Active Cellular Immunotherapy Research Articles

LL-37 as a Powerful Molecular Tool for Boosting the Performance of Ex Vivo-Produced Human Dendritic Cells for Cancer Immunotherapy.

Ex vivo-produced dendritic cells (DCs) constitute the core of active cellular immunotherapy (ACI) for cancer treatment. After many disappointments in clinical trials, the current protocols for their preparation are attempting to boost their therapeutic efficacy by enhancing their functionality towards Th1 response and capability to induce the expansion of cytotoxic tumor-specific CD8+ T cells. LL-37 is an antimicrobial peptide with strong immunomodulatory potential. This potential was previously found to either enhance or suppress the desired anti-tumor DC functionality when used at different phases of their ex vivo production. In this work, we show that LL-37 can be implemented during the whole process of DC production in a way that allows LL-37 to enhance the anti-tumor functionality of produced DCs. We found that the supplementation of LL-37 during the differentiation of monocyte-derived DCs showed only a tendency to enhance their in vitro-induced lymphocyte enrichment with CD8+ T cells. The supplementation of LL-37 also during the process of DC antigen loading (pulsation) and maturation significantly enhanced the cell culture enrichment with CD8+ T cells. Moreover, this enrichment was also associated with the downregulated expression of PD-1 in CD8+ T cells, significantly higher frequency of tumor cell-reactive CD8+ T cells, and superior in vitro cytotoxicity against tumor cells. These data showed that LL-37 implementation into the whole process of the ex vivo production of DCs could significantly boost their anti-tumor performance in ACI.

Abstract 4158: High dimensional analysis of sipuleucel T from metastatic castration resistant prostate cancer patients using mass cytometry

Abstract Background: Prostate cancer is the most commonly diagnosed non-skin cancer in men, and metastatic castration-resistant prostate cancer (mCRPC) represents the most lethal form of the disease. Sipuleucel T (sip T) is an autologous active cellular immunotherapy approved by FDA for patients with mCRPC and has been shown effective in improving overall survival (OS) among individuals with mCRPC. To date, detailed analysis of the sip T product has not been studied using an advanced mass cytometry approach. Here, we present high dimensional data describing the specific leukocyte subsets and phenotype of sip-T in detail. Methods: We performed a comprehensive assessment of sip T (n=14 samples, collected under an IRB approved protocol) from 11 different patients with mCRPC, using high-throughput mass cytometry (CyTOF) comprised of 37 different antibodies/markers. Sip T samples were then analyzed for various leukocyte subsets, as well as markers of activation and exhaustion. Furthermore, clinical correlations were drawn between immune cell subsets/markers and patient clinical data such, as prostate specific antigen (PSA) levels. Results: CyTOF analysis revealed that CD3+ T cells (including CD4+ and CD8+) constituted the highest proportion (median, range: 63%, 9-89%) of sip T, followed by CD19+ B cells (4%, 1-82%), CD3-CD14-CD56+ natural killer (NK) cells (4%, 1-18%), and CD3-CD19-CD56-HLA-DR+CD11c+CD14+ monocytes (1%, 1-37%) besides a small proportion (<1%) of conventional dendritic cells (cDC), innate lymphoid cells (ILC), plasmacytoid DC (pDC), myeloid-derived suppressor cells (MDSC) and NK T-cells (NKT). The majority of CD8+ T cells were either CD45RA+CCR7-effector (Teff: 43%, 9-70%), CD45RA-CCR7- effector memory (Tem: 23%, 11-59%) and CD45RA+CCR7+ naive (Tn: 22%, 4-50%). Contrarily, most CD4+ T cells belonged to CD45RA-CCR7+ central memory (Tcm: 45%, 24-78%), effector memory (Tem: 27%, 6-61%) and naive (Tn: 22%, 1-50%) subtypes. Interestingly, the majority of monocytes (86%, 78-97%) were CD86+ suggesting a role in inducing lymphocyte activation. Of activation/exhaustion markers, the highest proportion of PDL1+, PD1+, IFNg+ and ICOS+ cells were observed in monocytes, cDC, NK and CD4+ T cells respectively. Likewise, CTLA4+, VISTA+ and TIGIT+ cells were most commonly found among CD8+ T cells, suggesting a potential regulatory effect on sip T CD8+ T cell proliferation and effector function. Conclusion: This is the first comprehensive study to evaluate the composition of sip T from mCRPC patients using high dimensional CyTOF analysis. Given the increasing use of CyTOF in translational research, this data set serves as an important reference source, which could be used in future studies to help guide therapeutic modulation of activation/exhaustion markers to improve sip T efficacy and enhance beneficial immune responses. Citation Format: Muhammad Saeed, Kevin Kim, Ariel Borkowski, Russell Kent Pachynski. High dimensional analysis of sipuleucel T from metastatic castration resistant prostate cancer patients using mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4158.

