A Personalized Cancer Nanovaccine that Enhances T-Cell Responses and Efficacy Through Dual Interactions with Dendritic Cells and T Cells.

Conventional approaches to developing therapeutic cancer vaccines that primarily activate tumor-specific T cells via dendritic cells (DCs) often demonstrate limited efficacy due to the suboptimal activation of these T cells. To address this limitation, here a therapeutic cancer nanovaccine is developed that enhances T cell responses by interacting with both DCs and T cells. The nanovaccine is based on a cancer cell membrane nanoparticle (CCM-MPLA) that utilizes monophosphoryl lipid A (MPLA) as an adjuvant. To allow direct interaction between the nanovaccine and tumor-specific T cells, anti-CD28 antibodies (aCD28) are conjugated onto CCM-MPLA, resulting in CCM-MPLA-aCD28. This nanovaccine activates tumor-specific CD8+ T cells in both the presence and absence of DCs. Compared with nanovaccines that interact with either DCs (CCM-MPLA) or T cells (CCM-aCD28), CCM-MPLA-aCD28 induces more potent responses of tumor-specific CD8+ T cells and exhibits a higher antitumor efficacy in tumor-bearing mice. No differences in T cell activation efficiency and therapeutic efficacy are observed between CCM-MPLA and CCM-aCD28. This approach may lead to the development of effective personalized therapeutic cancer vaccines prepared from autologous cancer cells.

A Personalized Cancer Vaccine that Induces Synergistic Innate and Adaptive Immune Responses.

To demonstrate potent efficacy, a cancer vaccine needs to activate both innate and adaptive immune cells. Personalized cancer vaccine strategies often require the identification of patient-specific neoantigens; however, the clonal and mutational heterogeneity of cancer cells presents inherent challenges. Here, extracellular nanovesicles derived from alpha-galactosylceramide-conjugated autologous acute myeloid leukemia (AML) cells (ECNV-αGC) are presented as a personalized therapeutic vaccine that activates both innate and adaptive immune responses, bypassing the need to identify patient-specific neoantigens. ECNV-αGC vaccination directly engages with and activates both invariant natural killer T (iNKT) cells and leukemia-specific CD8+ T cells in mice with AML, thereby promoting long-term anti-leukemic immune memory. ECNV-αGC sufficiently serves as an antigen-presenting platform that can directly activate antigen-specific CD8+ T cells even in the absence of dendritic cells, thereby demonstrating a multifaceted cellular mechanism of immune activation. Moreover, ECNV-αGC vaccination results in a significantly lower AML burden and higher percentage of leukemia-free survivors among cytarabine-treated hosts with AML. Human AML-derived ECNV-αGCs activate iNKT cells in both healthy individuals and patients with AML regardless of responsiveness to conventional therapies. Together, autologous AML-derived ECNV-αGCs may be a promising personalized therapeutic vaccine that efficiently establishes AML-specific long-term immunity without requiring the identification of neoantigens.

Senescent cancer cell-derived nanovesicle as a personalized therapeutic cancer vaccine.

The development of therapeutic cancer vaccines (TCVs) that provide clinical benefits is challenging mainly due to difficulties in identifying immunogenic tumor antigens and effectively inducing antitumor immunity. Furthermore, there is an urgent need for personalized TCVs because only a limited number of tumor antigens are shared among cancer patients. Several autologous nanovaccines that do not require the identification of immunogenic tumor antigens have been proposed as personalized TCVs. However, these nanovaccines generally require exogenous adjuvants (e.g., Toll-like receptor agonists) to improve vaccine immunogenicity, which raises safety concerns. Here, we present senescent cancer cell-derived nanovesicle (SCCNV) as a personalized TCV that provides patient-specific tumor antigens and improved vaccine immunogenicity without the use of exogenous adjuvants. SCCNVs are prepared by inducing senescence in cancer cells ex vivo and subsequently extruding the senescent cancer cells through nanoporous membranes. In the clinical setting, SCCNVs can be prepared from autologous cancer cells from the blood of liquid tumor patients or from tumors surgically removed from solid cancer patients. SCCNVs also contain interferon-γ and tumor necrosis factor-α, which are expressed during senescence. These endogenous cytokines act as adjuvants and enhance vaccine immunogenicity, avoiding the need for exogenous adjuvants. Intradermally injected SCCNVs effectively activate dendritic cells and tumor-specific T cells and inhibit primary and metastatic tumor growth and tumor recurrence. SCCNV therapy showed an efficacy similar to that of immune checkpoint blockade (ICB) therapy and synergized with ICB. SCCNVs, which can be prepared using a simple and facile procedure, show potential as personalized TCVs.

