EG7 Tumor Research Articles

Abstract 3741: Helper/killer hybrid epitope long peptide (H/K-HELP) cancer vaccine augments Th1-dependent CTL induction at tumor-draining lymph node to eradicate solid tumor: its application to develop an innovative combination cancer immunotherapy(iCCI)

Abstract We have initially demonstrated the pivotal role of IFN-γ-producing tumor-antigen-specific Th1 cells for the complete eradication of established tumor mass by CTL. The accumulated evidence has clearly demonstrated that induction of Th1-dependent full activation of CTL at tumor-draining lymph nodes (TDLN) are crucial for inducing complete rejection of solid tumor by CTL. To induce Th1 immunity efficiently at TDLN in tumor-bearing host, we developed an artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP), which conjugated killer and helper epitope peptides of cancer antigen by a glycine-linker. In mouse model, OVA-H/K-HELP was selectively presented in vivo by professional DC in vaccinated TDLN and sustained antigen presentation capability of DC to stimulate both CTL and Th1 cells. Thus, in contrast to short peptide, vaccination of OVA-expressing EG7-bearing mice with OVA-H/K-HELP caused a complete eradication of EG7 tumor mass. To apply these basic findings to clinical treatment of cancer patients, we then developed Survivin-H/K-HELP. Treatment of a patient with a triple-negative breast cancer patient with Survivin-H/K-HELP induced a complete response in a triple-negative breast cancer patient concomitantly with the induction of Th1-dependent cellular and humoral immune responses. In this clinical study, the patients showed Th1-dependent C’-fixing IgG antibody production and exhibited IFN-γ-producing Th1 cellular responses. Finally, we also applied H/K-HELP-pulsing DC-vaccine to develop an innovative combination cancer immunotherapy (iCCI). The iCCI consisting of radiation, low dose of immune check point inhibitors (ICIs:Nibolumab, Ipilimumab) and H/K-HELP-pulsed DC, all of which have been shown to initially modulate antitumor immunity at TDLN. Here, we described a successful outcome of a case of the stage IVA inoperable advanced hypopharyngeal squamous cell cancer patient with lymph node-metastasis. The iCCI treatment of the patient caused a complete disappearance of all cancers and the patient has been cancer free for over 15 months so far. Although the destruction mechanism of cancer is not determined, we image this iCCI might improve patient’s antitumor immune capability in immune microenvironment around tumor (IMAT) including TDLN and TME. Our developed iCCI will become a promising strategy to overcome inoperable advanced cancers in future. Citation Format: Takanori Iwasaki, Yuichiro Yazaki, Takashi Masuko, Takashi Nishimura. Helper/killer hybrid epitope long peptide (H/K-HELP) cancer vaccine augments Th1-dependent CTL induction at tumor-draining lymph node to eradicate solid tumor: its application to develop an innovative combination cancer immunotherapy(iCCI) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3741.

Loss of Anti-Tumor Efficacy by Polyamine Blocking Therapy in GCN2 Null Mice.

GCN2 is one of the main sensors of amino acid starvation stress, and its activation in the stressful tumor microenvironment plays a crucial role in tumor survival and progression. We hypothesized that elevated polyamine biosynthesis and subsequent depletion of precursor arginine activates GCN2, thus rewiring metabolism to support tumor cell survival and drive myeloid immunosuppressive function. We sought to determine if the anti-tumor efficacy of a polyamine blocking therapy (PBT) may be mediated by its effect on GCN2. Unlike wild-type mice, PBT treatment in GCN2 knockout mice bearing syngeneic B16.F10 or EG7 tumors resulted in no tumor growth inhibition and no changes in the profile of infiltrating tumor immune cells. Studies with murine bone marrow cell cultures showed that increased polyamine metabolism and subsequent arginine depletion and GCN2 activation played an essential role in the generation and cytoprotective autophagy of myeloid derived suppressor cells (MDSCs) as well as the M2 polarization and survival of macrophages, all of which were inhibited by PBT. In all, our data suggest that polyamine-dependent GCN2 signaling in stromal cells promotes tumor growth and the development of the immunosuppressive tumor microenvironment, and that the PBT anti-tumor effect is mediated, at least in part, by targeting GCN2.