Biomaterial-enhanced cancer vaccines

Cancer vaccines are a promising strategy for preventing cancer progression through antitumor immunity activation. Some vaccines, such as Sipuleucel-T (an autologous active cellular immunotherapy), have been applied clinically. However, systemic toxicity and low immunogenicity have restricted their broad clinical application. Furthermore, the off-target effects induced by immunostimulatory adjuvants and cytokines also curtail cancer vaccine applications. Polymers, scaffolds, lipids, microneedles, and other biomaterials offer advantageous properties, such as excellent biocompatibility and biodegradability, flexible size, high loading capacity for immune-related components, and a large surface area for immune ligand binding. Thus, various biomaterials are applied as immune adjuvants in cancer vaccines to enhance cancer antigen immunogenicity, protect cargoes from the surrounding microenvironment, and promote antigen presentation. Herein, we divide biomaterial-enhanced cancer vaccines into peptide and protein vaccines, nucleic acid-based vaccines, tumor cell-based compound vaccine, and virus-based vaccines and summarize advances and challenges associated with the use of biomaterials in cancer vaccines.

Active Cellular Immunotherapy in the Desert of Advanced Prostate Cancer

Active Cellular Immunotherapy in the Desert of Advanced Prostate Cancer

Efficacy and Safety of Autologous Dendritic Cell–Based Immunotherapy, Docetaxel, and Prednisone vs Placebo in Patients With Metastatic Castration-Resistant Prostate Cancer

DCVAC/PCa is an active cellular immunotherapy designed to initiate an immune response against prostate cancer. To evaluate the efficacy and safety of DCVAC/PCa plus chemotherapy followed by DCVAC/PCa maintenance treatment in patients with metastatic castration-resistant prostate cancer (mCRPC). The VIABLE double-blind, parallel-group, placebo-controlled, phase 3 randomized clinical trial enrolled patients with mCRPC among 177 hospital clinics in the US and Europe between June 2014 and November 2017. Data analyses were performed from December 2019 to July 2020. Eligible patients were randomized (2:1) to receive DCVAC/PCa (add-on and maintenance) or placebo, both in combination with chemotherapy (docetaxel plus prednisone). The stratification was applied according to geographical region (US or non-US), prior therapy (abiraterone, enzalutamide, or neither), and Eastern Cooperative Oncology Group performance status (0-1 or 2). DCVAC/PCa or placebo was administered subcutaneously every 3 to 4 weeks (up to 15 doses). The primary outcome was overall survival (OS), defined as the time from randomization until death due to any cause, in all randomized patients. Survival was compared using 2-sided log-rank test stratified by geographical region, prior therapy with abiraterone and/or enzalutamide, and Eastern Cooperative Oncology Group performance status. A total of 1182 men with mCRPC (median [range] age, 68 [46-89] years) were randomized to receive DCVAC/PCa (n = 787) or placebo (n = 395). Of these, 610 (81.8%) started DCVAC/PCa, and 376 (98.4%) started placebo. There was no difference in OS between the DCVAC/PCa and placebo groups in all randomized patients (median OS, 23.9 months [95% CI, 21.6-25.3] vs 24.3 months [95% CI, 22.6-26.0]; hazard ratio, 1.04; 95% CI, 0.90-1.21; P = .60). No differences in the secondary efficacy end points (radiological progression-free survival, time to prostate-specific antigen progression, or skeletal-related events) were observed. Treatment-emergent adverse events related to DCVAC/PCa or placebo occurred in 69 of 749 (9.2%) and 48 of 379 (12.7%) patients, respectively. The most common treatment-emergent adverse events (DCVAC/PCa [n = 749] vs placebo [n = 379]) were fatigue (271 [36.2%] vs 152 [40.1%]), alopecia (222 [29.6%] vs 130 [34.3%]), and diarrhea (206 [27.5%] vs 117 [30.9%]). In this phase 3 randomized clinical trial, DCVAC/PCa combined with docetaxel plus prednisone and continued as maintenance treatment did not extend OS in patients with mCRPC and was well tolerated. ClinicalTrials.gov Identifier: NCT02111577.

