Improvement of STING-mediated cancer immunotherapy using immune checkpoint inhibitors as a game-changer.

Various cancer therapies, such as surgery, radiotherapy, chemotherapy, and immunotherapy, have been used to treat cancer. Among cancer immunotherapies, stimulators of interferon genes (STING) activate various immune cells and induce them to attack cancer cells. However, the secretion of type I interferon (IFN α and ß) increases after stimulation of the immune cell as a side effect of STING agonist, thereby increasing the expression of programmed death-ligand 1 (PD-L1) in the tumor microenvironment (TME). Therefore, it is necessary to reduce the side effects of STING agonists and maximize cancer treatment by administering combination therapy. Tumor-bearing mice were treated with cisplatin, tumor-specific peptide, neoantigen, DMXAA (STING agonist), and immune checkpoint inhibitor (ICI). The combination vaccine group showed a reduction in tumor mass, an increased survival rate, and IFN-γ+ (interferon gamma) CD8+ (cluster of differentiation 8) T cells in the spleen and TME. The distribution of immune cells in the spleen and TME was confirmed, and the number of active immune cells increased, whereas that of immunosuppressive cells decreased. When measuring cytokine levels in the tumor and serum, the levels of pro-inflammatory cytokines increased and anti-inflammatory cytokines decreased. This study demonstrated that when various cancer therapies are combined to treat cancer, it can lead to an anticancer immune synergistic effect by increasing the immune response and reducing side effects.

PAUF Induces Migration of Human Pancreatic Cancer Cells Exclusively via the TLR4/MyD88/NF-κB Signaling Pathway.

PAUF, a tumor-promoting protein secreted by cancer cells, exerts paracrine effects on immune cells through TLR4 receptors expressed on immune cell surfaces. This study aimed to investigate if PAUF elicits autocrine effects on pancreatic cancer (PC) cells through TLR4, a receptor that is overexpressed on PC cells. In this study, TLR4 expression was detected in PC cells only, but not normal pancreatic cells. The migration of TLR4 high-expressing PC cells (i.e., BxPC-3) was reduced by a selective TLR4 inhibitor, in a dose-dependent manner. Using TLR4 overexpressed and knockout PC cell lines, we observed direct PAUF-TLR4 binding on the PC cell surfaces, and that PAUF-induced cancer migration may be mediated exclusively through the TLR4 receptor. Further experiments showed that PAUF signaling was passed down through the TLR4/MyD88 pathway without the involvement of the TLR4/TRIF pathway. TLR4 knockout also downregulated PC membrane PD-L1 expression, which was not influenced by PAUF. To the best of our knowledge, TLR4 is the first receptor identified on cancer cells that mediates PAUF's migration-promoting effect. The results of this study enhanced our understanding of the mechanism of PAUF-induced tumor-promoting effects and suggests that TLR4 expression on cancer cells may be an important biomarker for anti-PAUF treatment.

Improvement of DC-based vaccines using adjuvant TLR4-binding 60S acidic ribosomal protein P2 and immune checkpoint inhibitors.

Cancer immunotherapy has fewer side effects and higher efficiency than conventional methods. Dendritic cell (DC)-based vaccine, a cancer immunotherapeutic, is prepared by processing mature DCs and pulsing with tumor antigen peptide ex vivo, to induce the activation of tumor-specific T lymphocytes followed by tumor clearance in vivo. Unfortunately, clinical trials of this method mostly failed due to low patient response, possibly due to the absence of novel adjuvants that induce DC maturation through Toll-like receptor (TLR) signals. Interestingly, immune checkpoint inhibitor (ICI) therapy has shown remarkable anti-tumor efficacy when combined with cancer vaccines. In this study, we identified 60S acidic ribosomal protein P2 (RPLP2) through pull-down assay using human cancer cells derived proteins that binds to Toll-like receptor 4 (TLR4). Recombinant RPLP2 induced maturation and activation of DCs in vitro. This DC-based vaccine, followed by pulsing with tumor-specific antigen, has shown to significantly increase tumor-specific CD8+IFN-γ+ T cells, and improved both tumor prevention and tumor treatment effects in vivo. The adjuvant effects of RPLP2 were shown to be dependent on TLR4 using TLR4 knockout mice. Moreover, ICIs that suppress the tumor evasion mechanism showed synergistic effects on tumor treatment when combined with these vaccines.