The glucocorticoid receptor modulator relacorilant reverses the immunosuppressive effects of cortisol

Cortisol, an endogenous glucocorticoid receptor (GR) agonist, controls a broad transcriptional program that affects T-cell activation, pro-inflammatory cytokine secretion, apoptosis, and immune-cell trafficking. The degree to which endogenous cortisol blunts the anti-tumor immune response checkpoint inhibitors stimulate had not been assessed. We addressed this question using relacorilant, a selective GR modulator (SGRM) that competitively antagonizes the effects of cortisol activity. GR expression in human tumor and immune cells positively correlated with PD-L1 expression and tumor infiltration of Th2 and Treg cells, and negatively correlated with Th1-cell infiltration. In vitro, cortisol inhibited, and relacorilant restored, T-cell activation and pro-inflammatory cytokine secretion in human peripheral blood mononuclear cells. In the ovalbumin-expressing EG7 and MC38 immune-competent tumor models, relacorilant significantly improved anti-PD-1 antibody efficacy and showed favorable effects on antigen-specific T-cells and systemic TNFα and IL-10. These data characterize the broad immunosuppressive effects of endogenous cortisol and highlight the potential of combining an SGRM with an immune checkpoint inhibitor.

Polyanhydride based nanovaccine promotes long term humoral and cellular response to PDAC associated protein MUC4β

Abstract Patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) have an unrelentingly poor prognosis, in part because of the lack of neoantigen targets associated with PDAC tumors. However, another approach would be to take advantage of differentially expressed antigens on PDAC tumor cells. It has been demonstrated that mucin 4 (MUC4) is overexpressed on the tumor cells of many PDAC patients and is considered a promising antigenic target for PC immunotherapy. Previous work by our lab has demonstrated that poly-anhydride (PA) nanovaccines based on 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy)hexane (CPH) nanoparticles (NPs) both induced a humoral immune response to the beta subunit of MUC4 (MUC4β) and an anti-tumor response against OVA in an EG-7 tumor model. In these studies, we show that both a PA-based nanovaccine formulation and a PLGA-based nanovaccine induced MUC4β-specific T cell response. Results demonstrated that restimulation of LN and splenic lymphocytes 40 days after initial vaccination induced both CD4+ and CD8+ MUC4β-specific T cells. The MUC4ß-specific T cells could be detected for at least 180 days post-vaccination. Cytokine secretions from the antigen specific T cells included IL-2, INFγ, and TNF-α; in addition, tetramer analysis detected MUC4ß-specific CD8+ T cells using the MUCβ peptide FTPVFYSQL. Collectively, these results demonstrate that a PA nanovaccine can function as an effective vaccine platform for induction of MUC4ß-specific cellular immunity, and lays the ground work for forthcoming studies in MUC4β-expressing tumor model systems. Supported by the National Institute of Health (U01 CA213862) and the Iowa State University Nanovaccine Institute. B. N. is grateful to the Vlasta Klima Balloun Faculty Chair.

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

BackgroundCytotoxic 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...

Distinct roles but cooperative effect of TLR3/9 agonists and PD-1 blockade in converting the immunotolerant microenvironment of irreversible electroporation-ablated tumors.

Irreversible electroporation (IRE) is a new cancer ablation technology, but methods to improve IRE-induced therapeutic immunity are only beginning to be investigated. We developed a mouse model bearing large primary (300 mm3) and medium distant (100 mm3) EG7 lymphomas engineered to express ovalbumin (OVA) as a nominal tumor antigen. We established experimental protocols including IRE alone and IRE combined with Toll-like receptor (TLR)3/9 agonists (poly I:C/CpG) (IRE + pIC/CpG), PD-1 blockade (IRE + PD-1 blockade), or both (IRE + Combo) to investigate therapeutic effects on primary and distant EG7 tumors and conversion-promoting effects on the immunotolerant tumor microenvironment (TME). We demonstrated that IRE alone simulated very weak OVA-specific CD8+ T cell responses and did not inhibit primary tumor growth. IRE + pIC/CpG synergistically stimulated more efficient OVA-specific CD8+ T cell responses and primary tumor growth inhibition than IRE + PD-1 blockade. IRE + pIC/CpG played a major role in the modulation of immune cell profiles but a minor role in the downregulation of PD-L1 expression in the TME and vice versa for IRE + PD-1 blockade. IRE + Combo cooperatively induced potent OVA-specific CD8+ T cell immunity and rescued exhausted intratumoral CD8+ T cells, leading to eradication of not only primary tumors but also untreated concomitant distant tumors and lung metastases. IRE + Combo efficiently modulated immune cell profiles, as evidenced by reductions in immunotolerant type-2 (M2) macrophages, myeloid-derived suppressor-cells, plasmacytoid dendritic cells, and regulatory T cells and by increases in immunogenic M1 macrophages, CD169+ macrophages, type-1 conventional dendritic cells, and CD8+ T cells, leading to conversion of immunotolerance in not only primary TMEs but also untreated distant TMEs. IRE + Combo also showed effective therapeutic effects in two breast cancer models. Therefore, our results suggest that IRE + Combo is a promising strategy to improve IRE ablation therapy in cancer.