Dendritic cell-based immunotherapy (DCVAC/OvCa) combined with second-line chemotherapy in platinum-sensitive ovarian cancer (SOV02): A randomized, open-label, phase 2 trial

ObjectiveDCVAC/OvCa is an active cellular immunotherapy designed to stimulate an immune response against ovarian cancer. We explored the safety and efficacy of DCVAC/OvCa plus carboplatin and gemcitabine in platinum-sensitive ovarian cancer. MethodsIn this open-label, parallel-group, phase 2 trial (ClinicalTrials.gov number NCT02107950), patients with platinum-sensitive ovarian cancer relapsing after first-line chemotherapy were randomized to DCVAC/OvCa and chemotherapy or chemotherapy alone. DCVAC/OvCa was administered every 3–6 weeks (10 doses). Endpoints included safety, progression-free survival (PFS; primary efficacy endpoint) and overall survival (OS; secondary efficacy endpoint). ResultsBetween November 2013 and May 2015, 71 patients were randomized to chemotherapy in combination with DCVAC/OvCa or to chemotherapy alone. Treatment-emergent adverse events related to DCVAC/OvCa, leukapheresis and chemotherapy occurred in six (16.2%), two (5.4%), and 35 (94.6%) patients in the DCVAC/OvCa group. Chemotherapy-related events occurred in all patients in the chemotherapy group. Seven patients in the DCVAC/OvCa group were excluded from primary efficacy analyses due to failure to receive ≥1 dose of DCVAC/OvCa. PFS was not improved (hazard ratio [HR] 0.73, 95% confidence interval [CI] 0.42–1.28, P = 0.274, data maturity 78.1%). Median OS was significantly prolonged (by 13.4 months) in the DCVAC/OvCa group (HR 0.38, 95% CI 0.20–0.74, P = 0.003; data maturity 56.3%). A signal for enhanced surrogate antigen-specific T-cell activity was seen with DCVAC/OvCa. ConclusionsDCVAC/OvCa combined with chemotherapy had a favorable safety profile in patients with platinum-sensitive ovarian cancer. DCVAC/OvCa did not improve PFS, but the exploratory analyses revealed OS prolongation and enhanced surrogate antigen-specific T-cell activity.

Autologous dendritic cell-based immunotherapy (DCVAC/LuCa) and carboplatin/paclitaxel in advanced non-small cell lung cancer: A randomized, open-label, phase I/II trial

PurposeTo investigate the efficacy and safety of an active cellular immunotherapy (DCVAC/LuCa) and chemotherapy in patients with stage IV non-small cell lung cancer (NSCLC). Patients and MethodsSLU01 was a multicenter, open-label, parallel-group, randomized, phase I/II trial. NSCLC patients were randomized in a ratio of 1:1:1 to receive: DCVAC/LuCa and chemotherapy (carboplatin and paclitaxel; Group A); DCVAC/LuCa, chemotherapy, pegylated interferon-α2b, and hydroxychloroquine (Group B); or chemotherapy alone (Group C). DCVAC/LuCa was administered subcutaneously every 3–6 weeks (up to 15 doses). The primary endpoint was overall survival (OS). During the study, enrollment into Group B was discontinued for strategic reasons. ResultsForty-five patients were randomized to Group A, 29 patients to Group B, and 38 patients to Group C. The median OS in the modified intention-to-treat (mITT) population was 3.7 months longer in Group A than in Group C (15.5 vs. 11.8 months; p = 0.0179; hazard ratio = 0.54; 95% confidence interval: 0.32–0.91). This OS effect was consistent across subgroups of the mITT population (females, males, current smokers, former smokers, and patients with non-squamous and squamous cell histology). The most common treatment-emergent adverse events of any grade reported in Groups A, B, and C, respectively, were neutropenia (50.0%, 29.6%, and 20.6%), fatigue (40.0%, 18.5%, and 20.6%), anemia (35.0%, 44.4%, and 32.4%), paresthesia (27.5%, 25.9%, and 17.6%), and alopecia (25.0%, 29.6%, and 41.2%). ConclusionDCVAC/LuCa in combination with carboplatin and paclitaxel extended OS and was well tolerated.

Efficacy and safety of DCVAC/LuCa Treatment plus standard chemotherapy and TCM in patients with advanced non-small cell lung cancer: A single-arm phase II study.

