Tumor Lysate-pulsed Dendritic Cell Vaccination Research Articles

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.

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.

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.

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-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.

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.

Abstract PR13: Adjuvant TLR-3 administration enhances proinflammatory immune responses and is associated with extended survival in glioblastoma patients treated with dendritic cell vaccination

Abstract Malignant glioma, associated with a poor prognosis, is the most common primary malignant brain tumor in adults. We and others have documented immune responses following dendritic cell (DC) vaccination as an active immunotherapeutic treatment for these patients. 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. TLRs are present on dendritic cells and serve to modulate immune responses. Twenty-three patients with WHO grade III or IV glioma were treated with three intradermal injections of autologous tumor lysate-pulsed DC on days 0, 14, and 28 followed by an adjuvant placebo, TLR-7 agonist (resiquimod), or TLR-3 agonist (Poly ICLC). Mass cytometry (CyTOF) was used to analyze immune cell populations of patient peripheral blood mononuclear cells (PBMC) before and following treatment. DC-vaccinated patients who received adjuvant Poly ICLC treatment had a significantly improved median survival of 54 months over placebo (11 months) and adjuvant resiquimod (17 months) groups (P&amp;lt;0.01). CyTOF and single-cell RNA sequencing (scRNseq) gene expression analysis of systemic PBMCs from patients demonstrated increased immune cell activation and expression of proinflammatory genes. Notably, the expansion of a heterogenous myeloid cell population was noted to correlate with increased survival, corroborating preclinical evidence that these cells modulate adaptive immunity in the tumor microenvironment. Overall, these findings suggest that adjuvant Poly ICLC treatment improves outcomes with autologous lysate-pulsed DC vaccine treatment via modulation of proinflammatory pathways. This abstract is also being presented as Poster B27. Citation Format: Joseph P. Antonios, Richard G. Everson, Aaron Mochizuki, Sara Khattab, Horacio Soto, Prashant Romiyo, Matthew Z. Sun, Diana Moughon, Emma Billingslea-Yoon, Sylvia Odesa, Gang Li, Eric Kawaguchi, Alex Salazar, William Yong, Jason Schlossman, Benjamin Ellingson, Anthony C. Wang, Timothy Cloughesy, Robert M. Prins, Linda M. Liau. Adjuvant TLR-3 administration enhances proinflammatory immune responses and is associated with extended survival in glioblastoma patients treated with dendritic cell vaccination [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr PR13.

ATIM-43. PLASMA EXTRACELLULAR VESICLE MIRNA SIGNATURES IN GBM PATIENTS RECEIVING AN EXPERIMENTAL IMMUNOTHERAPY

Abstract Patients with glioblastoma (GBM) have a median survival of 15 months despite aggressive treatment. Immunotherapies such as dendritic cell (DC) vaccines have modest clinical efficacy in small clinical trials. Treatment-related pseudo-progression confounds outcome assessment by MRI, particularly in patients receiving immunotherapy. Thus, there is a need for additional non-invasive methods to monitor treatment response. Extracellular vesicles (EVs), especially plasma exosomes, contain tumor-specific microRNA (miRNA) cargo that could serve as a liquid biopsy to distinguish true progression from treatment-related pseudo-progression. Plasma exosomes were isolated by serial density gradient ultracentrifugation from 20 newly diagnosed GBM patients enrolled in a clinical trial of allogeneic tumor lysate-pulsed autologous DC vaccination. Short non-coding RNA sequencing and bioinformatics analysis was performed for each patient at three time points (TP): pre-vaccine (TP1), post-initial vaccine (TP2), and at end of treatment (TP3). miRNA expression analysis revealed a total of 14 upregulated and 12 downregulated miRNAs across time points (p-value &lt; 0.05, |logFC| &gt;1), few of which have been previously reported to be differentially expressed in GBM. Interestingly, patients’ miRNA profile expression differed more at the beginning of treatment (e.g. TP1-vs-TP2) and at subsequent time points (e.g. TP2-vs-TP3). Ingenuity Pathway Analysis is in progress to identify pathways associated with immunotherapy treatment response in malignant gliomas. In conclusion, miRNA sequencing from GBM patients’ plasma exosomes enrolled in our DC clinical trial shows marked differential miRNA expression between time points. These results suggest that as patients progress through treatment, consistent differences in plasma exosomal miRNA expression profile can be identified that could be utilized as predictors of treatment response. Thus, plasma EVs may serve as a robust platform to monitor treatment outcome.

