα). EV-SIRPα showed minimal toxic effects on hematologic parameters and utilized RBCs as delivery vehicles to tumors rather than inducing anemia. EV-SIRPα inhibited ligation of residual CD47 molecules, which attribute to the EV-endocytosis-mediated CD47 depletion and steric hindrance of EV. In an immunologically cold tumor model, EV-SIRPα induced tumor-specific T-cell-mediated antitumor effects. When directly administered to the accessible lesions, EV-SIRPα monotherapy elicited an abscopal effect in the B16F10 tumor model by increasing immune cell infiltration and CD8+-mediated immunity against non-treated tumors. The combinational approach by loading doxorubicin into the EV-SIRPα dramatically reduced the tumor burden and led to 80% complete remission rate. Thus, a potent EV-based CD47 blockade that is hematologically safe, has efficient signaling blocking efficacy, and has systemic antitumor immunity against cancer is recommended.

Mechanically active integrins target lytic secretion at the immune synapse to facilitate cellular cytotoxicity.

Cytotoxic lymphocytes fight pathogens and cancer by forming immune synapses with infected or transformed target cells and then secreting cytotoxic perforin and granzyme into the synaptic space, with potent and specific killing achieved by this focused delivery. The mechanisms that establish the precise location of secretory events, however, remain poorly understood. Here we use single cell biophysical measurements, micropatterning, and functional assays to demonstrate that localized mechanotransduction helps define the position of secretory events within the synapse. Ligand-bound integrins, predominantly the αLß2 isoform LFA-1, function as spatial cues to attract lytic granules containing perforin and granzyme and induce their fusion with the plasma membrane for content release. LFA-1 is subjected to pulling forces within secretory domains, and disruption of these forces via depletion of the adaptor molecule talin abrogates cytotoxicity. We thus conclude that lymphocytes employ an integrin-dependent mechanical checkpoint to enhance their cytotoxic power and fidelity.

In situ immunogenic clearance induced by a combination of photodynamic therapy and rho-kinase inhibition sensitizes immune checkpoint blockade response to elicit systemic antitumor immunity against intraocular melanoma and its metastasis.

BACKGROUND: Uveal melanoma (UM) is the most frequent intraocular malignancy and is resistant to immunotherapy. Nearly 50% of patients with UM develop metastatic disease, and the overall survival outcome remains very poor. Therefore, a treatment regimen that simultaneously targets primary UM and prevents metastasis is needed. Here, we suggest an immunotherapeutic strategy for UM involving a combination of local photodynamic therapy (PDT), rho-kinase (ROCK) inhibitor, and PD-1/PD-L1 immune checkpoint blockade. METHODS: The antitumor efficacy and immune response of monotreatment or combinational treatment were evaluated in B16F10-bearing syngeneic mouse models. Abscopal antitumor immune responses induced by triple-combinational treatment were validated in syngeneic bilateral B16F10 models. After each treatment, the immune profiles and functional examinations were assessed in tumors and tumor draining lymph nodes by flow cytometry, ELISA, and immunofluorescence assays. In orthotopic intraocular melanoma models, the location of the immune infiltrate in the tumor microenvironment (TME) was evaluated after each treatment by multiplex immunohistochemistry and metastatic nodules were monitored. RESULTS: PDT with Ce6-embedded nanophotosensitizer (FIC-PDT) elicited immunogenic cell death and stimulated antigen-presenting cells. In situ immunogenic clearance induced by a combination of FIC-PDT with ripasudil, a clinically approved ROCK inhibitor, stimulated antigen-presenting cells, which in turn primed tumor-specific cytotoxic T cells. Moreover, local immunogenic clearance sensitized PD-1/PD-L1 immune checkpoint blockade responses to reconstruct the TME immune phenotypes of cold tumors into hot tumors, resulting in recruitment of robust cytotoxic CD8+ T cells in the TME, propagation of systemic antitumor immunity to mediate abscopal effects, and prolonged survival. In an immune-privileged orthotopic intraocular melanoma model, even low-dose FIC-PDT and ripasudil combined with anti-PD-L1 antibody reduced the primary tumor burden and prevented metastasis. CONCLUSIONS: A combination of localized FIC-PDT and a ROCK inhibitor exerted a cancer vaccine-like function. Immunogenic clearance led to the trafficking of CD8+ T cells into the primary tumor site and sensitized the immune checkpoint blockade response to evoke systemic antitumor immunity to inhibit metastasis, one of the major challenges in UM therapy. Thus, immunogenic clearance induced by FIC-PDT and ROCK inhibitor combined with anti-PD-L1 antibody could be a potent immunotherapeutic strategy for UM.