e21590 Background: Lung cancer remains the leading cause of cancer-related death in China. DCVAC/LuCa is an active autologous cellular immunotherapy consisting of autologous DCs loaded with NSCLC antigens from NSCLC cell lines. SQFZ is a kind of TCM injection commonly uesed for NSCLC, made from Ginseng and Astragalus membranaceus. Intent of this study is to evaluate the efficacy and safety of DCVAC/LuCa plus chemotherapy and TCM in patients with advanced NSCLC and wild type driving gene(EGFR/ALK). Methods: This study consist of screening/leukapheresis/treatment/follow-up period. On the treatment period, chemotherapy started after leukapheresis. Four cycles are recommended with limitation to maximum 6 cycles. Up to 12 subcutaneous doses of DCVAC/LuCa treatment were administrated on the 15th day of chemotherapy cycle every 3 weeks for 4 doses, and continued alone after chemotherapy finishes/stops every 6 weeks for the remaining 8 doses. TCM therapies were administered on the -3 to 3 days of DCVAC/LuCa. After completion of the total 12 doses DCVAC/LuCa or discontinuation of study treatment, patients will be followed up until experienced OS event or till 1 year of survival time. The primary efficacy endpoint included ORR, PFS and survival. Results: 23 pts were included, 19 males and 4 females, median age 63(48-80). Pathological type:16 adenocarcinoma, 5 squamous cell carcinoma and 1 other. 7 Ⅲb and 16 Ⅳ stage. ORR was 17.39%(4/23), DCR was 60.87%(14/23). Median PFS was 10.9 months, median follow-up in surviving patients was 22.3 months, 1-year survival is currently 92.86%, 18-months survival is currently 79.59%. 1 case was diagnosed as acute myelogenous leukemia myelodysplastic syndrome after 9 doses of DCVAC/LuCa. There were no other adverse events related to DCVAC/LuCa treatment TCM therapies in this study. Conclusions: DCVAC/LuCa, combined with standard chemotherapy and TCM had exhibited the potential to improve the survival in patients with advanced NSCLC, and decrease the occurrence of adverse events. But the sample size of this study is small, a further clinical investigation is needed.

Detection of tumor antigens and tumor-antigen specific T cells in NSCLC patients: Correlation of the quality of T cell responses with NSCLC subtype

Allogeneic cancer cell lines serve as universal source of tumor-associated antigens in cancer vaccines. Immunogenic high hydrostatic pressure-killed cancer cells derived from cell lines can be used for the generation of dendritic cell (DC)-based active cellular immunotherapy of non-small cell lung cancer (NSCLC). We investigated the expression of 12 known NSCLC tumor-associated antigens (TAA) (CEA, MAGE-A1, MAGE-A3, MAGE-A4, PRAME, hTERT, HER2, MUC1, Survivin, STEAP1, SOX2 and NY-ESO-1) in 6 NSCLC cell lines as candidates for the generation of DC-based lung cancer vaccine. We showed that the selected antigenic profile of these cell lines overlaps to various degrees with that of primary NSCLC tumors (n = 52), indicating that 4 out of 6 NSCLC cell lines would be suitable for DC-based vaccine generation. We further investigated the presence of TAA-specific T cells in blood of NSCLC patients (n = 32) using commercially available peptide mixes in an in vitro stimulation assay. IFN-γ+CD8+ and IFN-γ+CD4+ T cell responses to all antigens were detected in NSCLC patients. Interestingly, despite higher TAA expression in squamous cell carcinoma (SCC) the responsiveness of patients’ T cells to stimulation was significantly lower in SCC patients than in adenocarcinoma (AC) patients. This suggests qualitative differences in T cell functionality between NSCLC subtypes. Based on this study, and in order to maximize the amount of treatable patients, we selected a mix of H520 and H522 NSCLC cell lines for DC-based vaccine preparation. We also established a minimal panel of antigenic peptide mixes (CEA, hTERT, PRAME, HER2) for immunomonitoring of T cell responses during the DC-based lung cancer immunotherapy in Phase I lung cancer clinical trial (NCT02470468).

Case series of reports of pruritus and sipuleucel-T submitted to the Food and Drug Administration Adverse Event Reporting System

Sipuleucel-T, an autologous active cellular immunotherapy, is indicated for the treatment of asymptomatic or minimally symptomatic castration-resistant prostate cancer. The U.S. Food and Drug Administration Adverse Event Reporting System (FAERS) received a report of pruritus without rash following the second dose of sipuleucel-T in a patient who had otherwise not started any new medications concurrent with the first and second doses of sipuleucel-T. No further sipuleucel-T was administered, but symptoms persisted for at least 6 weeks despite treatment with several medications aimed at symptomatic relief of pruritus. Rash is the only dermatologic adverse event included in the sipuleucel-T U.S. package insert. A search of the FAERS database yielded seven additional U.S. reports of pruritus and sipuleucel-T identified as the primary suspect medication; two of these occurred prior to the administration of sipuleucel-T (following leukapheresis). In data mining analyses, pruritus following sipuleucel-T was not reported more frequently than expected when compared to all other adverse event-drug/biologic combinations in FAERS. Thus, pruritus following sipuleucel-T administration was rarely, but not disproportionately, reported to FAERS. Although we cannot exclude the possibility that diabetes, malignancy, or other conditions may have contributed to pruritus in our index patient, in view of the timing of sipuleucel-T therapy and onset of symptoms, a drug/biologic-related reaction is plausible. In the appropriate clinical scenario, sipuleucel-T (or its components) should not be overlooked as a potential etiological agent in pruritus.

Dendritic-cell vaccine (DCVAC) with first-line chemotherapy in patients with stage IV NSCLC: Final analysis of phase II, open label, randomized, multicenter trial.