First results on the DCVax phase III trial: raising more questions than providing answers.

Recently, the long-awaited report on efficacy data of the DCVax trial has appeared in the Journal of Translational Medicine.1 Patients and physicians alike are eager to critically evaluate the first results of this important trial, which is one of the earliest major attempts to improve outcome in patients with glioblastoma using immunotherapy based on an autologous tumor lysate-pulsed dendritic cell vaccine (DCVax). It is not only one of the first randomized phase III trials of its kind, but in light of recent successes with cancer immunotherapies outside neuro-oncology, there is a huge interest in any form of immune treatment for glioma patients.

First results on survival from a large Phase 3 clinical trial of an autologous dendritic cell vaccine in newly diagnosed glioblastoma

BackgroundStandard therapy for glioblastoma includes surgery, radiotherapy, and temozolomide. This Phase 3 trial evaluates the addition of an autologous tumor lysate-pulsed dendritic cell vaccine (DCVax®-L) to standard therapy for newly diagnosed glioblastoma.MethodsAfter surgery and chemoradiotherapy, patients were randomized (2:1) to receive temozolomide plus DCVax-L (n = 232) or temozolomide and placebo (n = 99). Following recurrence, all patients were allowed to receive DCVax-L, without unblinding. The primary endpoint was progression free survival (PFS); the secondary endpoint was overall survival (OS).ResultsFor the intent-to-treat (ITT) population (n = 331), median OS (mOS) was 23.1 months from surgery. Because of the cross-over trial design, nearly 90% of the ITT population received DCVax-L. For patients with methylated MGMT (n = 131), mOS was 34.7 months from surgery, with a 3-year survival of 46.4%. As of this analysis, 223 patients are ≥ 30 months past their surgery date; 67 of these (30.0%) have lived ≥ 30 months and have a Kaplan-Meier (KM)-derived mOS of 46.5 months. 182 patients are ≥ 36 months past surgery; 44 of these (24.2%) have lived ≥ 36 months and have a KM-derived mOS of 88.2 months. A population of extended survivors (n = 100) with mOS of 40.5 months, not explained by known prognostic factors, will be analyzed further. Only 2.1% of ITT patients (n = 7) had a grade 3 or 4 adverse event that was deemed at least possibly related to the vaccine. Overall adverse events with DCVax were comparable to standard therapy alone.ConclusionsAddition of DCVax-L to standard therapy is feasible and safe in glioblastoma patients, and may extend survival.Trial registration Funded by Northwest Biotherapeutics; Clinicaltrials.gov number: NCT00045968; https://clinicaltrials.gov/ct2/show/NCT00045968?term=NCT00045968&rank=1; initially registered 19 September 2002

Autologous tumor lysate-pulsed dendritic cell vaccination therapy after resection of stage IIA (T2N0, T3N0) esophageal cancer: An analysis of immunological responses.

96 Background: Immunotherapy using active immunization of tumor associated antigens has been expected to improve the prognosis of malignant tumor patients. We conducted phase I trial to investigate safety and efficacy of autologous tumor lysate-pulsed dendritic cell (DC) vaccination therapy after resection for esophageal cancer. We already reported the clinical results for enrolled 11 patients. All patients completed the protocol therapy and no treatment-related adverse events more than grade 3 was observed (primary endpoint), and recurrence rate within 2 years was 18% (n = 2). In this study, we further analyzed the immune responses in vaccinated patients. Methods: Patients with stage IIA (T2N0 or T3N0, UICC TNM classification 6th edition) esophageal cancer after curative (R0) resection were eligible. Tumor lysate we used for DC vaccines potentially contains mutated proteins (neoantigens) derived from somatic mutations. Patients received DC vaccines (more than 5.0×106 cells) 6 times every 2 weeks. We evaluated neutrophil-to-lymphocyte ratio (NLR) in each patient from CRF data. We identified candidate neoantigens by next generation sequencing and MHC class I binding prediction algorism. We screened the immune responses against those neoantigens using HLA transgenic mice and healthy human PBMCs. Results: Absolute lymphocyte counts, absolute neutrophil counts and NLR were not specifically different between patients with or without recurrence. For specific immune responses, we observed the reactivity in 6 out of 59 candidate neoepitopes identified from two patients in HLA transgenic mouse system. Two out of 6 peptides displayed reactivity in human healthy PBMCs. Conclusions: We analyzed immune responses to predicted candidate neoantigens. We are now investigating immune landscape in the tumor using RNA sequencing data and its relevance to the antigen responses. Clinical trial information: UMIN000002837.

