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Clinical characteristics of the SCC study population

Gating strategies of migratory cDC, APC, cDC1 and pDC

Purpose:Adenocarcinoma of the uterine cervix is the second most common type of cervical cancer after squamous cell carcinoma (SCC). Although both subtypes are treated similarly, patients with adenocarcinoma have a worse prognosis. In this study, immunologic features of the tumor microenvironment in these two subsets were pursued with potential therapeutic implications.Experimental Design:The immune microenvironment of primary tumors and nonmetastatic tumor-draining lymph nodes (TDLN) was compared between patients with cervical adenocarcinoma (n = 16) and SCC (n = 20) by polychromatic flow cytometry and by transcriptional profiling of the primary tumors (n = 299) using publicly available data from The Cancer Genome Atlas (TCGA).Results:Flow cytometric analyses revealed intact T-cell differentiation in TDLNs, but hampered effector T-cell trafficking to the primary tumors in adenocarcinoma, as compared with SCC. TCGA analysis demonstrated higher expression of chemokines involved in effector T-cell homing (CXCL9/10/11) in SCC primary tumors as compared with adenocarcinoma primary tumors, which was highly correlated to a transcriptional signature for type I conventional dendritic cells (cDC1). This was consistent with elevated frequencies of CD141/BDCA3+cDC1 in primary tumor SCC samples relative to adenocarcinoma and correspondingly elevated levels of CXCL9 and CXCL10 in 24-hour ex vivo cultures. Hampered cDC1 recruitment in adenocarcinoma was in turn related to lower transcript levels of cDC1-recruiting chemokines and an elevated β-catenin activation score and was associated with poor overall survival.Conclusions:Our data have identified an opportunity for the investigation of potentially novel therapeutic interventions in adenocarcinoma of the cervix, that is, β-catenin inhibition and cDC1 mobilization.

Novel T cell-based therapies for the treatment of B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), are thought to have strong potential. Progress, however, has been hampered by low efficacy and high toxicity. Tumor targeting by V gamma 9V delta 2 T cells, a conserved T-cell subset with potent intrinsic antitumor properties, mediated by a bispecific antibody represents a novel approach promising high efficacy with limited toxicity. Here, we describe the generation of a bispecific V gamma 9V delta 2 T-cell engager directed against CD40, which, due to its overexpression and biological footprint in malignant B cells, represents an attractive target. The CD40-targeting moiety of the bispecific antibody was selected because it can prevent CD4OL-induced prosurvival signaling and reduce CD40-mediated resistance of CLL cells to venetoclax. Selective activation of V gamma 9V delta 2 T cells in the presence of CD40(+) tumor cells induced potent V gamma 9V delta 2 T-cell degranulation, cytotoxicity against CLL and MM cells in vitro, and in vivo control of MM in a xenograft model. The CD40-bispecific gamma delta T-cell engager demonstrated lysis of leukemic cells by autologous V gamma 9V delta 2 T cells present in patient-derived samples. Taken together, our CD40 bispecific gamma delta T-cell engager increased the sensitivity of leukemic cells to apoptosis and induced a potent V gamma 9V delta 2 T cell-dependent antileukemic response. It may, therefore, represent a potential candidate for the development of novel treatments for B-cell malignancies.