Xenogenization of tumor cells by fusogenic exosomes in tumor microenvironment ignites and propagates antitumor immunity.

Many cancer patients not responding to current immunotherapies fail to produce tumor-specific T cells for various reasons, such as a lack of recognition of cancer cells as foreign. Here, we suggest a previously unidentified method for xenogenizing (turning self to non-self) tumors by using fusogenic exosomes to introduce fusogenic viral antigens (VSV-G) onto the tumor cell surface. We found that xenogenized tumor cells were readily recognized and engulfed by dendritic cells; thereby, tumor antigens were efficiently presented to T lymphocytes. Moreover, exosome-VSV-G itself acts as a TLR4 agonist and stimulates the maturation of dendritic cells, leading to CD8+ T cell cross-priming. The administration of these exosomes in multiple tumor mouse models xenogenized tumor cells, resulting in tumor growth inhibition. The combinatorial treatment with anti-PD-L1 exhibited complete tumor regression (30%) and better long-term overall survival. These results suggest that tumor xenogenization by fusogenic exosomes provides a previously unidentified novel strategy for cancer immunotherapy.

Dendritic cell activation by an E. coli-derived monophosphoryl lipid A enhances the efficacy of PD-1 blockade.

Cancer immunotherapy is a powerful approach for cancer treatment, but its clinical effects rely on the tumor's immune conditions. In particular, low response rates to PD-1 blockades are highly correlated with impaired T cell priming. Here, we demonstrate that E. coli-derived monophosphoryl lipid A (EcML) activates dendritic cells in a toll-like receptor-4 (TLR-4)-dependent manner and increases the sensitivity of cancer cells to anti-PD-1 immunotherapy. EcML is a mixture of 4'-monophosphoryl lipids A (MPLAs) produced directly by an engineered Escherichia coli strain; it has a unique congener composition that differentiates it from the well-established MPLA adjuvants, 3-O-desacyl-4'-monophosphoryl lipid A and glucopyranosyl lipid A. Given that active dendritic cells initiate adaptive immune responses, we investigated the anti-tumor activity of an aqueous formulation of EcML. Upon sensing EcML via TLR-4, dendritic cells matured into powerful antigen-presenting cells that could stimulate naïve T cells. EcML reduced tumor growth in the B16F10 mouse model via dendritic cell activation and potentiated PD-1 blockade therapy in the B16F10-OVA melanoma model. These data identify EcML as a promising TLR-4 agonist that can induce anti-tumor immune responses and potentiate PD-1 blockade therapy against tumors.

An optimized protocol to determine the engulfment of cancer cells by phagocytes using flow cytometry and fluorescence microscopy.

The engulfment of cancer cells by macrophages is an important cellular process in innate cancer immunity. Antitumor immunotherapy that utilizes the enhanced engulfment of cancer cells by phagocytic cells has attracted much attention. Therefore, there is a growing demand for methods of measuring cancer cell phagocytosis. Quantifying the various stages of phagocytosis is invaluable for elucidating cancer-immune responses during this process. Here, we describe two phagocytosis assays, a flow cytometric assay and a fluorescent microscopic assay; the flow cytometric method utilizing CellTracker dye provides a simple, measurable, and highly reproducible functional assay to measure the phagocytosis efficiency of cancer cells by bone marrow-derived macrophages. As an alternative method of evaluating various states of cancer cell phagocytosis, a fluorescent microscopic method that employs a pH-sensitive dye (pHrodo-SE dye) is also described in this paper. Image-based analysis using this labeling approach enables researchers to measure phagocytic indices that indicate the number of cancer cells engulfed by each macrophage. We have highlighted that these assays can be applied to multiple tumor types and used as selection tools for a variety of phagocytosis agonist types. The results of this study may facilitate a better understanding of the interactions between tumor cells and phagocytes, which could lead to the identification of new therapeutic targets against cancer.

Degradation of tumour stromal hyaluronan by small extracellular vesicle-PH20 stimulates CD103+ dendritic cells and in combination with PD-L1 blockade boosts anti-tumour immunity.