Abstract 1712: Neospora caninum: An immunotherapeutic protozoan against cancer

Abstract Background: Immunotherapy induces, provides, and/or reactivates anti-tumor immune responses. Some microorganisms also can initiate response that lyzes infected tumor and/or stimulates systemic immunity. Attenuated viruses or bacteria are well studied as oncotherapeutics, but no protozoa except Toxoplasma gondii. We assessed the effect on tumors of other protozoa that were naturally non-pathogenic to humans. Thus, we discovered the ability to use Neospora caninum (Nc) in a manner and form that demonstrated a synergistic array of pertinent immunotherapeutic characteristics against solid cancers. We report on the most recent data notably from Nc engineered to secrete human IL-15 within the tumor. Methods: In vitro, the immunostimulatory properties of Nc strains wildtype and engineered to secrete human IL-15 were studied. In vivo experiments of treatment with of Nc tachyzoites administered locally (intra and peri tumoral) or remotely (subcutaneous) in a murine thymoma EG7 tumor and in human Merkel cell carcinoma (MCC). Results: We demonstrated that the treatment of thymoma EG7 by Nc strongly inhibited tumor development. Analysis of immune responses and interactions between Nc and tumor cells showed that Nc had the ability to lyze infected cancer cells, reactivated immune competence within the tumor microenvironment (TME), and activated the systemic immune system by promoting the recruitment of immune cells to the site of tumor. We also established in a NOD/SCID mouse model that Nc was able to induce a strong regression of human MCC. Recently, to further enhance oncotherapeutic effect, we engineered an Nc strain to secrete human IL-15 (cross reactive with mouse cells), associated with alpha subunit of IL-15 receptor, increasing its stability. This strain induced proliferation of human PBMCs and their secretion of IFN-γ. In the EG7 model, human IL-15 secreting Nc showed greater protection against tumor development, confirming enhancement of immunotherapy by engineering Nc to deliver/secrete IL-15. Conclusion: These results highlight Neospora caninum as a potentially extremely efficient, and non-toxic anti-cancer agent, capable of being engineered to express at its surface or to secrete bio-drugs, like human IL-15 cytokine. Our work has identified the broad clinical possibilities of using N. caninum as an oncolytic protozoan in human medicine capable of vectoring molecular therapy, overcoming TME defenses. Citation Format: Louis Lantier, Agathe Poupée-Beaugé, Anne Di Tommaso, Céline Ducournau, Stéphanie Germon, Gordon S. Lee, Antoine Touze, Nathalie moiré, Isabelle Dimier-Poisson. Neospora caninum: An immunotherapeutic protozoan against cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1712.

Neospora caninum: a new class of biopharmaceuticals in the therapeutic arsenal against cancer

BackgroundMicroorganisms that can be used for their lytic activity against tumor cells as well as inducing or reactivating antitumor immune responses are a relevant part of the available immunotherapy strategies....