Iron Oxide Nanoparticle-Incorporated Mesenchymal Stem Cells for Alzheimer's Disease Treatment.

Alzheimer's disease (AD) is a neurodegenerative disease with multifactorial pathogenesis. However, most current therapeutic approaches for AD target a single pathophysiological mechanism, generally resulting in unsatisfactory therapeutic outcomes. Recently, mesenchymal stem cell (MSC) therapy, which targets multiple pathological mechanisms of AD, has been explored as a novel treatment. However, the low brain retention efficiency of administered MSCs limits their therapeutic efficacy. In addition, autologous MSCs from AD patients may have poor therapeutic abilities. Here, we overcome these limitations by developing iron oxide nanoparticle (IONP)-incorporated human Wharton's jelly-derived MSCs (MSC-IONPs). IONPs promote therapeutic molecule expression in MSCs. Following intracerebroventricular injection, MSC-IONPs showed a higher brain retention efficiency under magnetic guidance. This potentiates the therapeutic efficacy of MSCs in murine models of AD. Furthermore, human Wharton's jelly-derived allogeneic MSCs may exhibit higher therapeutic abilities than those of autologous MSCs in aged AD patients. This strategy may pave the way for developing MSC therapies for AD.

A Therapeutic Nanovaccine that Generates Anti-Amyloid Antibodies and Amyloid-specific Regulatory T Cells for Alzheimer's Disease.

Alzheimer's disease (AD), the most common cause of dementia, is a complex condition characterized by multiple pathophysiological mechanisms including amyloid-ß (Aß) plaque accumulation and neuroinflammation in the brain. The current immunotherapy approaches, such as anti-Aß monoclonal antibody (mAb) therapy, Aß vaccines, and adoptive regulatory T (Treg) cell transfer, target a single pathophysiological mechanism, which may lead to unsatisfactory therapeutic efficacy. Furthermore, Aß vaccines often induce T helper 1 (Th1) cell-mediated inflammatory responses. Here, a nanovaccine composed of lipid nanoparticles loaded with Aß peptides and rapamycin is developed, which targets multiple pathophysiological mechanisms, exhibits the combined effects of anti-Aß antibody therapy and adoptive Aß-specific Treg cell transfer, and can overcome the limitations of current immunotherapy approaches for AD. The Nanovaccine effectively delivers rapamycin and Aß peptides to dendritic cells, produces both anti-Aß antibodies and Aß-specific Treg cells, removes Aß plaques in the brain, alleviates neuroinflammation, prevents Th1 cell-mediated excessive immune responses, and inhibits cognitive impairment in mice. The nanovaccine shows higher efficacy in cognitive recovery than an Aß vaccine. Unlike anti-Aß mAb therapy and adoptive Treg cell transfer, both of which require complicated and costly manufacturing processes, the nanovaccine is easy-to-prepare and cost-effective. The nanovaccines can represent a novel treatment option for AD.

Tolerogenic nanoparticles induce type II collagen-specific regulatory T cells and ameliorate osteoarthritis.

Local inflammation in the joint is considered to contribute to osteoarthritis (OA) progression. Here, we describe an immunomodulating nanoparticle for OA treatment. Intradermal injection of lipid nanoparticles (LNPs) loaded with type II collagen (Col II) and rapamycin (LNP-Col II-R) into OA mice effectively induced Col II-specific anti-inflammatory regulatory T cells, substantially increased anti-inflammatory cytokine expression, and reduced inflammatory immune cells and proinflammatory cytokine expression in the joints. Consequently, LNP-Col II-R injection inhibited chondrocyte apoptosis and cartilage matrix degradation and relieved pain, while injection of LNPs loaded with a control peptide and rapamycin did not induce these events. Adoptive transfer of CD4+CD25+ T cells isolated from LNP-Col II-R-injected mice suggested that Tregs induced by LNP-Col II-R injection were likely responsible for the therapeutic effects. Collectively, this study suggests nanoparticle-mediated immunomodulation in the joint as a simple and effective treatment for OA.

A Senolytic-Eluting Coronary Stent for the Prevention of In-Stent Restenosis.