Detection of immune responses after immunotherapy in glioblastoma using PET and MRI

Contrast-enhanced MRI is typically used to follow treatment response and progression in patients with glioblastoma (GBM). However, differentiating tumor progression from pseudoprogression remains a clinical dilemma largely unmitigated by current advances in imaging techniques. Noninvasive imaging techniques capable of distinguishing these two conditions could play an important role in the clinical management of patients with GBM and other brain malignancies. We hypothesized that PET probes for deoxycytidine kinase (dCK) could be used to differentiate immune inflammatory responses from other sources of contrast-enhancement on MRI. Orthotopic malignant gliomas were established in syngeneic immunocompetent mice and then treated with dendritic cell (DC) vaccination and/or PD-1 mAb blockade. Mice were then imaged with [18F]-FAC PET/CT and MRI with i.v. contrast. The ratio of contrast enhancement on MRI to normalized PET probe uptake, which we term the immunotherapeutic response index, delineated specific regions of immune inflammatory activity. On postmortem examination, FACS-based enumeration of intracranial tumor-infiltrating lymphocytes directly correlated with quantitative [18F]-FAC PET probe uptake. Three patients with GBM undergoing treatment with tumor lysate-pulsed DC vaccination and PD-1 mAb blockade were also imaged before and after therapy using MRI and a clinical PET probe for dCK. Unlike in mice, [18F]-FAC is rapidly catabolized in humans; thus, we used another dCK PET probe, [18F]-clofarabine ([18F]-CFA), that may be more clinically relevant. Enhanced [18F]-CFA PET probe accumulation was identified in tumor and secondary lymphoid organs after immunotherapy. Our findings identify a noninvasive modality capable of imaging the host antitumor immune response against intracranial tumors.

Safety, efficacy, and immunogenicity of autologous tumor lysate-pulsed dendritic cell vaccination therapy after resection of stage IIA (T2N0, T3N0) esophageal cancer: A phase I trial.

153 Background: Immunotherapy using active immunization of tumor associated antigens has been expected to improve the prognosis of malignant tumor patients. We conducted a phase I trial to investigate safety and efficacy of autologous tumor lysate-pulsed dendritic cell (DC) vaccination therapy after resection for esophageal cancer. Methods: Patients (pts) with stage IIA (T2N0 or T3N0, UICC TNM classification 6th edition) esophageal cancer after curative (R0) resection, ECOG PS 0-2, 20 years or more and adequate organ function were eligible. We used tumor lysate for DC vaccines; the lysates potentially contain mutated proteins or neoantigens derived from somatic mutations in each patient's tumor. Patients received DC vaccines (more than 5.0×106 cells) 6 times every 2 weeks. Primary endpoint was safety and secondary endpoint were efficacy including recurrence rate and immunological responses. Results: A total of 11 pts (median age: 71 years) were enrolled until the trial stopped on March 31, 2016, because standard of therapy for objective patients had changed. No treatment related adverse events of grade 3 or 4 were reported. Recurrence rate within 2 years were 18% (n=2). We performed whole-exome sequence of tumors and adjacent normal tissues with 5 pts, and identified mutated peptide derived from their tumor specific missense mutations. To examine immune responses against those predicted candidate neoepitopes, we immunized HLA-A2 transgenic mice (HHD-II mice) with the synthesized peptides from the 2 pts harboring HLA-A2. Conclusions: This study showed that autologous tumor lysate-pulsed DC vaccination therapy is safe and have expectation of recurrence risk reduction after resection of T2N0 or T3N0 esophageal cancer. Clinical trial information: UMIN000002837.