Highly accumulated hyaluronan (HA) not only provides a physiological barrier but also supports an immune-suppressive tumour microenvironment. High-molecular-weight (HMW)-HA inhibits the activation of immune cells and their access into tumour tissues, whereas, low-molecular-weight oligo-HA is known to potentially activate dendritic cells (DCs). In this paper, we investigated whether small extracellular vesicle (EVs)-PH20 hyaluronidase induces tumour HA degradation, which, in turn, activates DCs to promote anti-cancer immune responses. Informed by our previous work, we used a small EV carrying GPI-anchored PH20 hyaluronidase (Exo-PH20) that could deeply penetrate into tumour foci via HA degradation. We found that Exo-PH20-treatment successfully activates the maturation and migration of DCs in vivo, particularly CD103+ DCs leading to the activation of tumour-specific CD8+ T cells, which work together to inhibit tumour growth. Moreover, combination with anti-PD-L1 antibody provided potent tumour-specific CD8+ T cell immune responses as well as elicited prominent tumour growth inhibition both in syngenic and spontaneous breast cancer models, and this anti-tumour immunity was durable. Together, these results present new insights for HA degradation by Exo-PH20, providing a better understanding of oligo HA-triggered immune responses to cancer.

Extracellular vesicles as a platform for membrane-associated therapeutic protein delivery.

Membrane proteins are of great research interest, particularly because they are rich in targets for therapeutic application. The suitability of various membrane proteins as targets for therapeutic formulations, such as drugs or antibodies, has been studied in preclinical and clinical studies. For therapeutic application, however, a protein must be expressed and purified in as close to its native conformation as possible. This has proven difficult for membrane proteins, as their native conformation requires the association with an appropriate cellular membrane. One solution to this problem is to use extracellular vesicles as a display platform. Exosomes and microvesicles are membranous extracellular vesicles that are released from most cells. Their membranes may provide a favourable microenvironment for membrane proteins to take on their proper conformation, activity, and membrane distribution; moreover, membrane proteins can cluster into microdomains on the surface of extracellular vesicles following their biogenesis. In this review, we survey the state-of-the-art of extracellular vesicle (exosome and small-sized microvesicle)-based therapeutics, evaluate the current biological understanding of these formulations, and forecast the technical advances that will be needed to continue driving the development of membrane protein therapeutics.

Comparison of exosomes and ferritin protein nanocages for the delivery of membrane protein therapeutics.

Exosomes are small membrane vesicles secreted by most cell types that play an important role in intercellular communication. Due to the characteristic of transferring their biomacromolecules, exosomes have potential as a new alternative for delivering protein therapeutics. Here, we investigate whether exosomes provide crucial advantages over other nanoparticles, in particular protein nanocage formulations, as a delivery system for membrane protein therapeutics. We characterized membrane-scaffold-based exosomes and protein-scaffold-based ferritin nanocages, both harboring SIRPα (signal regulatory protein α), an antagonist of CD47 on tumor cells. The efficacy of these two systems in delivering protein therapeutics was compared by testing their ability to enhance phagocytosis of tumor cells by bone-marrow-derived macrophages and subsequent inhibition of in vivo tumor growth. These analyses allowed us to comprehensively conclude that the therapeutic index of exosome-mediated CD47 blockade against tumor growth inhibition was higher than that of the same dose of ferritin-SIRPα. The results of this analysis reveal the importance of the unique characteristics of exosomes, in particular their membrane scaffold, in improving therapeutic protein delivery compared with protein-scaffold-based nanocages.

Nanocage-Therapeutics Prevailing Phagocytosis and Immunogenic Cell Death Awakens Immunity against Cancer.

A growing appreciation of the relationship between the immune system and the tumorigenesis has led to the development of strategies aimed at "re-editing" the immune system to kill tumors. Here, a novel tactic is reported for overcoming the activation-energy threshold of the immunosuppressive tumor microenvironment and mediating the delivery and presentation of tumor neoantigens to the host's immune system. This nature-derived nanocage not only efficiently presents ligands that enhance cancer cell phagocytosis, but also delivers drugs that induce immunogenic cancer cell death. The designed nanocage-therapeutics induce the release of neoantigens and danger signals in dying tumor cells, and leads to enhancement of tumor cell phagocytosis and cross-priming of tumor specific T cells by neoantigen peptide-loaded antigen-presenting cells. Potent inhibition of tumor growth and complete eradication of tumors is observed through systemic tumor-specific T cell responses in tumor draining lymph nodes and the spleen and further, infiltration of CD8+ T cells into the tumor site. Remarkably, after removal of the primary tumor, all mice treated with this nanocage-therapeutics are protected against subsequent challenge with the same tumor cells, suggesting development of lasting, tumor-specific responses. This designed nanocage-therapeutics "awakens" the host's immune system and provokes a durable systemic immune response against cancer.