Endoplasmic reticulum stress enhances the antigen-specific T cell immune responses and therapeutic antitumor effects generated by therapeutic HPV vaccines.

BACKGROUND: Endoplasmic reticulum stress has a profound effect on cancer cell proliferation and survival, and also has the capacity to activate cells of the adaptive immune system. Multimodal treatment methods that utilize and combine conventional cancer therapies with antigen-specific immunotherapies have emerged as promising approaches for the treatment and control of cancer. However, it is not well known whether endoplasmic reticulum stress-inducing agents can influence the efficacy of tumor antigen-targeting vaccines. METHODS: In the past, we developed a therapeutic human papillomavirus (HPV) DNA vaccine that encodes for calreticulin (CRT) linked to the HPV16 E7 antigen (CRT/E7). In this study, we utilize the CRT/E7 and further encode for an endoplasmic reticulum (ER) stress-inducing agent, 3-bromopyruvate (3-BrPA), in a preclinical model, by harnessing its potential to enhance HPV16 E7-specific CD8+ T cell immune responses as well as antitumor effects against E7-expressing tumors (TC-1 cells). E7-specific CD8+ T cells were added to evaluate the cytotoxicity of luciferase-expressing TC-1 tumor cells treated with 3-BrPA in vitro, as measured with an IVIS Luminescence Imaging System. We also determined the levels of ER stress markers in 3-BrPA-treated TC-1 cells. TC-1 tumor-bearing mice were treated with either 3-BrPA (10 mg/kg, intraperitoneal injection) and/or CRT/E7 DNA vaccine (30 µg/mouse). RESULTS: Treatment of E7-expressing TC-1 tumor cells with 3-BrPA induced significantly higher in vitro cytotoxicity and resulted in upregulation of endoplasmic reticulum stress markers (CHOP and GRP78). More importantly, combination treatment of 3-BrPA and the CRT/E7 DNA vaccine led to improved antigen-specific CD8+ T cell immune responses as well as therapeutic antitumor effects in TC-1 tumor-bearing mice. CONCLUSIONS: Our data indicate that 3-BrPA can enhance therapeutic HPV vaccine potency in generating improved antigen-specific immune responses and antitumor effects. These findings have important implications for future clinical translation and provide novel strategies for the treatment of HPV-associated diseases.

Tamarixetin Exhibits Anti-inflammatory Activity and Prevents Bacterial Sepsis by Increasing IL-10 Production.

Sepsis is a systemic inflammatory response to pathogenic infection that currently has no specific pharmaceutical interventions. Instead, antibiotics administration is considered the best available option, despite increasing drug resistance. Alternative strategies are therefore urgently required to prevent sepsis and strengthen the host immune system. One such option is tamarixetin (4'- O-methylquercetin), a naturally occurring flavonoid derivative of quercetin that protects against inflammation. The purpose of this study was to determine whether the anti-inflammatory effects of tamarixetin protect against the specific inflammatory conditions induced in lipopolysaccharide (LPS) or Escherichia coli K1 models of sepsis. Our study showed that tamarixetin reduced the secretion of various inflammatory cytokines by dendritic cells after activation with LPS. It also promoted the secretion of the anti-inflammatory cytokine interleukin (IL)-10 and specifically increased the population of IL-10-secreting immune cells in LPS-activated splenocytes. Tamarixetin showed general anti-inflammatory effects in mouse models of bacterial sepsis and decreased bacteria abundance and endotoxin levels. We therefore conclude that tamarixetin has superior anti-inflammatory properties than quercetin during bacterial sepsis. This effect is associated with an increased population of IL-10-secreting immune cells and suggests that tamarixetin could serve as a specific pharmaceutical option to prevent bacterial sepsis.

Neoagarooligosaccharides prevent septic shock by modulating A20-and cyclooxygenase-2-mediated interleukin-10 secretion in a septic-shock mouse model.

Analysis of the signaling mechanism triggered by endotoxin-mediated toll-like receptor-4 activation using immune cell systems or rodent models may help identify potential agents for the prevention of Gram-negative bacteria infection. ß-agarase cleaves the ß-1,4-linkages of agar to produce neoagarooligosaccharides (NAOs), which have various physiological functions. The aim of this study was to investigate the efficacy of NAOs in preventing experimental sepsis caused by the administration of endotoxin or Gram-negative bacteria. Organ damage and neutrophil infiltration in an endotoxemia and septic-shock mouse model were suppressed by NAOs. Pro-inflammatory cytokine level was decreased, but IL-10 level was increased by NAO-treatment. Further induction by NAOs in the presence of endotoxin was associated with a significant induction of A20 and cyclooxygenase (COX)-2 expressions. Our data suggest that NAOs have a beneficial preventive effect in septic shock correlated with the enhancement of IL-10 via the induction of A20 and COX-2.