Postoperative dendritic cell vaccine plus activated T-cell transfer improves the survival of patients with invasive hepatocellular carcinoma

The recurrence rate after surgery in patients with hepatocellular carcinoma (HCC) is very high, while prognosis is quite poor. However, there is no standard treatment to prevent recurrence of HCC after a curative operation. In this study, we investigated the clinical utilization of an autologous tumor lysate-pulsed dendritic cell vaccine plus ex vivo activated T cell transfer (ATVAC) in an adjuvant setting for postoperative HCC as a non-randomized controlled trial. Ninety-four patients with invasive HCC received informed consent information regarding the study, and 42 opted to have the ATVAC after surgery. Their recurrence-free survival (RFS) and overall survival (OS) were measured after 5 years and compared with those of 52 patients who selected to have the curative operation alone. The median RFS and OS were 24.5 months and 97.7 months in the patients receiving adjuvant ATVAC and 12.6 months and 41.0 months in the group receiving surgery alone (P = 0.011 and 0.029). In the treated group, patients with positive delayed-type hypersensitivity (DTH) had a better prognosis (RFS P = 0.019, OS P = 0.025). No adverse events of grade 3 or more were observed.A postoperative dendritic cell vaccine plus activated T cell transfer would be a feasible and effective treatment for preventing recurrence in HCC patients and achieving long-term survival especially in DTH positive patients.

Carthamus tinctoriusEnhances the Antitumor Activity of Dendritic Cell Vaccines via Polarization toward Th1 Cytokines and Increase of Cytotoxic T Lymphocytes

Carthamus tinctorius (CT), also named safflower, is a traditional Chinese medicine widely used to improve blood circulation. CT also has been studied for its antitumor activity in certain cancers. To investigate the effects of CT on the dendritic cell (DC)-based vaccine in cancer treatment, cytokine secretion of mouse splenic T lymphocytes and the maturation of DCs in response to CT were analyzed. To assess the antitumor activity of CT extract on mouse CD117+ (c-kit)-derived DCs pulsed with JC mammal tumor antigens, the JC tumor was challenged by the CT-treated DC vaccine in vivo. CT stimulated IFN-γ and IL-10 secretion of splenic T lymphocytes and enhanced the maturation of DCs by enhancing immunological molecule expression. When DC vaccine was pulsed with tumor antigens along with CT extract, the levels of TNF-α and IL-1β were dramatically increased with a dose-dependent response and more immunologic and co-stimulatory molecules were expressed on the DC surface. In addition, CT-treated tumor lysate-pulsed DC vaccine reduced the tumor weight in tumor-bearing mice by 15.3% more than tumor lysate-pulsed DC vaccine without CT treatment. CT polarized cytokine secretion toward the Th1 pathway and also increased the population of cytotoxic T lymphocytes ex vivo. In conclusion, CT activates DCs might promote the recognition of antigens and facilitate antigen presentation to Th1 immune responses.

Abstract 4749: Genetic signature of malignant glioma is associated with responsiveness to autologous tumor lysate-pulsed dendritic cell vaccination + TLR agonists