Combined Rho-kinase inhibition and immunogenic cell death triggers and propagates immunity against cancer.

Activation of T cell immune response is critical for the therapeutic efficacy of cancer immunotherapy. Current immunotherapies have shown remarkable clinical success against several cancers; however, significant responses remain restricted to a minority of patients. Here, we show a therapeutic strategy that combines enhancing the phagocytic activity of antigen-presenting cells with immunogenic cell death to trigger efficient antitumour immunity. Rho-kinase (ROCK) blockade increases cancer cell phagocytosis and induces antitumour immunity through enhancement of T cell priming by dendritic cells (DCs), leading to suppression of tumour growth in syngeneic tumour models. Combining ROCK blockade with immunogenic chemotherapy leads to increased DC maturation and synergistic CD8+ cytotoxic T cell priming and infiltration into tumours. This therapeutic strategy effectively suppresses tumour growth and improves overall survival in a genetic mouse mammary tumour virus/Neu tumour model. Collectively, these results suggest that boosting intrinsic cancer immunity using immunogenic killing and enhanced phagocytosis is a promising therapeutic strategy for cancer immunotherapy.

Cancer-derived exosomes as a delivery platform of CRISPR/Cas9 confer cancer cell tropism-dependent targeting.

An intracellular delivery system for CRISPR/Cas9 is crucial for its application as a therapeutic genome editing technology in a broad range of diseases. Current vehicles carrying CRISPR/Cas9 limit in vivo delivery because of low tolerance and immunogenicity; thus, the in vivo delivery of genome editing remains challenging. Here, we report that cancer-derived exosomes function as natural carriers that can efficiently deliver CRISPR/Cas9 plasmids to cancer. Compared to epithelial cell-derived exosomes, cancer-derived exosomes provide potential vehicles for effective in vivo delivery via selective accumulation in ovarian cancer tumors of SKOV3 xenograft mice, most likely because of their cell tropism. CRISPR/Cas9-loaded exosomes can suppress expression of poly (ADP-ribose) polymerase-1 (PARP-1), resulting in the induction of apoptosis in ovarian cancer. Furthermore, the inhibition of PARP-1 by CRISPR/Cas9-mediated genome editing enhances the chemosensitivity to cisplatin, showing synergistic cytotoxicity. Based on these results, tumor-derived exosomes may be very promising for cancer therapeutics in the future.

Virus-Mimetic Fusogenic Exosomes for Direct Delivery of Integral Membrane Proteins to Target Cell Membranes.

An efficient system for direct delivery of integral membrane proteins is successfully developed using a new biocompatible exosome-based platform. Fusogenic exosomes harboring viral fusogen, vascular stomatitis virus (VSV)-G protein, can fuse with and modify plasma membranes in a process called "membrane editing." This can facilitate the transfer of biologically active membrane proteins into the target cell membranes both in vitro and in vivo.

Exosome-SIRPα, a CD47 blockade increases cancer cell phagocytosis.

CD47, a "don't eat me" signal, is over-expressed on the surface of most tumors that interacts with signal regulatory protein α (SIRPα) on phagocytic cells. By engaging SIRPα, CD47 limits the ability of macrophages to engulf tumor cells, which acts as a major phagocytic barrier. In this study, we developed an exosome-based immune checkpoint blockade that antagonizes the interaction between CD47 and SIRPα. These exosomes harboring SIRPα variants (SIRPα-exosomes) were sufficient to induce remarkably augmented tumor phagocytosis, lead to prime effective anti-tumor T cell response. Given that clustering of native CD47 provides a high binding avidity to ligate dimerized SIRPα on macrophage, nature-derived exosomes could be appreciable platform to antagonize CD47. Disruption of CD47-SIRPα interaction by SIRPα-exosomes leads to an increase in cells being engulfed by macrophages and a concomitant inhibition of tumor growth in tumor-bearing mice. Moreover, SIRPα-exosomes therapy promotes an intensive T cell infiltration in syngeneic mouse models of cancer, raising the possibility of CD47-targeted therapies to unleash both an innate and adaptive anti-tumor response. Note that very small amount of exosomal SIRPα proteins could effectively lead to phagocytic elimination of tumor cells both in vitro and in vivo. Our results suggest that superlative exosome-based platform has broad potential to maximize the therapeutic efficacy of membrane-associated protein therapeutics.