Glycogen Synthase Kinase-3ß (GSK-3ß) Inhibition Enhances Dendritic Cell-based Cancer Vaccine Potency via Suppression of Interferon-γ-induced Indoleamine 2,3-Dioxygenase Expression.

Indoleamine 2,3-dioxygenase (IDO) functions as a crucial mediator of tumor-mediated immune tolerance by causing T-cell suppression via tryptophan starvation in a tumor environment. Glycogen synthase kinase-3ß (GSK-3ß) is also involved in immune and anti-tumor responses. However, the relativity of these proteins has not been as well defined. Here, we found that GSK-3ß-dependent IDO expression in the dendritic cell (DC) plays a role in anti-tumor activity via the regulation of CD8(+) T-cell polarization and cytotoxic T lymphocyte activity. By the inhibition of GSK-3ß, attenuated IDO expression and impaired JAK1/2-Stat signaling crucial for IDO expression were observed. Protein kinase Cδ (PKCδ) activity and the interaction between JAK1/2 and Stat3, which are important for IDO expression, were also reduced by GSK-3ß inhibition. CD8(+) T-cell proliferation mediated by OVA-pulsed DC was blocked by interferon (IFN)-γ-induced IDO expression via GSK-3ß activity. Specific cytotoxic T lymphocyte activity mediated by OVA-pulsed DC against OVA-expressing EG7 thymoma cells but not OVA-nonexpressing EL4 thymoma cells was also attenuated by the expressed IDO via IFN-γ-induced activation of GSK-3ß. Furthermore, tumor growth that was suppressed with OVA-pulsed DC vaccination was restored by IDO-expressing DC via IFN-γ-induced activation of GSK-3ß in an OVA-expressing murine EG7 thymoma model. Taken together, DC-based immune response mediated by interferon-γ-induced IDO expression via GSK-3ß activity not only regulates CD8(+) T-cell proliferation and cytotoxic T lymphocyte activity but also modulates OVA-pulsed DC vaccination against EG7 thymoma.

Annexin A5 increases survival in murine sepsis model by inhibiting HMGB1-mediated pro-inflammation and coagulation.

The identification of HMGB1 as a late-mediator in sepsis has highlighted HMGB1 as a promising therapeutic target for sepsis treatment. Recent studies have revealed that annexin A5, a 35 kDa Ca2+-dependent phospholipid binding protein, exerts anti-inflammatory effect by inhibiting LPS binding to TLR4/MD2 complex. Annexin A5 administration has been shown to protect against endotoxin lethality even when the treatment was given after the early cytokine response, which prompted our group to suspect that annexin A5 may inhibit the binding of HMGB1, as well as endotoxin to TLR4. Here we suggest annexin A5 as a new inhibitor of HMGB1-mediated pro-inflammatory cytokine production and coagulation in sepsis. We first confirmed the inhibitory role of annexin A5 in LPS-induced production of pro-inflammatory cytokines both in vitro and in vivo. We observed that annexin A5 protects against tissue damage and organ dysfunction during endotoxemia in vivo. We then assessed the inhibiting role of annexin A5 in HMGB1/TLR4 interaction, and showed that annexin A5 treatment reduces HMGB1-mediated cytokines IL6 and TNFα both in vitro and in vivo. Finally, we confirmed that anticoagulant property of annexin A5 persists in various septic conditions including elevated HMGB1. Overall, we suggest annexin A5 as an alternative therapeutic approach for controlling HMGB1-mediated pro-inflammation and coagulation in patients with sepsis.

Enhancement of tumor-specific T cell-mediated immunity in dendritic cell-based vaccines by Mycobacterium tuberculosis heat shock protein X.

