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.

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.

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.

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.

Glycan modification of glioblastoma-derived extracellular vesicles enhances receptor-mediated targeting of dendritic cells

ABSTRACTGlioblastoma is the most prevalent and aggressive primary brain tumour for which total tumour lysate-pulsed dendritic cell vaccination is currently under clinical evaluation. Glioblastoma extracellular vesicles (EVs) may represent an enriched cell-free source of tumour-associated (neo-) antigens to pulse dendritic cells (DCs) for the initiation of an anti-tumour immune response. Capture and uptake of EVs by DCs could occur in a receptor-mediated and presumably glycan-dependent way, yet the glycan composition of glioblastoma EVs is unknown. Here, we set out to characterize the glycocalyx composition of glioblastoma EVs by lectin-binding ELISA and comprehensive immunogold transmission electron microscopy (immuno-TEM). The surface glycan profile of human glioblastoma cell line-derived EVs (50–200 nm) was dominated by α-2,3- and α-2,6 linked sialic acid-capped complex N-glycans and bi-antennary N-glycans. Since sialic acids can trigger immune inhibitory sialic acid–binding Ig-like lectin (Siglec) receptors, we screened for Siglec ligands on the EVs. Glioblastoma EVs showed significant binding to Siglec-9, which is highly expressed on DCs. Surprisingly, however, glioblastoma EVs lack glycans that could bind Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), a receptor that mediates uptake and induction of CD4+ and CD8+ T cell activation. Therefore, we explored whether modification of the EV glycan surface could reduce immune inhibitory Siglec binding, while enhancing EV internalization by DCs in a DC-SIGN dependent manner. Desialylation with a pan-sialic acid hydrolase led to reduction of sialic acid expression on EVs. Moreover, insertion of a high-affinity ligand (LewisY) for DC-SIGN resulted in a four-fold increase of uptake by monocyte-derived DCs. In conclusion, we show that the glycocalyx composition of EVs is a key factor of efficient DC targeting and that modification of the EV glycocalyx potentiates EVs as anti-cancer vaccine.

Antigen-loaded dendritic cells triggers a specific cytotoxic Tlymphocytes immune response against hepatocellular carcinoma: in vitro study.

Hepatocellular carcinoma (HCC) is one of the common malignancies, characterized by poor response to conventional therapeutic options. Immunotherapy with dendritic cells (DCs)-vaccines is one of the most successful strategies used for the treatment of HCC. However, the methods applied in the preparation of antigen-loaded DCs are important factors for optimization of DCs vaccines. The present study was conducted to investigate the effect of HCC-whole tumor cell lysate prepared using rapid repetitive freeze-thaw cycles on the immunogenicity of DCs and evaluate the ability of whole tumor cell lysate-pulsed DCs vaccine to induce a specific cytotoxic Tlymphocytes (CTLs) response against HepG2 cell line. Immature DCs generated from peripheral blood monocytes were randomized into two groups: control DCs and whole tumor cell lysate-pulsed DCs. Phenotypic analysis of the DCs' cell maturation marker CD83 and co-stimulatory molecule CD86 was performed. HCC-specific cytotoxic activity of CD8+ CTLs was measured in vitro. Loading of DCs with necrotic whole cell lysate resulted in non-significant changes in DCs' expression of CD83, but a significant increase in expression of CD86. In addition, CD8+ CTLs stimulated with whole tumor cell lysate-pulsed DCs showed a high cytotoxic activity that specifically attack HepG2 cells. Our findings indicated that pulsation of DCs with whole tumor cell lysate prepared by repetitive freeze-thaw cycles could efficiently enhance the ability of DCs to induce proliferation and clonal expansion of CD8+ CTLs. Data herein, also indicated that whole tumor cell lysate-pulsed DCs triggers a specific CD8+ CTLs against HCC tumor cells.

Cancer Stem Cell Vaccination With PD-L1 and CTLA-4 Blockades Enhances the Eradication of Melanoma Stem Cells in a Mouse Tumor Model.

Immune checkpoint inhibitors and monoclonal antibodies reinvigorate cancer immunotherapy. However, these immunotherapies only benefit a subset of patients. We previously reported that ALDH tumor cells were highly enriched for cancer stem cells (CSCs), and ALDH CSC lysate-pulsed dendritic cell (CSC-DC) vaccine was shown to induce CSC-specific cytotoxic T lymphocytes. In this study, we investigated the CSC targeting effect of the CSC-DC vaccine combined with a dual blockade of programmed death-ligand 1 and cytotoxic T-lymphocyte-associated protein (CTLA-4) in B16-F10 murine melanoma tumor model. Our data showed that animals treated with the dual blockade of programmed death-ligand 1 and CTLA-4 and CSC-DC vaccine conferred significantly more tumor regression than the CSC-DC vaccine alone. Importantly, the triple combination treatment dramatically eliminated ALDH CSCs in vivo. We observed that CSC-DC vaccine in combination with anti-PD-L1 and anti-CTLA-4 administration resulted in ∼1.7-fold fewer PD-1CD8 T cells and ∼2.5-fold fewer CTLA-4CD8 T cells than the populations observed following the CSC-DC vaccination alone. Moreover, significant antitumor effects and dramatically eliminated ALDH CSCs following the triple combination treatment were accompanied by significantly enhanced T-cell expansion, suppressed transforming growth factor β secretion, enhanced IFN-γ secretion, and significantly enhanced host specific CD8 T-cell response against CSCs. Collectively, these data showed that administration of a-PD-L1 and a-CTLA-4 combined with CSC-DC vaccine may represent an effective immunotherapeutic strategy for cancer patients in clinical.