Purpose:Adenocarcinoma of the uterine cervix is the second most common type of cervical cancer after squamous cell carcinoma (SCC). Although both subtypes are treated similarly, patients with adenocarcinoma have a worse prognosis. In this study, immunologic features of the tumor microenvironment in these two subsets were pursued with potential therapeutic implications.Experimental Design:The immune microenvironment of primary tumors and nonmetastatic tumor-draining lymph nodes (TDLN) was compared between patients with cervical adenocarcinoma (n = 16) and SCC (n = 20) by polychromatic flow cytometry and by transcriptional profiling of the primary tumors (n = 299) using publicly available data from The Cancer Genome Atlas (TCGA).Results:Flow cytometric analyses revealed intact T-cell differentiation in TDLNs, but hampered effector T-cell trafficking to the primary tumors in adenocarcinoma, as compared with SCC. TCGA analysis demonstrated higher expression of chemokines involved in effector T-cell homing (CXCL9/10/11) in SCC primary tumors as compared with adenocarcinoma primary tumors, which was highly correlated to a transcriptional signature for type I conventional dendritic cells (cDC1). This was consistent with elevated frequencies of CD141/BDCA3+cDC1 in primary tumor SCC samples relative to adenocarcinoma and correspondingly elevated levels of CXCL9 and CXCL10 in 24-hour ex vivo cultures. Hampered cDC1 recruitment in adenocarcinoma was in turn related to lower transcript levels of cDC1-recruiting chemokines and an elevated β-catenin activation score and was associated with poor overall survival.Conclusions:Our data have identified an opportunity for the investigation of potentially novel therapeutic interventions in adenocarcinoma of the cervix, that is, β-catenin inhibition and cDC1 mobilization.

Background The sentinel lymph node (SLN) is the first node to receive lymphatic drainage from the primary tumor and the site where naive T cells are first primed. As such it is of great importance in initiating an effective anti-tumor immune response and an attractive target for immunomodulatory agents. Pre-clinical studies have reported that i.t. administration of anti-CTLA-4 is as effective in inducing tumor eradication as systemic delivery, without the risk of treatment related side effects. However, it remains unclear whether this is due primarily to modulation of the tumor microenvironment or of tumor-draining lymph nodes (TDLN). Here, we have evaluated the safety, tolerability and immunomodulatory effects in the SLN and peripheral blood mononuclear cells (PBMC) of anti-CTLA-4/tremelimumab, delivered locally at the tumor excision site in patients with early-stage melanoma. This unique setting (post tumor excision but prior to SLN biopsy) allowed us to clinically assess the role of TDLN in the biological efficacy of CTLA-4 blockade. Methods In this phase I dose-escalation trial, patients with clinical stage I-II melanoma received one intradermal injection of tremelimumab at four dose levels (2, 5, 10 [n=3 each] or 20 mg [n=4]) around the primary excision site of the tumor, seven days prior to re-excision and SLN biopsy. Flow cytometry was performed to study viable cells from melanoma SLN and PBMC (prior to tremelimumab administration [day 0], and at 7 days, 3 weeks and 3 months after tremelimumab injection). Systemic melanoma antigen (MART-1/NY-ESO-1)-specific T cells responses were assessed by IFN-γ ELISPOT assay. Results Intradermal delivery of tremelimumab was safe and well tolerated. In terms of biological efficacy it selectively induced profound and durable decreases in Treg frequencies in both SLN and PBMC, decreased systemic MDSC rates, activated migratory dendritic cell subsets in the SLN, and induced T cell activation (by HLA-DR and ICOS up-regulation), both in SLN and PBMC. Moreover, systemic anti-melanoma T cell responses were induced (n=5) or boosted (n=2), in association with T cell activation and central-memory T cell differentiation. Of note, tumor recurrences so far were only observed in two patients who did not develop a systemic anti-tumor T cell response. Conclusions These findings indicate that i.d. administration of anti-CTLA-4 may offer a safe and promising adjuvant treatment strategy for patients with early-stage melanoma. Moreover, they demonstrate a central role for TDLN in the biological efficacy of CTLA-4 blockade and warrant the development of TDLN-targeted delivery methods for anti-CTLA-4. Acknowledgements This study received funding from the Harry J. Lloyd Charitable Trust; tremelimumab was provided by Pfizer Inc. Trial Registration NCT04274816 Ethics Approval The study was approved by the Medical Ethics Committee of the VU University Medical Center and Spaarne Gasthuis.