Despite the potential for stimulation of robust antitumor immunity by dendritic cells (DCs), clinical applications of DC-based immunotherapy are limited by the low potency in generating tumor Ag-specific T cell responses. Therefore, optimal conditions for generating potent immunostimulatory DCs that overcome tolerance and suppression are key factors in DC-based tumor immunotherapy. In this study, we demonstrate that use of the Mycobacterium tuberculosis heat shock protein X (HspX) as an immunoadjuvant in DC-based tumor immunotherapy has significant potential in therapeutics. In particular, the treatment aids the induction of tumor-reactive T cell responses, especially tumor-specific CTLs. The HspX protein induces DC maturation and proinflammatory cytokine production (TNF-α, IL-1ß, IL-6, and IFN-ß) through TLR4 binding partially mediated by both the MyD88 and the TRIF signaling pathways. We employed two models of tumor progression and metastasis to evaluate HspX-stimulated DCs in vivo. The administration of HspX-stimulated DCs increased the activation of naive T cells, effectively polarizing the CD4(+) and CD8(+) T cells to secrete IFN-γ, as well as enhanced the cytotoxicity of splenocytes against HPV-16 E7 (E7)-expressing TC-1 murine tumor cells in therapeutic experimental animals. Moreover, the metastatic capacity of B16-BL6 melanoma cancer cells toward the lungs was remarkably attenuated in mice that received HspX-stimulated DCs. In conclusion, the high therapeutic response rates with tumor-targeted Th1-type T cell immunity as a result of HspX-stimulated DCs in two models suggest that HspX harnesses the exquisite immunological power and specificity of DCs for the treatment of tumors.

GM-CSF-loaded chitosan hydrogel as an immunoadjuvant enhances antigen-specific immune responses with reduced toxicity.

BACKGROUND: The application of vaccine adjuvants has been vigorously studied for a diverse range of diseases in order to improve immune responses and reduce toxicity. However, most adjuvants have limited uses in clinical practice due to their toxicity. METHODS: Therefore, to reduce health risks associated with the use of such adjuvants, we developed an advanced non-toxic adjuvant utilizing biodegradable chitosan hydrogel (CH-HG) containing ovalbumin (OVA) and granulocyte-macrophage colony-stimulating factor (GM-CSF) as a local antigen delivery system. RESULTS: After subcutaneous injection into mice, OVA/GM-CSF-loaded CH-HG demonstrated improved safety and enhanced OVA-specific antibody production compared to oil-based adjuvants such as Complete Freund's adjuvant (CFA) or Incomplete Freund's adjuvant (IFA). Moreover, CH-HG system-mediated immune responses was characterized by increased number of OVA-specific CD4(+) and CD8(+) INF-γ(+) T cells, leading to enhanced humoral and cellular immunity. CONCLUSIONS: In this study, the improved safety and enhanced immune response characteristics of our novel adjuvant system suggest the possibility of the extended use of adjuvants in clinical practice with reduced apprehension about toxic side effects.

MICA/B and ULBP1 NKG2D ligands are independent predictors of good prognosis in cervical cancer.

BACKGROUND: NKG2D (natural killer group 2, member D) is thought to play an important role in mediating the activation of anticancer immune response. Expression of NKG2D ligands (NKG2DLs) is pronounced in malignancies and the heterogeneity of NKG2DL expression remains unclear. Here, we investigate the expression and clinical significance of NKG2DLs in cervical cancer. METHODS: Immunohistochemical analyses of MICA/B, ULBP1, ULBP2, ULBP3, RAET1E, and RAET1G were performed using tissue microarray analysis of 200 cervical cancers, 327 high-grade cervical intraepithelial neoplasias (CINs), 99 low-grade CINs, and 541 matched nonadjacent normal cervical epithelial tissues and compared the data with clinicopathologic variables, including the survival of cervical cancer patients. RESULTS: MICA/B, ULBP1, and RAET1E expression was higher in cervical cancer than in low-grade CIN (p<0.001, p=0.012, p=0.013, respectively) and normal cervix (all p<0.001). Among these markers, expression of ULBP1 was significantly different depending on patient tumor stage (p=0.010) and tumor size (p=0.045). ULBP1 expression was correlated with MICA/B (p<0.001) and ULBP2 (p=0.002) expression in cervical cancer. While MICA/B+ or ULBP1+ patients had improved disease-free survival time (p=0.027 and p=0.009, respectively) relative to that of the low expression group, RAET1E+ or RAET1G+ was correlated with shorter survival time (p=0.018 and p=0.029, respectively). However, in terms of overall survival, the ULBP1+ group had significantly longer survival time than the low expression group (p=0.009). Multivariate analysis indicated that MICA/B+/ULBP1+ (HR=0.16, p=0.015) and ULBP1+ (HR=0.31, p=0.024) are independent prognostic factors of disease-free survival in cervical cancer. CONCLUSIONS: High expression of either ULBP1 or MICA/B and ULBP1 combined is an indicator of good prognosis in cervical cancer, suggesting their potential utility as prognostic tests in clinical assessment.