Abstract Purpose: We previously reported that the toll-like receptor (TLR)-7 agonist, imiquimod, could enhance dendritic cell activation and migration, as well as stimulate T cell-mediated anti-tumor immune responses in a murine glioma model. To translate these findings, a Phase I clinical trial was initiated to evaluate this strategy for its feasibility, safety, and induction of immune responses in patients with malignant glioma. Herein, we report the results of this phase I, dose escalation clinical trial using autologous tumor lysate-pulsed dendritic cell (DC) vaccination, supplemented with the toll-like receptor agonists, imiquimod or poly ICLC, for patients with malignant glioma. Patients and Methods: Twenty-two glioblastoma patients (15 newly diagnosed, 7 recurrent) were enrolled into a multi-cohort dose-escalation study and treated with 1, 5, or 10 million autologous DC pulsed with tumor lysate. Three biweekly intradermal vaccinations were initially administered. Subsequently, patients received booster vaccinations together with imiquimod or poly ICLC every three months until tumor progression or lysate was depleted. Patients were monitored for adverse events, survival, and immune responses. For patients with available pre-treatment tumor and/or PBMC, microarray-based hierarchical clustering, lymphocyte immunohistochemistry, FACS and cytokine bead arrays were performed for monitoring purposes. Results: DC vaccinations were not associated with any evidence of dose-limiting toxicity or serious adverse effects. The median survival was 35 months for newly diagnosed patients and 11 months for recurrent patients, but still increasing as several patients are still alive at this point. Typical glioblastoma gene expression signatures, previously characterized by our group, were found. Contrary to our previous reports, however, patients with proliferative (Pro) or mesenchymal (Mes) signatures exhibited prolonged survival, while patients with pro-neural (PN) signatures did not. Pre-treatment Pro, Mes or Pro/Mes tumor sections had increased CD3 and CD8 positive cells within the tumor compared with PN tumor sections. Conclusions: Pro, Mes or Pro/Mes gene expression signatures may identify patients with pre-existing immunity and who may benefit from immune-based vaccination studies. However, prospective studies will need to be performed to establish the validity of these findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4749.

A Pilot Study of Tumor Lysate-Pulsed Dendritic Cell Vaccine in Pediatric High-Risk Solid Tumor Patients Following HCT: Preliminary Results

Pediatric pts with metastatic or recurrent solid tumors often relapse even when remissions are consolidated with high dose chemotherapy and autologous hematopoietic cell transplant (HCT). We previously showed dendritic cell (DC) based tumor vaccines to be safe and produce anti-tumor responses in children with refractory solid tumors. Experimental evidence indicates that the period of lymphopenia that occurs after HCT may be an opportune time to improve the immune response to self tumor. Furthermore, a DC based tumor vaccine may be most effective when residual disease burden is minimal, as in the period immediately after HCT.

Phase Ib Trial Assessing Autologous, Tumor-pulsed Dendritic Cells as a Vaccine Administered With or Without IL-2 in Patients With Metastatic Melanoma

Twenty-four subjects with metastatic melanoma were treated on a randomized Phase Ib trial evaluating an autologous tumor lysate-pulsed dendritic cell (DC) vaccine with or without interleukin (IL)-2. The vaccine consisted of autologous DCs obtained from peripheral blood mononuclear cells (PBMCs) cultured in granulocyte macrophage-colony stimulating factor and IL-4 then pulsed with autologous tumor cell lysate and keyhole limpet hemocyanin (KLH). The primary end points of the trial were safety and immune response to vaccine. Subjects were randomized to vaccine administered every other week times 3, vaccine x 3 followed by low-dose IL-2, or vaccine x 3 followed by high-dose IL-2. Immune response was monitored pretreatment and at 2 and 4 weeks after the third vaccine administration. Disease evaluation was performed at 4 weeks after the third vaccination. Therapy was well tolerated with no local vaccine toxicity greater than grade 1 in any arm. IL-2 toxicity was as expected without additional toxicity from the addition of IL-2 to vaccine. Immune response defined as delayed-type hypersensitivity, PBMC interferon-gamma enzyme-linked immunosorbent spot, and PBMC proliferation, to both autologous tumor and KLH were detected in all arms. Interferon-gamma enzyme-linked immunosorbent spot response to KLH (7 of 10 patients) and autologous tumor (4 of 10 patients) were also detected in subjects with available vaccine draining lymph node cells. There were no differences in immune response between treatment arms. No clinical responses were seen. Autologous tumor lysate-pulsed DC vaccine with or without IL-2 was well tolerated and immunogenic but failed to induce clinical response in patients with advanced melanoma.