9039 Background: Immunotherapy aiming the induction of tumor cell specific immune responses controlling the tumor growth, has emerged as a promising treatment modality in lung cancer(LuCa). Autologous DCVAC can present tumor antigens to elicit a durable immune response. We hypothesized that adding DCVAC to the standard of care chemotherapy (ct) could prolong overall survival (OS) and progression-free survival (PFS). Methods: This study evaluated the efficacy and safety of DCVAC/LuCa (active cellular immunotherapy based on dendritic cells) concomitantly added to ct (carboplatin/paclitaxel) - Arm A (A) vs DCVAC/LuCa + immune modulators (IFN-α and hydroxychloroquine) - Arm B (B)+ct vs ct - Arm C (C) in NSCLC patients (pts). Randomization 1:1:1; pts in A and B received up to 15 doses of DCVAC, ct was given 4-6 cycles in A and C. Stage IV NSCLC was confirmed histologically or cytologically, ECOG 0-1 pts were eligible. Stratification was done by histology subtype and smoking history. Primary efficacy analysis compared A vs C only as enrollment to B was closed early based on Sponsor’s assessment of further clinical development potential, there were no safety concerns or signals. Results: 112 pts at 12 sites were randomized (A/45 B/29 C/38). Patients characteristics were comparable across the study groups with the exception of gender (m/f, %: 65/35 (A) and 74/26 (C) and smoking history (75 % of smokers in A, 97 % in C). Median follow up time was 25.8 months, range 0.1-41.8. Median OS was 15.5 months in A compared to 11.8 months in C, hazard ratio (HR) 0.55, p-value 0.0232, 95% CI [0.33, 0,93], data maturity 77%. Median PFS was 6.74 in A and 5.63 months in C, HR 0.59, p-value 0.0415, 95% CI [0.36, 0.99], data maturity 86%. Overall response rate was 45% in A vs 34% in C. Most TEAEs were related to ct (anemia [35%-A, 32%-C], neutropenia [48% in A, 21%-C], thrombocytopenia [25% in A, 27% in C]). There were no grade ≥ 3 TEAEs solely related to DCVAC. Most common leukapheresis (lp)-related AE was vomiting in 2 pts out of 67 pts undergone lp. Conclusions: Addition of DCVAC-based immunotherapy to the standard of care chemotherapy significantly improved OS in stage IV NSCLC. Clinical trial information: NCT02470468.

Cell-Based Immunotherapy of Gliomas.

Current cell-based immunotherapeutic strategies attempt to produce and maintain an immune response against glioma cells by artificially stimulating the immune system using passive and/or active approaches. Cellular immunotherapy is taken to mean the administration of live immune cells that either have immune effector capabilities themselves (passive immunotherapy) or engender a downstream antitumor response (active immunotherapy). Passive cellular immunotherapy most often takes the form of the adoptive transfer of a range of cell types, whereby antitumor immune cells from a patient (or allogeneic donor) are created, activated, and/or expanded ex vivo and subsequently administered back to the patient to directly attack the neoplasm. Active cellular immunotherapy approaches for the treatment of malignant gliomas have most often taken the form of dendritic cell (DC)-based vaccines.

High hydrostatic pressure affects antigenic pool in tumor cells: Implication for dendritic cell-based cancer immunotherapy

High hydrostatic pressure (HHP) can be used to generate dendritic cell (DC)-based active immunotherapy for prostate, lung and ovarian cancer. We showed here that HHP treatment of selected human cancer cell lines leads to a degradation of tumor antigens which depends on the magnitude of HHP applied and on the cancer cell line origin. Whereas prostate or ovarian cell lines displayed little protein antigen degradation with HHP treatment up to 300MPa after 2h, tumor antigens are hardly detected in lung cancer cell line after treatment with HHP 250MPa at the same time. On the other hand, quick reduction of tumor antigen-coding mRNA was observed at HHP 200MPa immediately after treatment in all cell lines tested. To optimize the DC-based active cellular therapy protocol for HHP-sensitive cell lines the immunogenicity of HHP-treated lung cancer cells at 150, 200 and 250MPa was compared. Lung cancer cells treated with HHP 150MPa display characteristics of immunogenic cell death, however cells are not efficiently phagocytosed by DC. Despite induction of the highest number of antigen-specific CD8+ T cells, 150 MPa-treated lung cancer cells survive in high numbers. This excludes their use in DC vaccine manufacturing. HHP of 200MPa treatment of lung cancer cells ensures the optimal ratio of efficient immunogenic killing and delivery of protein antigens in DC. These results represent an important pre-clinical data for generation of immunogenic killed lung cancer cells in ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa).

Generation of dendritic cell-based vaccine using high hydrostatic pressure for non-small cell lung cancer immunotherapy.