Targeted coating with antigenic peptide renders tumor cells susceptible to CD8(+) T cell-mediated killing.

The potency of immunotherapies targeting endogenous tumor antigens is hindered by immune tolerance. We created a therapeutic agent comprised of a tumor-homing module fused to a functional domain capable of selectively rendering tumor cells sensitive to foreign antigen-specific CD8(+) T cell-mediated immune attack, and thereby, circumventing concerns for immune tolerance. The tumor-homing module is comprised of a single-chain variable fragment (scFv) that specifically binds to mesothelin (Meso), which is commonly overexpressed in human cancers, including ovarian tumors. The functional domain is comprised of the Fc portion of IgG2a protein and foreign immunogenic CD8(+) T cell epitope flanked by furin cleavage sites (R), which can be recognized and cleaved by furin that is highly expressed in the tumor microenvironment. We show that our therapeutic protein specifically loaded antigenic epitope onto the surface of mesothelin-expressing tumor cells, rendering tumors susceptible to antigen-specific cytotoxic CD8(+) T lymphocytes (CTL)-mediated killing in vitro and in vivo. Our findings have important implications for bypassing immune tolerance to enhance cancer immunotherapy.

Intratumoral injection of therapeutic HPV vaccinia vaccine following cisplatin enhances HPV-specific antitumor effects.

Despite the conventional treatments of radiation therapy and chemotherapy, the 5-year survival rates for patients with advanced-stage cervical cancers remain low. Cancer immunotherapy has emerged as an alternative, innovative therapy that may improve survival. Here, we utilize a preclinical HPV-16 E6/E7-expressing tumor model, TC-1, and employ the chemotherapeutic agent cisplatin to generate an accumulation of CD11c+ dendritic cells in tumor loci making it an ideal location for the administration of therapeutic vaccines. Following cisplatin treatment, we tested different routes of administration of a therapeutic HPV vaccinia vaccine encoding HPV-16 E7 antigen (CRT/E7-VV). We found that TC-1 tumor-bearing C57BL/6 mice treated with cisplatin and intratumoral injection of CRT/E7-VV significantly increased E7-specific CD8+ T cells in the blood and generated potent local and systemic antitumor immune responses compared to mice receiving cisplatin and CRT/E7-VV intraperitoneally or mice treated with cisplatin alone. We further extended our study using a clinical grade recombinant vaccinia vaccine encoding HPV-16/18 E6/E7 antigens (TA-HPV). We found that intratumoral injection with TA-HPV following cisplatin treatment also led to increased E7-specific CD8+ T cells in the blood as well as significantly decreased tumor size compared to intratumoral injection with wild type vaccinia virus. Our study has strong implications for future clinical translation using intratumoral injection of TA-HPV in conjunction with the current treatment strategies for patients with advanced cervical cancer.

Enhancement of anticancer immunity by immunomodulation of apoptotic tumor cells using annexin A5 protein-labeled nanocarrier system.

Chemotherapy promotes phosphatidylserine (PS) externalization in tumors undergoing apoptosis, forms an immunosuppressive tumor microenvironment (TME), and inhibits dendritic cell (DC) maturation and antigen presentation by binding PS receptors expressed in DCs, thereby limiting naive T cell education and activation. In this study, we demonstrate a selective nanocarrier system composed of annexin A5-labeled poly (lactide-co-glycolide) nanoparticles (PLGA_NPs) encapsulating tumor specific antigen or neoantigen, to target apoptotic tumor cells expressing PS as an innate immune checkpoint inhibitor (ICI) that induces active cancer immunotherapy. Moreover, PLGA_NPs enhanced tumor-specific antigen-based cytotoxic T cell immunity via the original function of DCs by converting the tumor antigen-rich environment. Therefore, chemotherapy combined with an immunomodulatory nanocarrier system demonstrated an enhanced anticancer immune response by increasing survival rates, immune-activating cells, and pro-inflammatory cytokines in the spleen and TME. In contrast, the tumor mass, immune-suppressive cells, and anti-inflammatory cytokines were decreased. Furthermore, the combination of a nanocarrier system with other ICIs against large tumors showed therapeutic efficacy by immunosuppression in the TME and further amplified the anticancer immunity of interferon gamma+ (IFN-γ) CD8+ (cluster of differentiation 8) T cells. Taken together, our Annexin A5-labeled PLGA-NPs can be applied in various combination therapeutic techniques for cancer immunotherapy.