Autologous tumor-lysate pulsed dendritic cell vaccination, together with the TLR-7 agonist 5% imiquimod, and serum pro- inflammatory cytokine levels in glioblastoma patients

11021 Background: Standard therapy for glioblastoma, which includes surgery followed by radiation and concurrent chemotherapy, creates a low tumor burden environment that could be ideal for immunotherapeutic approaches. We conducted a Phase I study to assess the safety and immunologic responses of tumor lysate-pulsed dendritic cell (DC) therapy plus topical imiquimod, in combination with standard radio-chemotherapy. Methods: Thirteen patients with newly diagnosed glioblastoma were immunized using autologous tumor lysate-pulsed DC at 2-week intervals, following completion of radio-chemotherapy. The primary clinical endpoint was 2-year survival. Patients without tumor progression subsequently received booster vaccinations combined with topical administration of the TLR-7 agonist, imiquimod. Immunologic responses to tumor antigens were monitored by tetramer staining and intracellular cytokine staining. Measurement of serum cytokine levels (TH1/TH2: IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10) was performed using cytometric bead arrays (CBA). Global gene expression profiling was also performed on pre-treatment tumor samples. Results: All immunizations were well tolerated, with only mild side effects attributable to the DC vaccination and imiquimod adjuvant. Increased levels of CD8+ T cells reactive against tumor antigens, (e.g., gp100, TRP-2, her-2, survivin, and CMV antigens), were detected in 5 patients. CBA measurement of serum cytokine levels revealed small, but detectable levels of TNF-α following the initial DC vaccination. Robust increases in serum TNF-α and IL-6 were observed after booster vaccinations, temporally correlating with imiquimod administration. Clinically, 8 of the 13 patients have progressed, and 6 of those have died. The median PFS to date is 13.9 mos. and median OS is 33.8 mos. This compares favorably to the published literature, with a median PFS of 6.9 mos and OS of 14.6 mos. Conclusions: This study demonstrates the safety and clinical/immunologic effects of an autologous tumor lysate-pulsed DC vaccine for patients with newly diagnosed glioblastoma. Serum TNFα responses may portend therapeutic anti-tumor immune responses to DC vaccination. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Northwest Biotherapeutics, Inc. Northwest Biotherapeutics, Inc. Northwest Biotherapeutics, Inc.

Dendritic cell vaccination in combination with TLR-7 agonist, imiquimod, following radio-chemotherapy for newly diagnosed glioblastoma

2021 Background: Standard therapy for glioblastoma, which includes surgery followed by radiation and concurrent chemotherapy, creates a low tumor burden environment that could be ideal for immunotherapeutic approaches. We conducted a Phase I study to assess the safety and immunologic responses of tumor lysate-pulsed dendritic cell (DC) therapy plus topical imiquimod, in combination with standard radio-chemotherapy. Methods: Thirteen patients with newly diagnosed glioblastoma were immunized using autologous tumor lysate-pulsed DC. Each patient initially received 3 immunizations at 2-week intervals, following completion of a 6-week course of radio- chemotherapy. Four patients received 1 million DC, 4 received 5 million, and 5 received 10 million DC per immunization. Patients without tumor progression subsequently received booster vaccinations combined with topical administration of the TLR-7 agonist imiquimod. Immunologic responses to tumor antigens were monitored by HLA-restricted tetramer staining, CTL assays, and quantitation of T-regulatory cells. Clinical tumor growth was monitored by brain MRI scans every 2 months, and the primary clinical endpoint was 2-year survival. Results: All immunizations were well tolerated, with only mild side effects attributable to the DC vaccination and imiquimod adjuvant. Increased levels of CD8+ T cells reactive against tumor antigens, (e.g., gp100, TRP-2, her-2, survivin, and CMV antigens), were detected in 5 patients. Median PFS and OS have not been reached in this trial. To date, 6 of the 13 patients have progressed, and 4 of those have died. The median PFS to date is 18.1 mos. and median OS is 33.8 mos. This compares favorably with controls from the published literature, with a median PFS of 6.9 mos and OS of 14.6 mos. Conclusions: This study demonstrates the safety and clinical/immunologic effects of an autologous tumor lysate-pulsed DC vaccine for patients with newly diagnosed glioblastoma. The adjunctive use of the TLR-7 agonist imiquimod with DC vaccination appears to be non-toxic, and deserves further study. We demonstrate that this active immunotherapy strategy can generate antigen-specific immunologic responses in brain tumor patients following standard radio-chemotherapy. No significant financial relationships to disclose.