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.

Uric acid enhances the antitumor immunity of dendritic cell-based vaccine.

Uric acid (UA) released from dying cells has been recognized by the immune system as a danger signal. In response to UA, dendritic cells (DC) in the immune system mature and enhance the T cell response to foreign antigens. It is conceivable that the antitumor immunity of a tumor vaccine could be promoted by the administration of UA. To test this concept, we applied UA as an adjuvant to a DC-based vaccine, and discovered that the administration of UA as an adjuvant significantly enhanced the ability of the tumor lysate-pulsed DC vaccine in delaying the tumor growth. The antitumor activity was achieved with adoptively transferred lymphocytes, and both CD8+ T cells and NK cells were required to achieve effective immunity. This resulted in an increased accumulation of activated CD8+ T cells and an elevated production of IFN-γ. Collectively, our study shows that the administration of UA enhances the antitumor activity of tumor lysate-pulsed DC vaccine, thus providing the preclinical rationale for the application of UA in DC-based vaccine strategies.

Adjuvant inhibition of iAPC function in the tumor microenvironment promotes therapeutic immunity in the setting of vaccination-induced T cell anti-tumor response

Meeting abstracts Glioma tumor lysate-pulsed dendritic cell (DC) vaccination is an effective treatment modality. However, cure rates in the established tumor setting are not therapeutically significant in our preclinical models. We inferred that immunosuppressive antigen presenting cells (iAPCs)

Abstract 1071: Adoptive transfer of T cells overcomes barriers of homing to the tumor microenvironment in ovarian cancer

Abstract Objectives: Chemokine-dependent molecular mechanisms could play a key role in controlling tumor-specific T cell trafficking and the clinical success of adoptive transfer of ex vivo expanded T cells. We thus characterized the chemokine microenvironment in advanced stage serous ovarian cancer and evaluated whether CD3/CD28 co-stimulation of lysate vaccine-primed autologous T cells prepared for adoptive transfer led to the expansion of tumor antigen-reactive T cells able to home to ovarian cancer microenvironment. Methods: The expression of all known human chemokines was analyzed on a gene expression microarray from 63 patients with primary ovarian cancer and was validated on publicly available gene expression dataset and on tissue microarray constructed from a matching cohort of patients. Five HLA-A2 patients with recurrent ovarian cancer, who had previously received vaccination with autologous lysate-pulsed dendritic cell vaccine, underwent apheresis to harvest peripheral blood T cells, which were then expanded using beads coated with anti-CD3/anti-CD28 antibodies and IL-2. T cells were analyzed for 10 chemokine receptors that matched the chemokines expressed in ovarian cancer. Tumor-specific T cells were identified by a HER2/neu pentamer staining. Chemotaxis assays were performed to assess the functionality of these receptors. Results: Chemokines in general were found to be expressed at low level in advanced stage ovarian cancer, with the 10 most highly expressed ones being CCL2, CCL4, CCL5, CCL28, CXCL5, CXCL6 CXCL10, CXCL12, CXCL16 and CX3CL1. The two most commonly expressed chemokine receptors on the expanded cells were CXCR3 and CXCR4 (receptors for CXCL10 and CXCL12). At the end of expansion, 70% of output CD4+ and CD8+ cells expressed CXCR3 and CXCR4, compared to 20% of input T cells. The higher expression of CXCR3 and CXCR4 in output T cells resulted in significantly increased chemotaxis toward CXCL10 and CXCL12, suggesting a better homing ability to the tumor microenvironment. HER2/neu-specific T cells were detected in the expanded T cell population and showed migration toward CXCL10 and CXCL12. Conclusions: Adoptive transfer of ex vivo expanded tumor specific T cells that are capable of tumor engraftment holds a highly promising treatment approach for recurrent ovarian cancer. Chemokines are key regulators of circulation, homing and retention of these cells. Our data confirm that T cells prepared for adoptive transfer through CD3/CD28 bead expansion comprise tumor-specific cells, which upregulate appropriate chemokine receptors and show enhanced migration towards chemokines highly expressed by ovarian cancer. Citation Format: Emese Zsiros, Priyanka Duttagupta, Thomas Garrabrant, Janos L. Tanyi, Lana E. Kandalaft, George Coukos. Adoptive transfer of T cells overcomes barriers of homing to the tumor microenvironment in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1071. doi:10.1158/1538-7445.AM2014-1071