Immune checkpoint silencing using RNAi-incorporated nanoparticles enhances antitumor immunity and therapeutic efficacy compared with antibody-based approaches.

BACKGROUND: Cytotoxic CD8+ T cell-based cancer immunotherapy has been extensively studied and applied, however, tumor cells are known to evade immune responses through the expression of immune checkpoints, such as programmed death ligand 1 (PD-L1). To overcome these issues, antibody-based immune checkpoint blockades (eg, antiprogrammed cell death 1 (anti-PD-1) and anti-PD-L1) have been revolutionized to improve immune responses. However, their therapeutic efficacy is limited to 15%-20% of the overall objective response rate. Moreover, PD-L1 is secreted from tumor cells, which can interrupt antibody-mediated immune reactions in the tumor microenvironment. METHODS: We developed poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs) encapsulating PD-L1 small interfering RNA (siRNA) and PD-1 siRNA, as a delivery platform to silence immune checkpoints. This study used the TC-1 and EG7 tumor models to determine the potential therapeutic efficacy of the PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs, on administration twice per week for 4 weeks. Moreover, we observed combination effect of PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs and PLGA (antigen+adjuvant)-NPs using TC-1 and EG7 tumor-bearing mouse models. RESULTS: PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs boosted the host immune reaction by restoring CD8+ T cell function and promoting cytotoxic CD8+ T cell responses. We demonstrated that the combination of NP-based therapeutic vaccine and PLGA (siRNA)-NPs resulted in significant inhibition of tumor growth compared with the control and antibody-based treatments (p<0.001). The proposed system significantly inhibited tumor growth compared with the antibody-based approaches. CONCLUSION: Our findings suggest a potential combination approach for cancer immunotherapy using PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs and PLGA (antigen+adjuvant)-NPs as novel immune checkpoint silencing agents.

Enhancement of DNA vaccine potency by antigen linkage to IFN-γ-inducible protein-10.

DNA vaccines have emerged as an attractive approach to generate antigen-specific T-cell immune response. Nevertheless, the potency of DNA vaccines still needs to be improved for cancer immunotherapy. In this study, we explored whether functional linkage of a Th1-polarizing chemokine, IP-10, to a model tumor antigen, human papillomavirus type 16 (HPV-16) E7, enhanced DNA vaccine potency. IP-10 linkage changed the location of E7 from the nucleus to the endoplasmic reticulum and led to the secretion of functionally chemoattractive chimeric IP-10/E7 protein. In addition, this linkage drastically enhanced the endogenous processing of E7 antigen through MHC class I. More importantly, we found that C57BL/6 mice intradermally vaccinated with IP-10/E7 DNA exhibited a dramatic increase in the number of E7-specific CD4(+) Th1 T-cells and CD8(+) T-cells and, consequently, were strongly resistant over the long term to E7-expressing tumors compared to mice vaccinated with wild-type E7 DNA. Thus, because of the increase in tumor antigen-specific T-cell immune responses obtained through both enhanced antigen presentation and chemoattraction, vaccination with DNA encoding IP-10 linked to a tumor antigen holds great promise for treating tumors.

Combined administration with DNA encoding vesicular stomatitis virus G protein enhances DNA vaccine potency.

DNA vaccines have recently emerged at the forefront of approaches to harness the immune system in the prevention and treatment of viral infections, as well as the prevention and treatment of cancers. However, these vaccines suffer from limited efficacy since they often fail to produce significant antigen-specific CD8(+) T-cell responses. We report here a novel concept for DNA vaccine design that exploits the unique and powerful ability of viral fusogenic membrane glycoproteins (FMGs) to couple concentrated antigen transfer to dendritic cells (DCs) with local induction of the acute inflammatory response. Intramuscular administration into mice by electroporation technology of a plasmid containing the FMG gene from vesicular stomatitis virus (VSV-G)-together with DNA encoding the E7 protein of human papillomavirus type 16, a model cervical cancer antigen-elicited robust E7-specific CD8(+) T-cell responses, as well as therapeutic control of E7-expressing tumors. This effect could potentially be mediated through the immunogenic form of cellular fusion and necrosis induced by VSV-G, which in a concerted fashion provokes leukocyte infiltration into the inoculation site, enhances cross-presentation of antigen to DCs, and stimulates them to mature efficiently. Thus, the incorporation of FMGs into DNA vaccines holds promise for the successful control of viral infections and cancers in the clinic.

