Lysate-pulsed Dendritic Cells Research Articles

TLR agonists polarize interferon responses in conjunction with dendritic cell vaccination in malignant glioma: a randomized phase II Trial

In this randomized phase II clinical trial, we evaluated the effectiveness of adding the TLR agonists, poly-ICLC or resiquimod, to autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination in patients with newly-diagnosed or recurrent WHO Grade III-IV malignant gliomas. The primary endpoints were to assess the most effective combination of vaccine and adjuvant in order to enhance the immune potency, along with safety. The combination of ATL-DC vaccination and TLR agonist was safe and found to enhance systemic immune responses, as indicated by increased interferon gene expression and changes in immune cell activation. Specifically, PD-1 expression increases on CD4+ T-cells, while CD38 and CD39 expression are reduced on CD8+ T cells, alongside an increase in monocytes. Poly-ICLC treatment amplifies the induction of interferon-induced genes in monocytes and T lymphocytes. Patients that exhibit higher interferon response gene expression demonstrate prolonged survival and delayed disease progression. These findings suggest that combining ATL-DC with poly-ICLC can induce a polarized interferon response in circulating monocytes and CD8+ T cells, which may represent an important blood biomarker for immunotherapy in this patient population.Trial Registration: ClinicalTrials.gov Identifier: NCT01204684.

Comparing the efficacy of combined versus single immune cell adaptive therapy targeting colorectal cancer.

Colorectal cancer (CRC) is the most frequent cancer with limited therapeutic achievements. Recently, adoptive cellular immunotherapy has been developed as an antitumor therapy. However, its efficacy has not been tested in CRC. This study investigated the ability of an immune cell cocktail of dendritic cells (DCs), T cells, and natural killer (NK) cells to overcome immunological hurdles and improve the therapeutic efficacy of cell therapy for CRC. CRC lysate-pulsed monocyte-derived DCs (Mo-DCs), CRC antigen-specifically expanded T cells (CTL), and in vitro-expanded NK cells were cultured from patient peripheral blood mononuclear cells (PBMC). The ability of the combined immune cells to kill autologous tumor cells was investigated by co-culturing the combined immune cells with patient-derived tumor cells. The Mo-DCs produced expressed T cell co-stimulating molecules like CD80, CD86, human leukocyte antigen (HLA)-DR and HLA-ABC, at high levels and were capable of activating naive T cells. The expanded T cells were predominantly CD8 T cells with high levels of CD8 effector memory cells and low levels of regulatory T cells. The NK cells expressed high levels of activating receptors and were capable of killing other cancer cell lines (K562 and HT29). The immune cell cocktail demonstrated a higher ability to kill autologous tumor cells than single types. An in vivo preclinical study confirmed the safety of the combined immune cell adaptive therapy showing no therapy-related death or general toxicity symptoms. The results suggested that combined immune cell adaptive therapy could overcome the limited efficacy of cell immunotherapy.

SiRNA-Mediated B7H7 Knockdown in Gastric Cancer Lysate-Loaded Dendritic Cells Amplifies Expansion and Cytokine Secretion of Autologous T Cells.