The vast majority of drug-eluting stents (DES) elute either sirolimus or one of its analogues. While limus drugs stymie vascular smooth muscle cell (VSMC) proliferation to prevent in-stent restenosis, their antiproliferative nature is indiscriminate and limits healing of the endothelium in stented vessels, increasing the risk of late-stent thrombosis. Oxidative stress, which is associated with vascular injury from stent implantation, can induce VSMCs to undergo senescence, and senescent VSMCs can produce pro-inflammatory cytokines capable of inducing proliferation of neighboring nonsenescent VSMCs. We explored the potential of senolytic therapy, which involves the selective elimination of senescent cells, in the form of a senolytic-eluting stent (SES) for interventional cardiology. Oxidative stress was modeled in vitro by exposing VSMCs to H2O2, and H2O2-mediated senescence was evaluated by cytochemical staining of senescence-associated ß-galactosidase activity and qRT-PCR. Quiescent VSMCs were then treated with the conditioned medium (CM) of H2O2-treated VSMCs. Proliferative effects of CM were analyzed by staining for proliferating cell nuclear antigen. Senolytic effects of the first-generation senolytic ABT263 were observed in vitro, and the effects of ABT263 on endothelial cells were also investigated through an in vitro re-endothelialization assay. SESs were prepared by dip coating. Iliofemoral arteries of hypercholesteremic rabbits were implanted with SES, everolimus-eluting stents (EESs), or bare-metal stents (BMSs), and the area of stenosis was measured 4 weeks post-implantation using optical coherence tomography. We found that a portion of H2O2-treated VSMCs underwent senescence, and that CM of H2O2-treated senescent VSMCs triggered the proliferation of quiescent VSMCs. ABT263 reverted H2O2-mediated senescence and the proliferative capacity of senescent VSMC CM. Unlike everolimus, ABT263 did not affect endothelial cell migration and/or proliferation. SES, but not EES, significantly reduced stenosis area in vivo compared with bare-metal stents (BMSs). This study shows the potential of SES as an alternative to current forms of DES.

Nanovesicle-Mediated Targeted Delivery of Immune Checkpoint Blockades to Potentiate Therapeutic Efficacy and Prevent Side Effects.

Despite the clinically proven efficacies of immune checkpoint blockades, including anti-cytotoxic T lymphocyte-associated protein 4 antibody (αCTLA-4), the low response rate and immune-related adverse events (irAEs) in cancer patients represent major drawbacks of the therapy. These drawbacks of αCTLA-4 therapy are mainly due to the suboptimal activation of tumor-specific cytotoxic T lymphocytes (CTLs) and the systemic nonspecific activation of T cells. To overcome such drawbacks, αCTLA-4 is delivered by dendritic cell-derived nanovesicles presenting tumor antigens (DCNV-TAs) that exclusively interact with tumor-specific T cells, leading to selective activation of tumor-specific CTLs. Compared to conventional αCTLA-4 therapy, treatment with αCTLA-4-conjugated DCNV-TAs significantly inhibits tumor growth and reduces irAEs in syngeneic tumor-bearing mice. This study demonstrates that the spatiotemporal presentation of both αCTLA-4 and tumor antigens enables selective activation of tumor-specific T cells and potentiates the antitumor efficacy of αCTLA-4 without inducing systemic irAEs.

Local Delivery of Senolytic Drug Inhibits Intervertebral Disc Degeneration and Restores Intervertebral Disc Structure.

Intervertebral disc (IVD) degeneration (IVDD) is a leading cause of chronic low back pain. There is a strong clinical demand for more effective treatments for IVDD as conventional treatments provide only symptomatic relief rather than arresting IVDD progression. This study shows that senolytic therapy with local drug delivery can inhibit IVDD and restore IVD integrity. ABT263, a senolytic drug, is loaded in poly(lactic-co-glycolic acid) nanoparticles (PLGA-ABT) and intradiscally administered into injury-induced IVDD rat models. The single intradiscal injection of PLGA-ABT may enable local delivery of the drug to avascular IVD, prevention of potential systemic toxicity caused by systemic administration of senolytic drug, and morbidity caused by repetitive injections of free drug into the IVD. The strategy results in the selective elimination of senescent cells from the degenerative IVD, reduces expressions of pro-inflammatory cytokines and matrix proteases in the IVD, inhibits progression of IVDD, and even restores the IVD structure. This study demonstrates for the first time that local delivery of senolytic drug can effectively treat senescence-associated IVDD. This approach can be extended to treat other types of senescence-associated degenerative diseases.

Nanocomplex-Mediated In Vivo Programming to Chimeric Antigen Receptor-M1 Macrophages for Cancer Therapy.