High hydrostatic pressure (HHP) induces immunogenic death of tumor cells which confer protective anti-tumor immunity in vivo. Moreover, DC pulsed with HHP-treated tumor cells induced therapeutic effect in mouse cancer model. In this study, we tested the immunogenicity, stability and T cell stimulatory activity of human monocyte-derived dendritic cell (DC)-based HHP lung cancer vaccine generated in GMP compliant serum free medium using HHP 250 MPa. DC pulsed with HHP-killed lung cancer cells and poly(I:C) enhanced DC maturation, chemotactic migration and production of pro-inflammatory cytokines after 24h. Moreover, DC-based HHP lung cancer vaccine showed functional plasticity after transfer into serum-containing media and stimulation with LPS or CD40L after additional 24h. LPS and CD40L stimulation further differentially enhanced the expression of costimulatory molecules and production of IL-12p70. DC-based HHP lung cancer vaccine decreased the number of CD4+CD25+Foxp3+ T regulatory cells and stimulated IFN-γ-producing tumor antigen-specific CD4+ and CD8+ T cells from non-small cell lung cancer (NSCLC) patients. Tumor antigen specific CD8+ and CD4+ T cell responses were detected in NSCLC patient’s against a selected tumor antigens expressed by lung cancer cell lines used for the vaccine generation. We also showed for the first time that protein antigen from HHP-killed lung cancer cells is processed and presented by DC to CD8+ T cells. Our results represent important preclinical data for ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa) in combination with chemotherapy and immune enhancers.

Precise manipulation of biophysical particle parameters enables control of proinflammatory cytokine production in presence of TLR 3 and 4 ligands.

The manuscript describes a systematic investigation into the effects of biophysical parameters of nanoparticles (NPs) on immune cells. Modulation of the biophysical character of the NP surface can be achieved by introduction of amino acids on monodisperse silica NPs, introducing a range of tunable biophysical parameters of interest, i.e. distinct sizes, different surface charges and varying degrees of surface hydrophobicity. We examine internalization of the NP in dendritic cells (DCs) and measure a myriad of cytokines, including IL-1β and IFNγ, which prognosticate the efficacy of the NPs as a potential vaccine (IL-1β metric) or active cellular immunotherapy carrier (IFNγ metric). Two different TLR ligands (a viral TLR3 ligand and a bacterial TLR4 ligand) were used along with the PAA NPs to compare their costimulatory immunogenicity. We strongly believe that this study will provide crucial information to many readers of Acta Biomaterialia and further drive the use of nanoparticle platforms in modulating immune responses.

P3.06-001 Phase I/II Study to Evaluate Safety and Efficacy DCVAC/LuCa with 1st Line Chemotherapy +/- Immune Enhancers vs Chemotherapy, Stage IV NSCLC: Topic: Clinical Studies

Lung cancer (LuCa) has been the most common cancer in the world for several decades and also the most common cause of death from cancer worldwide. Immunotherapy, for induction of tumor cell specific immune responses destroying tumor cells, has emerged as a promising treatment modality in solid malignant tumors. Studies have shown that chemotherapy can be combined with vaccine without impairing the immune response. SLU01 is a randomized, open, parallel-group, multicenter, international phase I/II study to evaluate the efficacy and safety of DCVAC/LuCa (active cellular immunotherapy based on dendritic cells) added to standard first line chemotherapy with carboplatin and paclitaxel +/- immune enhancers (interferon-α and hydroxychloroquine) vs chemotherapy alone in patients with Stage IV NSCLC. The study was initiated in December 2014 and plans to enroll 105 patients at approximately 12 sites in Czech and Slovak Republics. Eligible patients are required to present with metastatic NSCLC defined by histologically or cytologically confirmation and ECOG score 0-1. All patients will receive Standard of Care (SoC) carboplatin and paclitaxel, and will be randomized 1:1:1 to DCVAC/LuCa or DCVAC/LuCa + immune enhancers (pegylated IFN-α2b and hydroxychloroquine) or SoC only. Patients will be stratified by histology subtype adenomatous or squamous cell carcinoma and smoking history. The primary objective is to compare efficacy of DCVAC/LuCa + chemotherapy +/- immune enhancers vs. chemotherapy alone in patients with stage IV NSCLC, as measured by progression free survival (PFS). Secondary objectives include assessments of safety, objective response rate (ORR), duration of response (DoR) and overall survival (OS). Exploratory objectives include comparison of changes in immune responses and search for prognostic biomarkers. Clinical trial information: EudraCT number 2014-003084-37. Section is not applicable. Section is not applicable.