Gastric cancer, ranked as the fifth most common cancer worldwide, presents multiple treatment challenges. These obstacles often arise due to cancer stem cells, which are associated with recurrence, metastasis, and drug resistance. While dendritic cell (DC)-based immunotherapy has shown promise as a therapeutic strategy, its efficacy can be limited by the tumor microenvironment and certain inhibitory immune checkpoint molecules, such as B7H7. SiRNA-medicated knockdown of B7H7 in tumor cell lysate-pulsed DCs can increase cytokine secretion and autologous T lymphocyte expansion. This study aimed to evaluate the impact of B7H7 suppression in gastric cancer cell lysate-pulsed DCs on the stimulatory potential of autologous CD3+ T lymphocytes. Peripheral blood mononuclear cells (PBMCs) were isolated and monocytes were obtained; then, they were differentiated to immature DCs (iDCs) by GM-CSF and IL-4. Tumor cell lysates from human gastric cancer cell lines were harvested, and iDCs were transformed into mature DCs (mDCs) by stimulating iDCs with tumor cell lysate and lipopolysaccharide. B7H7-siRNA was delivered into mDCs using electroporation, and gene silencing efficiency was assessed. The phenotypic characteristics of iDCs, mDCs, and B7H7-silenced mDCs were evaluated using specific surface markers, an inverted light microscope, and flow cytometry. CD3+ T cells were isolated via magnetically activated cell sorting. They were labeled with CFSE dye and co-cultured with mDCs and B7H7-silenced mDCs to evaluate their ability to induce T-cell proliferation. T-cell proliferation was assessed using flow cytometry. The concentration of TGF-β, IL-4, and IFN-γ secreted from CD3+ T cells in the co-cultured supernatant was evaluated to investigate the cytokine secretory activity of the cells. Transfection of B7H7 siRNA into mDCs was performed in optimal conditions, and the siRNA transfection effectively reduced B7H7 mRNA expression in a dose-dependent manner. SiRNA-mediated B7H7 knockdown in mDCs enhanced maturation and activation of the DCs, as demonstrated by an increased surface expression of CD11c, CD86, and CD40. Co-culture experiments revealed that B7H7-silenced mDCs had more capacity to induce T cell proliferation compared to non-transfected mDCs. The cytokine production patterns of T cells were also altered. Upon examining the levels of TGF-β, IL-4, and IFN-γ released by CD3+ T cells in the co-culture supernatant, we found that silencing B7H7 in mDCs resulted in a rise in IL-4 secretion and a reduction in TGF-β levels compared to mDCs that were not transfected. The study found that suppressing B7H7 expression in DCs significantly enhances their maturation and stimulatory activity when exposed to gastric cancer cell lysate. These B7H7-silenced DCs can substantially increase cytokine production and promote co-cultured T-cell expansion. Consequently, inhibiting B7H7 in DCs may offer a practical strategy to enhance the ability of DCs to initiate T lymphocyte responses and improve the effectiveness of DC-based cell therapy for cancer patients.

IMMU-13. DEVELOPMENT OF ADAPTIVE IMMUNE RESISTANCE IN A MURINE GLIOMA MODEL TREATED WITH DENDRITIC CELL VACCINATION AND PD-1 CHECKPOINT BLOCKADE

Abstract Immunotherapies such as immune checkpoint inhibitors and active vaccination have shown great potential in unleashing antitumor immunity for cancer treatment. However, their effectiveness in treating glioblastoma (GBM) is limited. In a preclinical glioma model, administration of both autologous tumor lysate-pulsed dendritic cell vaccination (ATL-DC) and anti-PD-1 checkpoint blockade (aPD1) significantly increased survival and enhanced intratumoral T cell activation. We hypothesize that the combination of these two treatments will substantially increase the abundance and activity of tumor-reactive cytotoxic T cells. As such, we recently started a phase I trial administering ATL-DC and neoadjuvant aPD1 to recurrent GBM patients (NCT ID: NCT04201873). To understand the local antitumor immune response in greater depth, we performed single-cell RNA sequencing analysis of the CD45+ immune compartment of explanted gliomas from twelve mice, four of which were treated with ATL-DC and aPD1 while the other eight received no treatment. We found that the combination therapy increased tumor-infiltrating T cells, monocytes and DCs. We also observed the expansion and differentiation of CD8+ effector T cells under the effect of combination therapy. These T cells exhibited a differentiation trajectory from a Tcf7+Il2ra+ early activated phenotype to a terminally exhausted state marked by high expression of Pdcd1, Tox and Tigit. Interestingly, combination therapy specifically increased a macrophage subset that overexpressed Ch25h, an interferon-stimulated gene encoding for the oxysterol lipid 25-hydroxycholesterol (25-HC). Preliminary results from in vitro studies demonstrated that 25-HC restricted T cell proliferation and tumor cell cytolysis, suggesting its immunosuppressive function. In summary, our study reveals the impact of ATL-DC + aPD1 combination therapy on the immune microenvironment of GBM and a potential maladaptive mechanism mediated by 25-HC. Future studies will seek to exploit these targets therapeutically to develop more effective GBM treatment regimens.