A novel form of immunotherapy using antigen peptides conjugated on PD-L1 antibody.

Immune checkpoint inhibitors (ICIs), including programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 have shown promising cancer clinical outcomes. However, IC therapy has low patient response rates (10%-15%). Thus, ICIs and sufficient antigen combinations into the tumor microenvironment (TME) is important to produce strong tumor-specific adaptive immune responses. Mice were treated with cisplatin, and human cancer cells were exposed to inflammatory cytokines, to confirm increased PD-L1 and major histocompatibility complex (MHC) I expression by tumor cells or dendritic cells. TC-1, CT26, B16-F1, or B16-F10 tumor cells, and bone marrow-derived dendritic cells, were treated with interferon (IFN)-ß, IFN-γ, or tumor necrosis factor-α to identify the molecular mechanisms underlying tumor PD-L1 and MHC I upregulation, and to examine MHC I, CD40, CD80, CD86, or PD-L1 levels, respectively. For synergistic combination therapy, αPD-L1 monoclonal antibody (mAb) covalently linked to the long E7 peptide was generated. Chemotherapy shifted the TME to express high PD-L1 and MHC I, resulting in targeted ICI cargo delivery and enhanced generation and activation of tumor antigen-specific T cells. Synergistic effects of vaccination and IC blockade in the TME were demonstrated using an anti-PD-L1 mAb covalently conjugated to the E7 long peptide.

NIR irradiation-controlled drug release utilizing injectable hydrogels containing gold-labeled liposomes for the treatment of melanoma cancer.

Drug-based chemotherapy is associated with serious side effects. We developed a chemotherapeutic system comprising a chitosan hydrogel (CH-HG) containing gold cluster-labeled liposomal doxorubicin (DOX) (CH-HG-GLDOX) as an injectable drug depot system. CH-HG-GLDOX can be directly injected into tumor tissue without a surgical procedure, allowing this system to act as a reservoir for liposomal DOX. CH-HG-GLDOX enhanced the retention time of DOX in tumor tissue and controlled its release in response to near-infrared (NIR) irradiation, resulting in significant inhibition of tumor growth and reduced DOX-related toxicity. The combined effect of CH-HG-GLDOX and poly (D,L-lactide-co-glycolic acid) nanoparticle-based vaccines increased cytotoxic CD8+ T cell immunity, leading to enhanced synergistic therapeutic efficacy. CH-HG-GLDOX provides an advanced therapeutic approach for local drug delivery and controlled release of DOX, resulting in reduced toxicity. Here, we suggest a combination strategy for chemo- and immunotherapies, as well as in nanomedicine applications. STATEMENT OF SIGNIFICANCE: We developed an injectable hydrogel containing gold cluster-labeled liposomes for sustained drug release at the tumor site. Moreover, we demonstrated the combined therapeutic efficacy of a hydrogel system and a nanoparticle-based immunotherapeutic vaccine for melanoma cancer. Thus, we show a potential combination approach for chemo- and immunotherapies for cancer treatment.

Activation of Akt as a mechanism for tumor immune evasion.

Immune evasion is an important reason why the immune system cannot control tumor growth. To elucidate the mechanism for tumor immune evasion, we generated an immune-resistant human papillomavirus type 16 (HPV-16) E7-expressing tumor cell line by subjecting a susceptible tumor cell line to multiple rounds of in vivo immune selection with an E7-specific vaccine. Comparison of parental and immune-resistant tumors revealed that Akt is highly activated in the immune-resistant tumors. Retroviral transfer of a constitutively active form of Akt into the parental tumor significantly increased its resistance against E7-specific CD8(+) T-cell mediated apoptosis. The observed resistance against apoptosis was found to be associated with the upregulation of antiapoptotic molecules. We also observed that intratumoral injection of an Akt inhibitor enhanced the therapeutic efficacy of E7-specific vaccine or E7-specific CD8(+) T-cell adoptive transfer against the immune-resistant tumors. Thus, our data indicate that the activation of PI3K/Akt pathway represents a new mechanism of immune escape and has important implications for the development of a novel strategy in cancer immunotherapy against immune-resistant tumor cells.