Background Cervical cancer (CxCa) is mainly a locally invading disease that metastasizes to loco-regional lymph node basins before involving distant organs in more advanced stages. Local immune potentiation of tumor-draining lymph nodes (TDLN) may thus protect against tumor progression. Methods To identify therapeutic targets for local immune modulation, multi-parameter flow cytometric T-cell profiling of primary cervical tumors (PT) and TDLN (n = 37) was performed. The in-vitro effect of PD-1 blockade on T-cell reactivity to HPV16 E6 oncoproteins was determined in cultures of TDLN and PT single cell suspensions (n = 19). Also, intracellular cytokine staining (ICS) upon anti-CD3 stimulation was performed in metastatic TDLN (LN+) and PT (n = 7), as well as multiplexed immunofluorescence histochemistry staining (n = 8). Results Our data revealed elevated rates of activated regulatory T cells (aTregs) and of central or effector memory CD8+ T cells in metastatic TDLN (LN+) as compared to tumor-free TDLN (LN-), and equally high or even higher rates of these subsets in PT. Both memory subsets co-expressed multiple immune checkpoints. PD-1 blockade significantly enhanced detectable E6-specific T-cell responses in 4/5 HPV16+ LN+ and in 1/5 HPV16+ PT. Whereas aTreg rates were higher in anti-PD-1 non-responders, in responders elevated levels of CD8+FoxP3+CD25+ T cells were observed, which correlated with the efficacy of PD-1 blockade (P = 0.018). This subset was characterized by an early effector memory phenotype with particularly high levels of co-expressed PD-1, CTLA-4, TIM-3 and LAG-3 checkpoints, but, rather than exhausted, was shown upon polyclonal activation to produce higher levels of Granzyme-B and effector cytokines as compared to its CD8+FoxP3− counterparts. Conclusion These observations support local PD-(L)1 blockade to interrupt loco-regional immune suppression in CxCa and control metastatic spread to TDLN. Furthermore, our data identify CD8+FoxP3+CD25+ T cells as therapeutic targets, which may also serve as predictive biomarker for PD-(L)1 checkpoint blockade. Electronic supplementary material The online version of this article (10.1186/s40425-019-0526-z) contains supplementary material, which is available to authorized users.

To mount a strong anti-tumor immune response, non T cell inflamed (cold) tumors may require combination treatment encompassing vaccine strategies preceding checkpoint inhibition. In vivo targeted delivery of tumor-associated antigens (TAA) to dendritic cells (DCs), relying on the natural functions of primary DCs in situ, represents an attractive vaccination strategy. In this study we made use of a full-length MART-1 expressing C/B-chimeric adenoviral vector, consisting of the Ad5 capsid and the Ad3 knob (Ad5/3), which we previously showed to selectively transduce DCs in human skin and lymph nodes. Our data demonstrate that chimeric Ad5/3 vectors encoding TAA, and able to target human DCs in situ, can be used to efficiently induce expansion of functional tumor-specific CD8+ effector T cells, either from a na&iuml, ve T cell pool or from previously primed T cells residing in the melanoma-draining sentinel lymph nodes (SLN). These data support the use of Ad3-knob containing viruses as vaccine vehicles for in vivo delivery. &ldquo, Off-the-shelf&rdquo, DC-targeted Ad vaccines encoding TAA could clearly benefit future immunotherapeutic approaches.

Irreversible electroporation (IRE) may result in a form of immunogenic tumor cell death that converts the tumor microenvironment from immune suppressive to immune permissive. The resulting influx of pro-inflammatory innate immune cells and subsequent uptake of apoptotic cell fragments could ultimately result in the priming of tumor-specific killer T cells in the lymph nodes directly draining the tumor ablation site. These T cells in turn will recirculate through peripheral blood and may thus provide systemic protection against the outgrowth of distant metastases. Local tumor ablation through IRE may thus be translated into a systemic antitumor treatment by virtue of secondarily induced T cell priming. These processes may be further facilitated by the primarily nonthermal mechanism of action of IRE, resulting in the conservation of the intra- and peritumoral vasculature and the preserved chemical structure of damage-associated molecular patterns (DAMPs) that are vital for proper immune activation. In this chapter, different clinically explored local tumor ablation techniques are reviewed in relation to their ability to prime or boost an antitumor immune response. Evidence for similar antitumor immune effects of IRE are discussed, as well as ways in which this antitumor immune efficacy may be enhanced through combination treatment with immune modulatory regimens.