Enhancing cholangiocarcinoma immunotherapy with adoptive T cells targeting HLA-restricted neoantigen peptides derived from driver gene mutations

Precision immunotherapy, driven by genomic and bioinformatic advancements, has emerged as a promising and viable approach to combat cancer. Targeting neoantigens offers the advantage of specific immune responses with minimal off-tumor toxicity. In this study, we investigated the potential of adoptive T cells activated by HLA-restricted neoantigen peptides from driver gene mutations for treating cholangiocarcinoma (CCA), a highly aggressive cancer with poor prognosis and high mortality rates. Through whole exome sequencing of CCA cell lines, KKU-213A and KKU-100, we identified mutations in common driver genes and predicted corresponding HLA-restricted peptides. Peptides from KRAS, RNF43, and TP53 mutations exhibited strong binding affinity to HLA-A11, as validated through molecular docking and T2-cell binding assays. Dendritic cells (DCs) from healthy donors expressing HLA-A* 11:01, pulsed with individual or pooled peptides, showed comparable levels of costimulatory molecules (CD11c, CD40, CD86, and HLA-DR) to conventional DCs but higher expression of maturation markers, CD80 and CD86. Autologous HLA-A* 11:01-restricted T cells, activated by peptide-pulsed DCs, effectively lysed KKU-213A (HLA-A*11:01) cells, outperforming conventional tumor lysate-pulsed DCs. This effect was specific to HLA-A* 11:01-restricted T cells and not observed in KKU-100 (HLA-A*33:03) cells. Moreover, HLA-A* 11:01-restricted T cells exhibited elevated levels of IFN-gamma, granulysin, and granzyme B, indicating their potent anti-tumor capabilities. These findings underscore the specificity and efficiency of HLA-A* 11:01-restricted T cells targeting KRAS, RNF43, TP53 mutated CCA cells, and offer valuable insights for developing immunotherapeutic strategies and therapeutic peptide-vaccines for CCA treatment.

Engineering an active immunotherapy for personalized cancer treatment and prevention of recurrence.

Breast cancer has been shown to be resistant to immunotherapies. To overcome this challenge, we developed an active immunotherapy for personalized treatment based on a smart nanovesicle. This is achieved by anchoring membrane-bound bioactive interleukin 2 (IL2) and enriching T cell-promoting costimulatory factors on the surface of the dendritic cell-derived small extracellular vesicles. This nanovesicle also displays major histocompatibility complex-bound antigens inherited from tumor lysate-pulsed dendritic cell. When administrated, the surface-bound IL2 is able to guide the nanovesicle to lymphoid organs and activate the IL2 receptor on lymphocytes. Furthermore, it is able to perform antigen presentation in the replacement of professional antigen-presenting cells. This nanovesicle, named IL2-ep13nsEV, induced a strong immune reaction to rescue 50% of the mice in our humanized patient-derived xenografts, sensitized cancer cells to immune checkpoint inhibitor treatment, and prevented the recurrence of resected tumors. This paradigm presents a feasible strategy for the treatment and prevention of metastatic breast cancer.

Differential centrifugation enhances the anti-tumor immune effect of tumor lysate-pulsed dendritic cell vaccine against glioblastoma*

Abstract Objective This study aimed to improve the antitumor immunocompetence of a tumor lysate-pulsed dendritic cell (DC) vaccine through differential centrifugation and provide a theoretical basis for its clinical application in glioblastoma. Methods Peripheral blood mononuclear cells were extracted using Ficoll-Paque PLUS and induced into mature DCs in vitro with a cytokine cocktail. The modified tumor lysate was generated by differential centrifugation. The maturity markers of DCs in each group, namely the modified tumor lysate, tumor lysate, and negative and positive control groups, were assessed using flow cytometry. Furthermore, their ability to stimulate lymphocyte proliferation and in vitro antitumor effects were assessed using Cell Trace TM CFSE. IFN-γ secretion levels were measured with ELISA. Intracellular reactive oxygen species were measured using 2′,7′-dichlorofluorescein diacetate (DCFDA) staining. The results were statistically analyzed using an unpaired Student’s t-test and were considered significant at P < 0.05. Results Compared with tumor lysate-pulsed DCs, modified tumor lysate-pulsed DCs had a higher expression of maturity markers: CD1a (7.38 ± 0.53% vs. 4.47 ± 0.75%) and CD83 (19.81 ± 4.09% vs. 9.64 ± 1.50%), were better capable of stimulating lymphocyte proliferation [proliferation index (PI): 8.54 ± 0.16 vs. 7.35 ± 0.05], secreting IFN-γ, and inducing stronger in-vitro cytotoxic T lymphocyte (CTL) cytotoxicity against glioblastoma cells. In addition, we found that the level of ROS in modified tumor lysate-pulsed DCs was lower than that in tumor lysate-pulsed DCs. Conclusion Differential centrifugation of tumor lysates can improve the antitumor immunocompetence of DC vaccines, and reactive oxygen species may be the key to affecting DC function in the whole tumor lysate.