Abstract 3398: PTPN22 is a systemic target for augmenting antitumor immunity

Abstract Remarkable progress in cancer immunology has revolutionized cancer therapy. The majority of patients, however, do not respond to immunotherapeutic options, warranting the ongoing search for better strategies. Leveraging the established role of protein tyrosine phosphatase non-receptor type 22 (PTPN22) in autoimmune diseases, we hypothesized that PTPN22 is a novel target for cancer immunotherapy. PTPN22 is a physiologic regulator of T cell receptor (TCR) signaling acting by dephosphorylating activating tyrosine residues in Lck and Zap70. We first confirmed the relevance of PTPN22 expression by exploring its expression in multiple human cancer types using The Cancer Genome Atlas (TCGA). PTPN22 expression positively correlated with T cell and M1 macrophage gene signatures and immune regulatory genes, especially inflamed tumor types. Next, we directly investigated the role of PTPN22 in antitumor immunity by comparing in vivo tumor characteristics in wild-type (WT) and PTPN22 knockout (KO) mice. Consistent with our hypothesis, PTPN22 KO mice resisted MC38 and EG7 tumors significantly compared with WT. Mass cytometry (CyTOF) profiling of the immune tumor microenvironment demonstrated that MC38 tumors in PTPN22 KO mice were infiltrated with greater numbers of T cells, particularly CD8+ T cells expressing granzyme B and PD1. To further delineate the effects of PTPN22 KO on TCR signaling, we established an optimized CyTOF panel of 9 phosphorylation sites involved in the TCR signaling pathway, including two enzymatic substrates of PTPN22 (Lck Y394 and Zap70 Y493) and 15 immune subtyping markers. CyTOF phospho-profiling of CD8 T cells from tumor-bearing mouse spleens and the peripheral blood of immunotherapy-naïve cancer patients showed that the phosphorylated state of Zap70 Y493 correlated strongly with granzyme B expression. Furthermore, phospho-profiling of tumor-infiltrating CD8+ T cells (a measure of T cell activation) revealed the highest TCR-pathway phosphorylation levels in memory CD8+ T cells that express PD1. The difference in phosphorylation levels between WT and PTPN22 KO was most pronounced for Lck Y394. Based on these findings, we then hypothesized that PD1 inhibition will further enhance the antitumor immune responses promoted by the lack of PTPN22. Indeed, PTPN22 KO mice bearing MC38 and EG7 tumors responded more significantly to anti-PD1 therapy when compared with tumor-bearing WT mice. Finally, we treated WT tumor bearing mice with two different small molecule inhibitors of PTPN22, one previously published compound, LTV1, and one novel compound, L1 (discovered through structure based synthesis). While both inhibitors phenocopied the PTPN22 KO mice in resisting MC38 tumor growth, L1 treatment gave an immune profile that resembled what was observed in tumor-bearing PTPN22 KO mice. Taken together, our results demonstrate that PTPN22 is a novel systemic target for augmenting antitumor immunity. Citation Format: Won Jin Ho, Jianping Lin, Ludmila Danilova, Zaw Phyo, Soren Charmsaz, Aditya Mohan, Todd Armstrong, Ben H. Park, Elana J. Fertig, Zhong-Yin Zhang, Elizabeth M. Jaffee. PTPN22 is a systemic target for augmenting antitumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3398.

Abstract 6615: Peroxynitrite mediates immune escape of tumor cells from cytotoxic T cells in situ

Abstract Despite improved ability to generate strong tumor-specific T cell responses, clinical benefits remain relatively moderate. Only a small proportion of predicted neoepitopes was shown to induce specific cytotoxic T cells (CTLs) or serve as an efficient target for T cell-based therapy. We suggest here that low efficacy of CTLs could be at least partially explained by the fact that tumor microenvironment (TME) modulates antigen presentation and epitope profile expressed by tumor. As a result, tumor escapes recognition by CTLs. TME is enriched in peroxynitrite (PNT), a potent oxidant. It is mostly produced by infiltrating myeloid cells, but also by some tumors (as melanoma). We hypothesized that PNT is able to affect antigen presentation on tumor cells and facilitates tumor immune escape. To check this hypothesis, we treated murine EG7 tumor cells with PNT, isolated MHC I peptides and compared their expression on treated versus non-treated cells using SILAC mass spectrometry. We found that PNT treatment significantly decreased expression of a portion of MHC I peptides presented by tumor cells, whereas other peptides remained relatively intact. We found that PNT-sensitive and resistant peptides demonstrated similar binding capacity to MHC I. However, the off-rate of PNT-sensitive peptides was significantly higher. PNT strongly affected proteasomal activity of tumor cells suggesting the mechanism for under-representation of MHC I peptides with high off-rate level. To investigate the biological role of this effect, we generated CTLs specific to a pool of PNT-sensitive or resistant peptides. In vitro we observed that both types of CTLs effectively killed tumor cells. Pre-treatment of tumor cells with PNT significantly reduced killing only by CTLs specific to PNT-sensitive peptides. By ELISPOT we showed that most of antitumor-specific CTL responses in mice with different tumors were induced against PNT-resistant, but not PNT-sensitive species. Moreover, the adoptive transfer of CTLs specific to PNT-resistant peptides combined with immune checkpoint inhibition (ICI) was able to induce tumor rejection in 70% of mice, whereas similar setting for CTLs specific to PNT-sensitive peptides didn't affect tumor growth. PNT inhibition combined with ICI significantly delayed the tumor growth in mice. In addition, we showed that PNT had similar effect on MHC I peptide profile for human melanoma cells. Finally, we measured nitrotyrosine (NT) level (marker of PNT presence) in tumor tissues resected from melanoma patients before the start of ICI therapy. We retrospectively correlated NT levels with patient ability to respond to ICI therapy. We found that high NT level was a predictor of poor prognosis and patient un-responsiveness to ICI. Overall, our study demonstrates PNT ability to affect antigen presentation in tumor site. In addition, we suggest PNT as a promising target for ICI-combined therapy as well as a biomarker for resistance to ICI. Citation Format: Evgenii N. Tcyganov, Dmitry I. Gabrilovich. Peroxynitrite mediates immune escape of tumor cells from cytotoxic T cells in situ [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6615.