Chimeric antigen receptor-T (CAR-T) cell immunotherapy has shown impressive clinical outcomes for hematologic malignancies. However, its broader applications are challenged due to its complex ex vivo cell-manufacturing procedures and low therapeutic efficacy against solid tumors. The limited therapeutic effects are partially due to limited CAR-T cell infiltration to solid tumors and inactivation of CAR-T cells by the immunosuppressive tumor microenvironment. Here, a facile approach is presented to in vivo program macrophages, which can intrinsically penetrate solid tumors, into CAR-M1 macrophages displaying enhanced cancer-directed phagocytosis and anti-tumor activity. In vivo injected nanocomplexes of macrophage-targeting nanocarriers and CAR-interferon-γ-encoding plasmid DNA induce CAR-M1 macrophages that are capable of CAR-mediated cancer phagocytosis, anti-tumor immunomodulation, and inhibition of solid tumor growth. Together, this study describes an off-the-shelf CAR-macrophage therapy that is effective for solid tumors and avoids the complex and costly processes of ex vivo CAR-cell manufacturing.

Nanoparticle-Mediated Blocking of Excessive Inflammation for Prevention of Heart Failure Following Myocardial Infarction.

Severe cardiac damage following myocardial infarction (MI) causes excessive inflammation, which sustains tissue damage and often induces adverse cardiac remodeling toward cardiac function impairment and heart failure. Timely resolution of post-MI inflammation may prevent cardiac remodeling and development of heart failure. Cell therapy approaches for MI are time-consuming and costly, and have shown marginal efficacy in clinical trials. Here, nanoparticles targeting the immune system to attenuate excessive inflammation in infarcted myocardium are presented. Liposomal nanoparticles loaded with MI antigens and rapamycin (L-Ag/R) enable effective induction of tolerogenic dendritic cells presenting the antigens and subsequent induction of antigen-specific regulatory T cells (Tregs). Impressively, intradermal injection of L-Ag/R into acute MI mice attenuates inflammation in the myocardium by inducing Tregs and an inflammatory-to-reparative macrophage polarization, inhibits adverse cardiac remodeling, and improves cardiac function. Nanoparticle-mediated blocking of excessive inflammation in infarcted myocardium may be an effective intervention to prevent the development of post-MI heart failure.

T-Cell-Derived Nanovesicles for Cancer Immunotherapy.

Although T-cell therapy is a remarkable breakthrough in cancer immunotherapy, the therapeutic efficacy is limited for solid tumors. A major cause of the low efficacy is T-cell exhaustion by immunosuppressive mechanisms of solid tumors, which are mainly mediated by programmed death-ligand 1 (PD-L1) and transforming growth factor-beta (TGF-ß). Herein, T-cell-derived nanovesicles (TCNVs) produced by the serial extrusion of cytotoxic T cells through membranes with micro-/nanosized pores that inhibit T-cell exhaustion and exhibit antitumoral activity maintained in the immunosuppressive tumor microenvironment (TME) are presented. TCNVs, which have programmed cell death protein 1 and TGF-ß receptor on their surface, block PD-L1 on cancer cells and scavenge TGF-ß in the immunosuppressive TME, thereby preventing cytotoxic-T-cell exhaustion. In addition, TCNVs directly kill cancer cells via granzyme B delivery. TCNVs successfully suppress tumor growth in syngeneic-solid-tumor-bearing mice. Taken together, TCNV offers an effective cancer immunotherapy strategy to overcome the tumor's immunosuppressive mechanisms.

Multilayered Cell Sheets of Cardiac Reprogrammed Cells for the Evaluation of Drug Cytotoxicity.

BACKGROUND: Various cell-culture systems have been used to evaluate drug toxicity in vitro. However, factors that affect cytotoxicity outcomes in drug toxicity evaluation systems remain elusive. In this study, we used multilayered sheets of cardiac-mimetic cells, which were reprogrammed from human fibroblasts, to investigate the effects of the layer number on drug cytotoxicity outcomes. METHODS: Cell sheets of cardiac-mimetic cells were fabricated by reprogramming of human fibroblasts into cardiac-mimetic cells via coculture with cardiac cells and electric stimulation, as previously described. Double-layered cell sheets were prepared by stacking the cell sheets. The mono- and double-layered cell sheets were treated with 5-fluorouracil (5-FU), an anticancer drug, in vitro. Subsequently, apoptosis and lipid peroxidation were analyzed. Furthermore, effects of cardiac-mimetic cell density on cytotoxicity outcomes were evaluated by culturing cells in monolayer at various cell densities. RESULTS: The double-layered cell sheets exhibited lower cytotoxicity in terms of apoptosis and lipid peroxidation than the mono-layered sheets at the same 5-FU dose. In addition, the double-layered cell sheets showed better preservation of mitochondrial function and plasma membrane integrity than the monolayer sheets. The lower cytotoxicity outcomes in the double-layered cell sheets may be due to the higher intercellular interactions, as the cytotoxicity of 5-FU decreased with cell density in monolayer cultures of cardiac-mimetic cells. CONCLUSION: The layer number of cardiac-mimetic cell sheets affects drug cytotoxicity outcomes in drug toxicity tests. The in vitro cellular configuration that more closely mimics the in vivo configuration in the evaluation systems seems to exhibit lower cytotoxicity in response to drug.