Choroid Plexus-Infiltrating T Cells as Drivers of Murine Neuropsychiatric Lupus.

T cells are critical in the pathogenesis of systemic lupus erythematosus (SLE) in that they secrete inflammatory cytokines, help autoantibody production, and form autoreactive memory T cells. Although the contribution of T cells to several forms of organ-mediated damage in SLE has been previously demonstrated, the role of T cells in neuropsychiatric SLE (NPSLE), which involves diffuse central nervous system manifestations and is observed in 20-40% of SLE patients, is not known. Therefore, we conducted this study to evaluate how behavioral deficits are altered after depletion or transfer of T cells, to directly assess the role of T cells in NPSLE. MRL/lpr mice, an NPSLE mouse model, were either systemically depleted of CD4+ T cells or intracerebroventricularly injected with choroid plexus (CP)-infiltrating T cells and subsequently evaluated for alterations in neuropsychiatric manifestations. Our study end points included evaluation of systemic disease and assessment of central nervous system changes. Systemic depletion of CD4+ T cells ameliorated systemic disease and cognitive deficits. Intracerebroventricular injection of CP-infiltrating T cells exacerbated depressive-like behavior and worsened cognition in recipient mice compared with mice who received injection of splenic lupus T cells or phosphate buffered saline. Moreover, we observed enhanced activation in CP-infiltrating T cells when cocultured with brain lysate-pulsed dendritic cells in comparison to the activation levels observed in cocultures with splenic T cells. T cells, and more specifically CP-infiltrating antigen-specific T cells, contributed to the pathogenesis of NPSLE in mice, indicating that, in the development of more targeted treatments for NPSLE, modulation of T cells may represent a potential therapeutic strategy.

A randomised phase II clinical trial of low-dose cyclophosphamide and transarterial chemoembolization (TACE) with or without vaccination with dendritic cells (DC) pulsed with HepG2 lysate ex vivo in patients with hepatocellular carcinoma (HCC): The ImmunoTACE trial.

4012 Background: A previous study by our group using autologous monocyte-derived DC pulsed ex vivo with HepG2 cell lysate showed some clinical benefit with evidence of antigen-specific T-cell responses in patients with advanced HCC. The current trial reports the activity of this vaccine in combination with TACE in patients with HCC. All patients also received low-dose cyclophosphamide to deplete regulatory T cells and thereby enhance vaccination. Methods: Patients with intermediate stage HCC (performance status 0-2, Child Pugh A/B7) were randomised 1:1 to TACE plus low-dose cyclophosphamide (Group 1) or TACE plus low-dose cyclophosphamide plus dendritic cell vaccination (Group 2). Cyclophosphamide was administered on Day 1 and 29 followed by TACE on Day 31 (+/- DC infusion), with further cyclophosphamide on Days 60, 90 and 120 (+/- additional DC infusions on Days 62, 92 and 122). The primary endpoint was progression free survival (PFS) by RECIST v1.1. Secondary endpoints included radiological response by RECIST v1.1, PFS and radiological response according to modified (m) RECIST, overall survival (OS), immune response and toxicity. Target recruitment was 48 evaluable patients (24 patients in each arm) to detect a 20% increase in PFS rate at 1 year (30% vs 50%) with a relaxed one-sided statistical significance level of 20% and 80% power using a logrank test. Results: Between March 2016 and October 2019, 55 patients from 3 UK centres were randomised of whom 48 were evaluable (24 each arm). Median PFS by RECIST criteria was significantly longer in Group 2 compared to Group 1 (18.6 vs 10.4 months: hazard ratio (HR) 0.43, 80% CI -∞-0.59; one-sided p = 0.02). Median PFS using mRECIST criteria showed a similar magnitude of benefit (18.6 vs 10.8 months: HR 0.48, 95% CI 0.22-1.02). Median OS was 25.7 months in Group 2 vs 21.5 months in Group 1 (HR 0.61, 95% CI 0.27-1.38). Group 2 showed a higher overall response rate (complete and partial response) by RECIST (54% vs 29%) and mRECIST (75% vs 54%) and a higher disease control rate (complete and partial response and stable disease) by RECIST (92% vs 67%) and mRECIST (88% vs 67%). Treatment with DC infusions was well tolerated; the most common adverse events were chills (30%), fatigue (22%) and nausea (22%), all of which were low grade. Immune response analyses are currently ongoing. Conclusions: The addition of tumour lysate pulsed DC infusions to treatment with TACE plus low-dose cyclophosphamide significantly increased PFS in patients with HCC. To the best of our knowledge, this is the first randomised study to demonstrate efficacy using DC in HCC. Further investigation of the role of DC infusions in the treatment of HCC are warranted but will need to take into account the current evolving immunotherapy landscape. Clinical trial information: 11889464.