Galactosylated chitosan-modified ethosomes combined with silk fibroin nanofibers is useful in transcutaneous immunization

Transcutaneous immunization (TCI) has the advantages of avoiding the liver first-pass effect, good compliance and convenient use compared with the traditional oral or injection vaccination. However, the stratum corneum (SC) of the skin is the main obstacle that limits the entry of antigen molecules into the epidermis for activating dendritic cells (DCs). In the present study, the hyaluronic acid (HA) and galactosylated chitosan (GC) modified ethosome (Eth-HA-GC) was prepared through layer-by-layer self-assembly method. Eth-HA-GC has good stability and can be effectively phagocytosed by the bone-marrow-derived DCs (BMDCs) in vitro. The ovalbumin (OVA) loaded Eth-HA-GC (OVA@Eth-HA-GC) can promote BMDCs' expression of CD80, CD86 (DCs maturation-associated marker molecules), TNF-α, IL-2 and IL-6. Subsequently, a novel OVA@Eth-HA-GC-loaded silk fibroin (OVA@Eth-HA-GC/SF) nanofibrous mats were fabricated through green electrospinning. The OVA@Eth-HA-GC/SF mats exhibit good transdermal performance in vitro. Transdermal administration with OVA@Eth-HA-GC/SF mats induced the serum anti-OVA-specific IgG and increased the expression of IFN-γ, IL-2 and IL-6 by spleen cells in vivo. Furthermore, the use of OVA@Eth-HA-GC/SF mats evidently inhibited the growth of EG7 tumor in the murine model. These results demonstrate the OVA@Eth-HA-GC/SF mats can effectively stimulate the immune response to OVA through transdermal administration. In conclusion, the antigens@Eth-HA-GC/SF mats is a promising TCI system.

Enhanced anti-tumor immunotherapy by dissolving microneedle patch loaded ovalbumin.

The skin is a very suitable organ for the induction of immune responses to vaccine antigens. Antigen delivery systems to the skin by needle and syringe directly deposit the antigen into the epidermal-dermal compartment, one of the most immunocompetent sites due to the presence of professional antigen-presenting cells aimed at the induction of antigen-specific T cells. In this study, we analyzed the amount of ovalbumin as an antigen delivered to the skin by a microneedle. When ovalbumin protein as an antigen was delivered to the skin of mice using a dissolving microneedle, it induced an immune response through the enhanced proliferation and cytokines production by the splenocytes and lymph nodes. Also, it effectively increased the ovalbumin-specific CD8+ T cell and CD4+ T cell population and induced an ovalbumin-specific CTL response against the graft of ovalbumin-expressing EG7 tumor cells in the immunized mice. Also, we identified the inhibition of tumor growth and prevention of tumor formation in the context of the therapeutic and prophylactic vaccine, respectively through EG-7 tumor mouse model. Finally, these data show the potential of patches as attractive antigen delivery vehicles.

TLR9 agonist enhances radiofrequency ablation-induced CTL responses, leading to the potent inhibition of primary tumor growth and lung metastasis.