Abstract A039: Caspase-2 and oxidative-ER stress crosstalk regulates the exposure of “eat me” signal calreticulin by high hydrostatic pressure treated cancer cells

Abstract Immunogenic cell death (ICD) is a type of cell death induced by various chemotherapeutics, physical modalities such as photodynamic therapy, radiotherapy and high hydrostatic pressure (HHP). HHP induces immunogenic cell death of cancer cell is currently tested in running II. and III. phase of clinical trial for preparation of active cellular cancer immunotherapy (DCVAC). Previous studies have shown that the immunogenicity of ICD mainly relies on surface exposed calreticulin (ecto-CRT); CRT is normally located in the lumen of the endoplasmic reticulum (ER) and may translocate to the cell surface within hours after treatment. Although ICD can be triggers by HHP, underlying danger signaling pathways remain unclear. Therefore ovarian (OV90) cancer cell line was treated by HHP, UV-B and anthracycline (Idarubicin). The kinetics of ICD markers and key components of ER stress-mediated apoptotic pathway were analyzed by flow cytometry, western blotting and confocal microscopy. In this work we described the ability of HHP triggers oxidative ER-stress (the production of reactive oxygen species (ROS) together with ER stress response) which results in phosphorylation of PERK and eIF2α. We also observed a rapid caspase-8 activation subsequently followed by BAP31 cleavage, Bid activation and caspase-3 activation. While caspase-8 activation was necessary for cell surface exposure of calreticulin (ecto-CRT) after HHP treatment, proapoptotic proteins Bax and Bak were dispensable for this process. Moreover, we identified that HHP treatment leads to caspase-2 activation. Whereas Idarubicin treatment has no effect on ecto-CRT exposure in Caspase-2 knockdown tumor cells, ecto-CRT was significantly decreased in these cells after HHP treatment. In addition, phagocytosis of HHP-treated Caspase-2 knockdown tumor cells was compromised in contrast to Idarubicin-treated knockout tumor cells. In conclusion, we characterized the molecular mechanism of ecto-CRT exposure pathway induced by HHP which is analogous to that induced by anthracycline treatment. For the first time we provide evidence that caspase-2 might play an important role in the HHP-mediated ecto-CRT exposure. Citation Format: Irena Kusova Moserova, Iva Truxova, Abhishek G, Garg, Patrizia Agostinis, Piere Francois Cartron, Sarka Vosahlikova, Radek Spisek, Jitka Fucikova. Caspase-2 and oxidative-ER stress crosstalk regulates the exposure of “eat me” signal calreticulin by high hydrostatic pressure treated cancer cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A039.

Abstract B18: Combining selective toll-like receptor 9 (TLR9) agonists and GM-CSF activity for potentiating cellular activation in active cell immunotherapy (ACI).

Abstract Sipuleucel-T (Provenge®), indicated for the treatment of asymptomatic or minimally symptomatic metastatic castration resistant prostate cancer, is the first FDA-approved Active Cellular Immunotherapy (ACI). Here we describe the development of a therapeutic ACI for the treatment of renal, lung, colon, and cervical cancer. Like PROVENGE®, this new ACI utilizes a recombinant antigen consisting of carbonic anhydrase IX (CA9) linked to GM-CSF (CA9:GM-CSF). In these studies, we investigated the effects of incorporating selective TLR9 agonists on in vitro measures of ACI potency. Initially, employing a panel of proprietary TLR9 agonists, we compared the phenotype of antigen presenting cells (APC) following culture of PBMC from normal healthy donors with either CA9:GM-CSF or CA9:GM-CSF plus TLR9 agonist. While antigen derived GM-CSF activity matured APC, characterized by elevated cell surface expression of CD40, CD54, CD80 and CD86, proprietary agonists for TLR9 further enhanced expression of CD40, CD80, and CD86. By extension, stimulation via TLR9 elicited increased costimulatory capacity of CD14+ large APC in allogeneic mixed lymphocyte response assays. Elevated levels of MCP1-3 in culture supernatant were consistent with APC activation, and notably, viability of CD14+ large APC was unaffected following TLR9 stimulation within the ACI product. Interestingly, using an HLA class II restricted CA9-specific T cell hybridoma reporter assay, staggering the addition of CA9:GM-CSF prior to TLR9 agonist was important to maximize antigen uptake and peptide presentation. Collectively, antigen derived GM-CSF activity is robust at activating APC and certain TLR9 agonists strengthen this effect. Next, as determined by cell surface expression of activation markers (CD27, CD38, CD40, CD54, CD86, IgD), the potential for TLR9 agonists to activate B cells was examined. Relative to cultures supplemented with only CA9:GM-CSF, TLR9 agonists were sufficient to produce a generalized B cell activation pattern consisting of enhanced cell surface expression of CD38, CD40, CD86, and CD54; while expression of CD27 was decreased. Expression of IgD remained unchanged. Associated with the prominent pattern of B cell activation was a marked increase in the accumulation of proinflammatory Type-1 like growth factors (IFN-α, IFN-γ, CXCL9, CXCL10, CCL3, and CCL4) in response to each TLR9 agonist. Despite the degree of B cell activation, this phenotype did not correlate with cellular activation of any T cell subset; suggesting the inflammatory signature resulting from TLR9 stimulation is primarily driven by B cells and/or APC in the ACI setting. Importantly, IL-10 levels were significantly enhanced following TLR9 stimulation, raising the possibility for enhanced regulatory cell activity, but changes in the frequency of regulatory CD4+ T cells were not observed. However, continuing studies are aimed at examining whether regulatory B cell numbers may be enhanced. Thus, in the ACI product, TLR9 agonists potentiate B cell activation and represent powerful polarizers of predominantly Type 1-like cytokine responses. Overall, agonists of TLR9 are compatible with the ACI platform for potentially enhancing antigen specific immunotherapy of cancer. In current experiments, the capacity of TLR9 agonists to enhance cross-priming and generate cytotoxic T cell responses is under investigation. Citation Format: Jason Chinn, Crystal Cummings, Felecia Wagener, Shaarwari Sridhar, Kien Khuu-Duong, Xinhui Ge, Karen Yoshino, Sam Li, Lisa Martel, Chris Ramsborg, Ken Brasel, James Trager, Craig Meagher. Combining selective toll-like receptor 9 (TLR9) agonists and GM-CSF activity for potentiating cellular activation in active cell immunotherapy (ACI). [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B18.