Chemotherapy Can Synergize With Adoptive Immunotherapy to Inhibit Medulloblastoma Growth.

In the age of ever-increasing developments in targeted cancer treatments, new immune-based approaches for brain tumor therapy represent an attractive avenue. Despite encouraging pre-clinical data, results in patients have been sub-optimal, likely due to tumor-induced immune suppression and intrinsic resistance to immune attack. Chemotherapy and biologic agents may be able to disrupt these mechanisms and restore tumor sensitivity to immune attack. In this study, we explore whether a combination of gemcitabine and rapamycin can sensitize medulloblastoma cells to immunotherapy in vitro and in vivo. With the commercial medulloblastoma cell line, Daoy, we explored the concentrations of combinations of Gemcitabine with rapamycin needed to induce cytotoxicity. Next, we used flow cytometry to assess the cytotoxicity of chemotherapy-treated Daoy cells with the addition of anti-tumor T-cells, generated from naive T-cells stimulated in the presence of Daoy lysate-pulsed dendritic cells. Then, we examined the efficacy of chemotherapy alone versus chemotherapy plus immunotherapy in tumor growth inhibition of subcutaneous medulloblastoma xenografts. Rapamycin alone at <1,000 nM had moderate activity against Daoy cells in vitro and IC50 was >1,000 nM. Gemcitabine had a 3-day IC50 alone of 10 nM but in combination with 100 nM rapamycin, it decreased to 1 nM, suggesting increased cytotoxicity with combined therapy. Stimulated T-cells mediated in-vitro cytotoxicity, although background cytotoxicity of unstimulated "naïve" T-cells was also significant. Finally, established subcutaneous Daoy cell xenografts in SCID mice were treated with chemotherapy alone or chemotherapy plus adoptive immunotherapy (stimulated and non-stimulated). Gemcitabine and rapamycin alone significantly slowed tumor growth, but the addition of immunotherapy further augmented inhibition. Combining immunotherapy and chemo-biologic therapy inhibit medulloblastoma cell and xenograft growth, and may offer an effective treatment for patients with medulloblastoma.

49P A phase II randomized trial with adjuvant autologous tumor lysate-pulsed dendritic cells (DC) in resected stage III-IV melanoma patients: Preliminary immunological results

49P A phase II randomized trial with adjuvant autologous tumor lysate-pulsed dendritic cells (DC) in resected stage III-IV melanoma patients: Preliminary immunological results

Contribution of pre-existing neoantigen-specific T cells to a durable complete response after tumor-pulsed dendritic cell vaccine plus nivolumab therapy in a patient with metastatic salivary duct carcinoma

ABSTRACT Although immune checkpoint inhibitors (ICIs) have emerged as new therapeutic options for refractory cancer, they are only effective in select patients. Tumor antigen-pulsed dendritic cell (DC) vaccine therapy activates tumor-specific cytotoxic T lymphocytes, making it an important immunotherapeutic strategy. Salivary ductal carcinoma (SDC) carries a poor prognosis, including poor long-term survival after metastasis or recurrence. In this study, we reported a case of refractory metastatic SDC that was treated with a tumor lysate-pulsed DC vaccine followed by a single injection of low-dose nivolumab, and a durable complete response was achieved. We retrospectively analyzed the immunological factors that contributed to these long-lasting clinical effects. First, we performed neoantigen analysis using resected metastatic tumor specimens obtained before treatment. We found that the tumor had 256 non-synonymous mutations and 669 class I high-affinity binding neoantigen peptides. Using synthetic neoantigen peptides and ELISpot analysis, we found that peripheral blood mononuclear leukocytes cryopreserved before treatment contained pre-existing neoantigen-specific T cells, and the cells obtained after treatment exhibited greater reactivity to neoantigens than those obtained before treatment. Our results collectively suggest that the rapid and long-lasting effect of this combination therapy in our patient may have resulted from the presence of pre-existing neoantigen-specific T cells and stimulation and expansion of those cells following tumor lysate-pulsed DC vaccine and ICI therapy.