Radiofrequency ablation (RFA) is the most common approach to thermal ablation for cancer therapy. Unfortunately, its efficacy is limited by incomplete ablation, and further optimization of RFA is required. Here, we demonstrate that incubation at 65 °C triggers more EG7 tumor cell death by necrosis than treatment at 45 °C, and the 65 °C-treated cells are more effective at inducing antigen-specific CD8+ cytotoxic T lymphocyte (CTL) responses after injection in mice than the 45 °C-treated ones. Dendritic cells (DCs) that phagocytose 65 °C-treated EG7 cells become mature with upregulated MHCII and CD80 expression and are capable of efficiently inducing effector CTLs in mouse tumor models. RFA (65 °C) therapy of EG7 tumors induces large areas of tumor necrosis and stimulates CTL responses. This leads to complete regression of small (~100 mm3) tumors but fails to suppress the growth of larger (~350 mm3) tumors. The administration of the Toll-like receptor-9 (TLR9) agonist unmethylated cytosine-phosphorothioate-guanine oligonucleotide (CpG) to DCs phagocytosing 65 °C-treated EG7 cells enhances the expression of MHCII and CD40 on DCs as well as DC-induced stimulation of CTL responses. Importantly, the intratumoral administration of CpG following RFA also increases the frequencies of tumor-associated immunogenic CD11b-CD11c+CD103+ DC2 and CD11b+F4/80+MHCII+ M1 macrophages and increases CD4+ and CD8+ T-cell tumor infiltration, leading to enhanced CD4+ T cell-dependent CTL responses and potent inhibition of primary RFA-treated or distant untreated tumor growth as well as tumor lung metastasis in mice bearing larger tumors. Overall, our data indicate that CpG administration, which enhances RFA-induced CTL responses and ultimately potentiates the inhibition of primary tumor growth and lung metastasis, is a promising strategy for improving RFA treatment, which may assist in optimizing this important cancer therapy.

Surface assembly of poly(I:C) on polyethyleneimine‐modified gelatin nanoparticles as immunostimulatory carriers for mucosal antigen delivery

The mucosal immune system is the host's first line of defense against invasion by foreign pathogens. Gelatin nanoparticles (GNPs) are suitable carriers for the delivery of antigens via various routes of administration. In the present study, GNPs were modified with polyethyleneimine (PEI), a positively charged polymer. Then, ovalbumin (OVA) and polyinosinic:polycytidylic acid (poly(I:C)), an immunostimulant, were adsorbed onto the surface of the positively charged GNPs. We assessed whether GNPs could act as an effective mucosal vaccine that is capable of inducing both mucosal and systemic immune responses. The results showed that GNPs effectively adsorbed OVA/poly(I:C), facilitated cellular uptake by RAW 264.7 macrophage cells and murine bone marrow-derived dendritic cells (BMDCs) in vitro, and led to increased expression of the maturation markers CD80 and CD86 on BMDCs. Furthermore, GNPs induced increased secretion of proinflammatory cytokines in both RAW 264.7 and BMDCs. C57BL/6 mice that were intranasally twice-immunized with OVA/poly(I:C)-loaded GNPs produced high levels of serum OVA-specific IgG antibodies and secretory IgA in nasal and lung lavage. Spleen cells from immunized mice were collected and re-stimulated with OVA, and results showed significantly augmented production of IFN-γ, IL-4, IL-5, and IL-6 in mice that received OVA/poly(I:C)-loaded GNPs. Moreover, intranasal immunization with OVA/poly(I:C)-loaded GNPs resulted in the inhibition of EG7 tumor growth in C57BL/6 mice. Taken together, these results indicate that nasal administration of OVA/poly(I:C)-loaded GNPs elicited effective mucosal and systemic immune responses, which might be useful for further applications of antigen delivery. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1228-1237, 2019.

Regulatory effects of dexamethasone on NK and T cell immunity

Glucocorticoids (GCs) act via the intracellular glucocorticoid receptor (GR), which can regulate the expression of target genes. With regard to the immune system, GCs may affect both innate and adaptive immunity. Our study analyzed the immunoregulatory effects of dexamethasone (Dex) treatment on splenic T, Treg, NK and NKT cells by treating C57Bl6 mice with various doses of Dex. We observed that treatment with Dex decreased the number of NK cells in the spleen and suppressed their activity. In particular, the expression of both Ly49G and NKG2D receptors was decreased by Dex. However, Dex did not affect the population of NKT cells. With regard to splenic T cells, our results show a dose-dependent reduction in CD3+, CD4+, CD8+, CD44+ and CD8+CD122+ T cells, but a stimulatory effect on CD4+CD25+ regulatory T cells by Dex treatment. In addition, treatment with Dex suppressed anti-tumor immune response in a mouse EG7 tumor model. We conclude that Dex may suppress both T- and NK-mediated immunity.