The Senolytic Drug JQ1 Removes Senescent Cells via Ferroptosis.

BACKGROUND: Ferroptosis is an iron-dependent, non-apoptotic programmed cell death. Cellular senescence contributes to aging and various age-related diseases through the expression of a senescence-associated secretory phenotype (SASP). Senescent cells are often resistant to ferroptosis via increased ferritin and impaired ferritinophagy. In this study, we investigated whether treatment with JQ1 could remove senescent cells by inducing ferroptosis. METHODS: Senescence of human dermal fibroblasts was induced in vitro by treating the cells with bleomycin. The senolytic effects of JQ1 were evaluated using a SA-ß gal assay, annexin V analysis, cell counting kit-8 assay, and qRT-PCR. Ferroptosis following JQ1 treatment was evaluated with qRT-PCR and BODIPY staining. RESULTS: At a certain range of JQ1 concentrations, JQ1 treatment reduced the viability of bleomycin-treated cells (senescent cells) but did not reduce that of untreated cells (non-senescent cells), indicating that JQ1 treatment can selectively eliminate senescent cells. JQ1 treatment also decreased SASP expression only in senescent cells. Subsequently, JQ1 treatment reduced the expression of ferroptosis-resistance genes in senescent cells. JQ1 treatment induced lipid peroxidation in senescent cells but not in non-senescent cells. CONCLUSION: The data indicate that JQ1 can eliminate senescent cells via ferroptosis. This study suggests ferroptosis as a new mechanism of senolytic therapy.

T-Cell-Mimicking Nanoparticles for Cancer Immunotherapy.

Cancer immunotherapies, including adoptive T cell transfer and immune checkpoint blockades, have recently shown considerable success in cancer treatment. Nevertheless, transferred T cells often become exhausted because of the immunosuppressive tumor microenvironment. Immune checkpoint blockades, in contrast, can reinvigorate the exhausted T cells; however, the therapeutic efficacy is modest in 70-80% of patients. To address some of the challenges faced by the current cancer treatments, here T-cell-membrane-coated nanoparticles (TCMNPs) are developed for cancer immunotherapy. Similar to cytotoxic T cells, TCMNPs can be targeted at tumors via T-cell-membrane-originated proteins and kill cancer cells by releasing anticancer molecules and inducing Fas-ligand-mediated apoptosis. Unlike cytotoxic T cells, TCMNPs are resistant to immunosuppressive molecules (e.g., transforming growth factor-ß1 (TGF-ß1)) and programmed death-ligand 1 (PD-L1) of cancer cells by scavenging TGF-ß1 and PD-L1. Indeed, TCMNPs exhibit higher therapeutic efficacy than an immune checkpoint blockade in melanoma treatment. Furthermore, the anti-tumoral actions of TCMNPs are also demonstrated in the treatment of lung cancer in an antigen-nonspecific manner. Taken together, TCMNPs have a potential to improve the current cancer immunotherapy.

M1 Macrophage-Derived Nanovesicles Potentiate the Anticancer Efficacy of Immune Checkpoint Inhibitors.

Cancer immunotherapy modulates immune cells to induce antitumor immune responses. Tumors employ immune checkpoints to evade immune cell attacks. Immune checkpoint inhibitors such as anti-PD-L1 antibody (aPD-L1), which is being used clinically for cancer treatments, can block immune checkpoints so that the immune system can attack tumors. However, immune checkpoint inhibitor therapy may be hampered by polarization of macrophages within the tumor microenvironment (TME) into M2 tumor-associated macrophages (TAMs), which suppress antitumor immune responses and promote tumor growth by releasing anti-inflammatory cytokines and angiogenic factors. In this study, we used exosome-mimetic nanovesicles derived from M1 macrophages (M1NVs) to repolarize M2 TAMs to M1 macrophages that release pro-inflammatory cytokines and induce antitumor immune responses and investigated whether the macrophage repolarization can potentiate the anticancer efficacy of aPD-L1. M1NV treatment induced successful polarization of M2 macrophages to M1 macrophages in vitro and in vivo. Intravenous injection of M1NVs into tumor-bearing mice suppressed tumor growth. Importantly, injection of a combination of M1NVs and aPD-L1 further reduced the tumor size, compared to the injection of either M1NVs or aPD-L1 alone. Thus, our study indicates that M1NV injection can repolarize M2 TAMs to M1 macrophages and potentiate antitumor efficacy of the checkpoint inhibitor therapy.