A Polysaccharide From the Whole Plant of Plantago asiatica L. Enhances the Antitumor Activity of Dendritic Cell-Based Immunotherapy Against Breast Cancer.

Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) that mediate T-cell immune responses. Breast cancer is one of the most commonly diagnosed diseases and its mortality rate is higher than any other cancer in both humans and canines. Plantain polysaccharide (PLP), extracted from the whole plant of Plantago asiatica L., could promote the maturation of DCs. In this research, we found that PLP could upregulate the maturation of DCs both in vitro and in vivo. PLP-activated DCs could stimulate lymphocytes’ proliferation and differentiate naive T cells into cytotoxic T cells. Tumor antigen-specific lymphocyte responses were enhanced by PLP and CIPp canine breast tumor cells lysate-pulsed DCs, and PLP and CIPp-cell-lysate jointly stimulated DCs cocultured with lymphocytes having the great cytotoxicity on CIPp cells. In the 4T1 murine breast tumor model, PLP could control the size of breast tumors and improve immunity by recruiting DCs, macrophages, and CD4+ and CD8+ T cells in the tumor microenvironment. These results indicated that PLP could achieve immunotherapeutic effects and improve immunity in the breast tumor model.

Oxidized curdlan activates dendritic cells and enhances antitumor immunity

Curdlan activates dendritic cells (DCs) and enhances DC-based antitumor immunity. However, hydrophobicity and heterogeneity of curdlan particulates hinder perfect binding of curdlan to dectin-1 receptor, resulting in the reduced activation of antigen presenting cells and limited antitumor effects. Herein, we synthesized partially oxidized curdlan derivative (β-1,3-polyglucuronic acid, denote PGA). PGA-45 polymer, the reaction product prepared from curdlan by oxidation with 4-acetamido-TEMPO/NaClO/NaClO2 systems under acid conditions for 45 min, activated DCs, induced the expression of co-stimulatory molecules and cytokines, and promoted allogenic T cell proliferation as well as the expression of IL-2. Mechanistically, PGA-45 polymer strongly enhanced phosphorylation of IKK-β and reduced the expression of phosphorylated Akt, suggesting that PGA-45 may activate multiple cell surface receptors such as TLR4 and dectin-1. Administration of tumor lysate pulsed DCs pre-treated with PGA-45 particles induced strong antitumor activity in B16F10 melanoma model. Our data suggest that PGA-45 have strong adjuvant effects for anti-cancer immunity and the design of PGA polymers may provide insights in the development of novel adjuvants for cancer immunotherapy.

Personalized cancer vaccine strategy elicits polyfunctional T cells and demonstrates clinical benefits in ovarian cancer

T cells are important for controlling ovarian cancer (OC). We previously demonstrated that combinatorial use of a personalized whole-tumor lysate-pulsed dendritic cell vaccine (OCDC), bevacizumab (Bev), and cyclophosphamide (Cy) elicited neoantigen-specific T cells and prolonged OC survival. Here, we hypothesize that adding acetylsalicylic acid (ASA) and low-dose interleukin (IL)-2 would increase the vaccine efficacy in a recurrent advanced OC phase I trial (NCT01132014). By adding ASA and low-dose IL-2 to the OCDC-Bev-Cy combinatorial regimen, we elicited vaccine-specific T-cell responses that positively correlated with patients’ prolonged time-to-progression and overall survival. In the ID8 ovarian model, animals receiving the same regimen showed prolonged survival, increased tumor-infiltrating perforin-producing T cells, increased neoantigen-specific CD8+ T cells, and reduced endothelial Fas ligand expression and tumor-infiltrating T-regulatory cells. This combinatorial strategy was efficacious and also highlighted the predictive value of the ID8 model for future ovarian trial development.

90K predicts the prognosis of glioma patients and enhances tumor lysate-pulsed DC vaccine for immunotherapy of GBM in vitro.