Development of a Curative Therapeutic Vaccine (TheraVac) for the Treatment of Large Established Tumors

Harnessing immune system to treat cancer requires simultaneous generation of tumor-specific CTLs and curtailment of tumor immunosuppressive environment. Here, we developed an immunotherapeutic regimen capable of eliminating large established mouse tumors using HMGN1, a DC-activating TLR4 agonist capable of inducing anti-tumor immunity. Intratumoral delivery of HMGN1 with low dose of Cytoxan cured mice bearing small (∅ ≈ 0.5 cm), but not large (∅ ≈ 1.0 cm) CT26 tumors. Screening for activators capable of synergizing with HMGN1 in activating DC identified R848. Intratumoral delivery of HMGN1 and R848 plus Cytoxan eradicated large established CT26 tumors. The resultant tumor-free mice were resistant to subsequent challenge with CT26, indicating the generation of CT26-specific protective immunity. This immunotherapeutic regimen caused homing of tumor-infiltrating DC to draining lymph nodes and increased infiltration of T cells into tumor tissues. Cytoxan in this regimen could be replaced by anti-CTLA4) or anti-PD-L1. Importantly, this immunotherapeutic regimen was also curative for large established mouse Renca and EG7 tumors. Thus, we have developed a curative therapeutic vaccination regimen dubbed ‘TheraVac’ consisting of HMGN1 and R848 plus a checkpoint inhibitor, that can, without using exogenous tumor-associated antigen(s), eliminate various large tumors and induce tumor-specific immunity.

Modification of anti-tumor immunity by tolerogenic dendritic cells

Immunosuppressive functions of glucocorticoids (GC) can be mediated via various mechanisms, including the modulation of dendritic cells (DC). Our study investigates the effects of tolerogenic GC-treated DCs on NK and T cell anti-tumor responses in OT-1/Rag−/− mice, expressing a transgenic TCR in CD8+ T cells. The effects caused by GC-treated DCs were compared to the responses to immunogenic, CpG-activated DCs. The effects of DCs on anti-tumor immune responses were analyzed using the EG7 tumor model, where the tumor cells express the peptide epitope recognized by OT-1 T cells. We observed that immunization with CpG and peptide-treated DCs protected against tumor growth by activation of NK cell response. Also, immunogenic DCs induced the expansion of cytotoxic CD8+OT-1 cells, expressing activation markers CD44 and CD69 and producing IFNγ. In contrast, the peptide and GC-treated DCs in OT-1 mice increased the numbers of immature Mac-1+CD27− NK cells as well as Foxp3+ and IL-10 secreting CD8+OT-1 cells with suppressive properties. We conclude that the generation of tolerogenic DCs is one of many immunosuppressive mechanisms that can be induced by GC. Our study demonstrated that tolerogenic DCs modify anti-tumor immune response by suppressing NK cell activity and stimulating the formation of IL-10-secreting CD8+ Tregs.

Abstract 4598: Triggering of OX40 on T cells by a novel monoclonal antibody elicits robust antitumor immunity<!–EndFragment–>