Systematic review/Meta-analysis Sipuleucel-T immunotherapy for castration-resistant prostate cancer. A systematic review and meta-analysis

IntroductionSipuleucel-T is a novel active cellular immunotherapy for the treatment of asymptomatic or minimally symptomatic metastatic castrate-resistant prostate cancer (mCRPC). It is assumed to be associated with less adverse events than conventional docetaxel-based chemotherapy.Material and methodsA systematic review of literature published between January, 1 1966 and February, 6 2012 was performed to assess the efficacy and safety of sipuleucel-T in patients with mCRPC. Databases were searched: Medline, EMBASE, Cochrane, CancerLit as well as ASCO and ESCO websites.ResultsThree randomized clinical trials with a total of 737 participants fulfilled established criteria. The overall survival of patients who received sipuleucel-T in comparison to the control group was significantly longer with a hazard ratio (HR) of 0.73 (95% CI: 0.61-0.88; p = 0.001). Time to disease progression was not prolonged using sipuleucel-T compared to placebo, HR = 0.89 (95% CI: 0.75-1.05; p = 0.18). Relative benefit (RB) of serum PSA level reduction of at least 50% for sipuleucel-T compared to placebo did not meet statistical significance, RB = 1.97 (95% CI: 0.48-8.14; p = 0.38). The safety population consisted of 729 patients with mCRPC. Compared to the control group, the pooled relative risks (RR) of all adverse events – RR = 1.03 (95% CI: 1.00-1.05; p = 0.06), grade 3 to 5 adverse events – RR = 0.98 (95% CI: 0.79-1.22; p = 0.86) and cerebrovascular events – RR = 1.93 (95% CI: 0.73-5.09; p = 0.18) were not significantly higher for men treated with sipuleucel-T.ConclusionsThe use of sipuleucel-T prolonged the overall survival among men with mCRPC. No effect on time to disease progression was observed and the safety profile was acceptable.

Sipuleucel-T in Metastatic Castration-Resistant Prostate Cancer†

Sipuleucel-T is an autologous active cellular immunotherapy used in the treatment of men with asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (CRPC). It is the first therapeutic cancer vaccine to receive US FDA approval. Approximately 3 days prior to each infusion of sipuleucel-T, patients undergo a leukapheresis procedure for collection of autologous peripheral blood mononuclear cells. Preparation of sipuleucel-T involves enrichment for antigen-presenting cells from the leukapheresis product and activation ex vivo with a recombinant fusion protein (PA2024). In the randomized, double-blind, placebo-controlled IMPACT study in patients with metastatic CRPC, sipuleucel-T was associated with a 22% relative reduction in the risk of death (hazard ratio 0.78; p = 0.03), which was the primary endpoint of the trial. After a median follow-up period of 34.1 months, median survival was 4.1 months longer with sipuleucel-T than placebo (25.8 vs 21.7 months). There was no significant between-group difference for the median time to objective disease progression (a secondary endpoint). Almost all patients treated with sipuleucel-T in clinical trials reported an adverse event, although these were mild or moderate in severity (grade 1 or 2) in most patients. The most common adverse events (e.g. infusion-related events, such as chills and fever) generally occurred within the first day after administration of sipuleucel-T and resolved within 2 days.