Objectives: This study aimed to investigate the relationship between 90K expression with glioma malignancy and prognosis. Additionally, the enhancement effect of 90K in the Dendritic cell (DC) vaccine for Immunotherapy of glioblastoma (GBM) was evaluated in vitro.Methods: The expression of 90K protein in glioma tissues was detected by western blot. The relationship between the 90K expression and the tumor grade as well as the prognosis of patients was further analyzed by mining TCGA and CGGA database. The concentration of IL-12p70 and IL-10 was detected by ELISA. T lymphocyte proliferation and lethal effect of cytotoxic T cell (CTL) were detected by CCK-8.Results: The expression of 90K was significantly higher in glioma than normal tissue and increased with tumor grade (P< 0.05). Higher 90K expression was observed in IDH wildtype glioma than IDH mutant and predicted worse overall survival for glioma patients. The concentration of IL-12p70 and IFN-γ was the highest in the Apoptosis U251-90K-DC group, in which group the ability to kill U251 cells by CTL was also the strongest.Conclusion: 90K was a useful biomarker for glioma malignancy and patient prognosis. The appearance of 90K enhanced the effect of Apoptosis U251-DC vaccine for immunotherapy of GBM.

CTIM-06. DENDRITIC CELL VACCINE STUDY FOR RECURRENT, HIGH-GRADE GLIOMA: TISSUE-BASED RNA SEQUENCING AND SERUM CYTOKINE LEVELS AS POTENTIAL BIOMARKERS

Abstract BACKGROUND Glioblastoma is the most common primary malignant brain tumor. Despite multimodality treatment approach, median progression-free survival (PFS) is only 8 months, median overall-survival (OS) 14 months and 5-year survival rate of under 10%. Dendritic cells (DCs) are the professional antigen presenting cells of the immune system. The rationale for sensitizing dendritic cells to a pool of non-selected tumor antigens is based on the marked heterogeneity present within glioblastoma tumor cells. METHODS Phase 1/feasibility study of DC vaccine for recurrent high-grade glioma was conducted. Pooled, non-selected tumor antigens collected via tumor cell lysate were used for DC sensitization. RNA sequencing analysis was performed on all tumor samples. Cytokine levels in serum were detected using a Luminex cytokine panel. RESULTS A total of 20 patients were enrolled onto this study (median age 58yrs, range: 39–74, 65% male). Pathology showed WHO grade IV glioblastoma in 14 (70%) and grade III anaplastic astrocytoma in 6 (30%) patients. IDH wild type in 19 (95%) patients. Treatment emergent adverse events (all grades, regardless of attribution) occurred in more than 15% of the patients (20% fatigue, 15% dizziness, 15% headache, none leading to treatment discontinuation). There were five grade 3–4 and none grade 5 events. One grade 4 event (seizure) probable related to investigational treatment leading to treatment discontinuation. Four grade 3 events (dysphasia, possible related; intracranial hemorrhage unrelated; muscle weakness, unlikely related and hematoma, unrelated). Median PFS was 3.8 months. Median OS was 11 months. RNA sequencing in tumor samples and correlation with cytokine levels in serum is currently been analyzed. CONCLUSION Tumor lysate pulsed DC vaccination demonstrates acceptable safety and tolerability in high-grade glioma patients. Evaluations of integrating molecular profiling RNA sequencing information and cytokine levels to identify potential subset of patients with significant clinical benefit will be provided.

CTIM-18. DENDRITIC CELL VACCINATION IN CONJUNCTION WITH ADJUVANT TLR-3 AGONIST ADMINISTRATION ENHANCES PRO-INFLAMMATORY IMMUNE RESPONSES AND IS ASSOCIATED WITH EXTENDED SURVIVAL IN MALIGNANT GLIOMA PATIENTS