Abstract T cell costimulation is an attractive strategy for cancer treatment in addition to check point inhibitors. Costimulatory molecule OX40 is a member of the TNF receptor superfamily that is transiently expressed on activated T cells. Activation of OX40 leads to enhanced T cell proliferation and cytokine secretion and in turn results in better anti-tumor efficacy as shown by multiple mouse syngeneic tumor models. PF-04518600 (PF-8600) is a fully human IgG2 agonist antibody to human OX40 with high affinity and specificity. We used in vitro and in vivo methods to evaluate the co-stimulatory functions of PF-8600. PF-8600 demonstrated better agonist activity in a luciferase reporter cell line expressing OX40 and NFκB as compared to a human IgG1 antibody. In human primary T cells, PF-8600 dose dependently increased T cell proliferation and cytokine secretion in vitro. Furthermore in a mouse line expressing human OX40, PF-8600 in a mouse IgG1 framework increased the proliferation of OT1 cells and inhibited EG7 tumor growth as compared to isotype control antibody, further confirming the mechanism of action as an OX40 agonist. As regulatory T cells are a major inhibitory population in the tumor microenvironment, their specific depletion in the tumor is highly desirable. In an in vitro assay using monocyte derived macrophages as effector cells, PF-8600 mediated the depletion of OX40 expressing cells similar to a human IgG1 antibody, and this activity is dependent on the binding to hFcγRIIA. In human FcγR knock-in mice, anti-OX40-hIgG1 showed increased Treg reduction in the tumor comparing to anti-OX40-hIgG2, but less T cell proliferation and activation in the periphery. In combination with an anti-PD-L1 antibody, both anti-OX40-hIgG1 and hIgG2 showed similar enhanced anti-tumor activity in the MC38 colon carcinoma model as comparing to anti-PD-L1 alone. Both in vitro and in vivo results demonstrated PF-8600 enhanced T cells functions and inhibited tumor growth in a mouse syngeneic tumor model. The activity of the hIgG2 antibody is similar to that of an hIgG1 in both phagocytosis assays and hFcγR KI in vivo assays. Based on the mechanism of action and robust anti-tumor efficacy in preclinical models, PF-8600 is currently in clinical development in a broad spectrum of malignancies. Citation Format: Hua Long, Ann White, Jie Wei, Brittany Jiang, Reid Feldman, Danielle Pappas, Aymen Al-Shamkhani, John Lin. Triggering of OX40 on T cells by a novel monoclonal antibody elicits robust antitumor immunity<!–EndFragment–> [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4598. doi:10.1158/1538-7445.AM2017-4598

Development of a curative therapeutic vaccine (TheraVac) for the treatment of large established tumors

Abstract Harnessing immune system to treat cancer requires simultaneous generation of tumor-specific CTLs and curtailment of tumor immunosuppressive environment. Here, we developed an immunotherapeutic regimen capable of eliminating large established mouse tumors using HMGN1, a DC-activating TLR4 agonist capable of inducing anti-tumor immunity. Intratumoral delivery of HMGN1 with low dose of Cytoxan that acts as a functional checkpoint inhibitor cured mice bearing small (diameter ~ 0.5 cm), but not large (diameter ~ 1.0 cm) CT26 tumors. Screening for activators capable of synergizing with HMGN1 in activating DC identified R848. Intratumoral delivery of HMGN1 and R848 plus Cytoxan eradicated large established CT26 tumors. The resultant tumor-free mice were resistant to subsequent challenge with CT26, indicating the generation of CT26-specific protective immunity. This immunotherapeutic regimen caused homing of tumor-infiltrating DC to draining lymph nodes and increased infiltration of T cells into tumor tissues. Cytoxan in this regimen could be replaced by anti-CTLA4) or anti-PD-L1. Importantly, this immunotherapeutic regimen was also curative for large established mouse Renca and EG7 tumors. Thus, we have developed a curative therapeutic vaccination regimen dubbed ‘TheraVac’ consisting of HMGN1 and R848 plus a checkpoint inhibitor, that can, without using exogenous tumor-associated antigen(s), eliminate various large tumors and induce tumor-specific immunity.

In vitro assessment of deferoxamine on mesenchymal stromal cells from tumor and bone marrow.

Deferoxamine (DFO), an iron chelator, is commonly used to remove excess iron from the body. DFO has also been demonstrated to have anti-tumor effect. However, there is no available report on the effect of deferoxamine on mesenchymal stromal cells (MSCs). In this study, we first isolated tumor-associated MSCs (TAMSCs) from EG-7 tumors, which were positive for CD29, CD44, CD73, CD90 and CD105. Ex vivo cultured stem cells derived from tumor and bone marrow compartment were exposed to DFO. We demonstrated that DFO had growth-arresting and apoptosis-inducing effect on TAMSCs and bone marrow MSCs (BMMSCs). DFO also influenced the expression pattern of adhesion molecule VCAM-1 on both TAMSCs and BMMSCs. Notwithstanding its widespread use, our results here warrants caution in the application of DFO, and also highlights the need for careful evaluation of the bone marrow compartment in patients receiving DFO treatment.