Abstract Despite recent advances with immunotherapy in other tumor types, malignant glioma patients have not shown a therapeutic benefit, potentially due to the insufficient activation of an immune response. We and others have documented immune responses and extended survival following dendritic cell (DC) vaccination in small clinical trials. In this Phase II clinical trial, we randomized malignant glioma patients to receive autologous tumor lysate pulsed DC vaccination with and without adjuvant toll-like receptor (TLR) agonists. Treatment with TLRs in cancer patients has been shown to activate the innate immune response by inducing the expression of pro-inflammatory factors and cytokines. Twenty-three patients with WHO grade III or IV glioma received three intradermal injections of autologous tumor lysate-pulsed DC on days 0, 14, and 28 in conjunction with either a placebo adjuvant, TLR-7 agonist (Resiquimod), or TLR-3 agonist (Poly ICLC). We observed a difference in survival for the DC-vaccinated patients who received adjuvant Poly ICLC treatment of 54 months over adjuvant placebo (20 months) and adjuvant Resiquimod (28 months) groups (P = 0.04). The patient cohorts were balanced for WHO grade, IDH mutation, and MGMT methylation status. Mass cytometry (CyTOF) analysis of patient peripheral blood mononuclear cells (PBMCs) showed increased levels of the monocyte populations CD14+CD16- and CD14dimCD16+ after Poly ICLC treatment. In addition, gene expression analysis of the PBMC populations using single cell RNA sequencing demonstrated increased expression of pro-inflammatory genes after adjuvant Poly ICLC and Resiquimod treatment. Nonetheless, a greater fold change increase and a larger pro-inflammatory repertoire was observed in the Poly ICLC group. Overall, these findings demonstrate that adjuvant Poly ICLC increases the number of circulating monocytes and induces a large pro-inflammatory response, which may account for the survival differences observed over adjuvant Resiquimod and placebo.

Immunological responses through miRNA signatures in GBM plasma extracellular vesicles from patients receiving experimental immunotherapy

Abstract Glioblastoma (GBM) patients have a median survival of 15 months despite aggressive treatment. Treatment-related pseudo-progression confounds outcome assessment by MRI, particularly in patients receiving immunotherapy. Extracellular vesicles (EVs) contain tumor-specific miRNA that could serve as a liquid biopsy to distinguish true progression from treatment-related pseudo-progression. Small plasma EVs were isolated by serial density gradient ultracentrifugation from 20 newly-diagnosed GBM patients enrolled in a clinical trial of allogeneic tumor lysate-pulsed autologous dendritic cell (DC) vaccination. Short non-coding RNA sequencing was performed for each patient at three time points (TP): pre-vaccine (TP1), post-initial vaccine (TP2), and at end of treatment (TP3). Ingenuity Pathway Analysis from 31 differentially expressed miRNAs across time points (p-value &amp;lt;0.05, |logFC|&amp;gt;1) revealed that glioma-related signaling pathways previously seen in small EVs from glioma patients compared to normal donors were replicated when comparing patients at the beginning of the treatment (TP1-vs-TP2) and between early to late time points (TP1-vs-TP3). In addition, pathways corresponding to immunological responses typically seen in GBM patients undergoing treatment, such as IL-10, IL-6, and Toll-like Receptor (TLR) signaling, were exclusively found after the initial vaccine towards the end of treatment (TP2-vs-TP3). In conclusion, signaling pathways derived from differentially expressed miRNAs across time points suggest that as patients progress through treatment, consistent differences in plasma small EVs miRNA expression profile and immunological responses could be utilized as predictors of treatment response.

A phase I trial of surgical resection with Gliadel Wafer placement followed by vaccination with dendritic cells pulsed with tumor lysate for patients with malignant glioma

High grade gliomas are associated with poor prognosis and high mortality. Conventional treatments and management of high grade gliomas have shown little improvement in 5-year overall survival. This phase I trial evaluated the safety, immunogenicity, and potential synergy of surgical resection with Gliadel Wafer implantation, followed by autologous tumor lysate-pulsed dendritic cell (DC) vaccine in patients with malignant glioma. Primary end points of this study were safety and surrogate markers of immunogenicity, overall survival, and progression free survival. Following surgical resection, Gliadel Wafers were placed along the resection cavity. Patients subsequently received intradermal injections of autologous tumor lysate-pulsed DC vaccines 3 times at 2 week intervals. Treatment response was evaluated clinically and through MRI at regular intervals. Twenty-eight patients received Gliadel Wafers and DC vaccination: 11 newly diagnosed (8 glioblastoma [GBM], 2 anaplastic astrocytoma [AA], and 1 anaplastic oligodendroglioma [AO]) and 17 recurrent (15 GBMs, 1 AA, and 1 AO) high grade gliomas. Immunogenicity data was collected for 20 of the 28 patients. Five of 20 patients showed elevated IFN-γ responses following vaccination. Median progression-free survival and overall survival for all GBM patients in the trial from the start of vaccination were 3.6 months and 16.9 months respectively. Comparisons between vaccine responders and non-vaccine responders were not statistically significant. Adjuvant autologous dendritic cells pulsed with tumor-lysate following resection and Gliadel Wafer placement is safe, elicits modest immunogenicity and shows similar clinical outcomes in patients who had DC vaccination in previous studies.