Though Vγ9Vδ2-T cells constitute only a small fraction of the total T cell population in human peripheral blood, they play a vital role in tumor defense and are therefore of major interest to explore for cancer immunotherapy. Vγ9Vδ2-T cell-based cancer immunotherapeutic approaches developed so far have been generally well tolerated and were able to induce significant clinical responses. However, overall results were inconsistent, possibly due to the fact that these strategies induced systemic activation of Vγ9Vδ2-T cells without preferential accumulation and targeted activation in the tumor. Here we show that a novel bispecific nanobody-based construct targeting both Vγ9Vδ2-T cells and EGFR induced potent Vγ9Vδ2-T cell activation and subsequent tumor cell lysis both in vitro and in an in vivo mouse xenograft model. Tumor cell lysis was independent of KRAS and BRAF tumor mutation status and common Vγ9Vδ2-T cell receptor sequence variations. In combination with the conserved monomorphic nature of the Vγ9Vδ2-TCR and the facile replacement of the tumor-specific nanobody, this immunotherapeutic approach can be applied to a large group of cancer patients.

Autologous tumor cell-based vaccines provide a wide range of tumor antigens and personalized neo-epitopes based on individual tumors’ unique antigenic mutanome signatures. However, tumor-derived factors may hamper in situ maturation of dendritic cells (DC) and thus interfere with the generation of effective anti-tumor immunity. As the skin is a preferred site for tumor vaccine delivery, we investigated the influence of primary colon carcinoma-derived soluble factors on the maturation state of migrating DC in a human skin explant model. Primary tumor-derived supernatants (TDSN) enhanced the phenotypic maturation state of skin-emigrated DC, resulting in an increased T-cell stimulatory ability in an allogeneic mixed leukocyte response. In case of monocyte-derived DC a similar TDSN-induced maturation induction was found to entirely depend on cyclooxygenase (COX)-regulated prostaglandins. In contrast, the increase in skin-emigrated DC maturation was completely prostaglandin-independent, as evidenced by the inability of the COX inhibitor indomethacin to abrogate this TDSN-induced effect. Although TDSN conditioning affected a drop in IL-12p70 release by the skin-emigrated DC and induced a predominant Th17/Th22 transcriptional profile in subsequently stimulated T-cells, Th cell subset differentiation, as assessed by intracellular cytokine expression upon polyclonal priming and re-stimulation, was not affected. Comparative analysis of phenotypic and transcriptional profiles suggests that the observed maturational effects in skin-derived DC may have been induced by tumor-derived GM-CSF. In conclusion, soluble factors derived from whole-cell colon tumor vaccines will not negatively impact DC migration and maturation in human skin, but rather induce DC maturation that will facilitate the priming of a poly-functional Th cell response.

Purpose To (a) investigate the safety of percutaneous irreversible electroporation (IRE) for locally advanced pancreatic cancer and (b) evaluate the quality of life (QOL), pain perception, and efficacy in terms of time to local progression, event-free survival, and overall survival (OS). Materials and Methods The study was approved by the local review board (NL42888.029.13). All patients provided written informed consent for study participation, the ablation procedure, and data usage. Between January 2014 and June 2015, 25 patients with histologically proved locally advanced pancreatic cancer 5 cm or smaller (13 women, 12 men; median age, 61 years; age range, 41-78 years) were prospectively included to undergo percutaneous computed tomographic-guided IRE. Patients with a metallic biliary Wallstent, epilepsy, or ventricular arrhythmias were excluded. Kaplan-Meier estimates were used to investigate time to local progression, event-free survival, and OS. Safety was assessed on the basis of adverse events, which were graded according to the Common Terminology Criteria for Adverse Events. Pain perception and QOL were evaluated by using specific questionnaires. Results All patients underwent IRE. The median largest tumor diameter was 4.0 cm (range, 3.3-5.0 cm). After a median follow-up of 12 months (interquartile range: 7-16 months), median event-free survival after IRE was 8 months (95% confidence interval [CI]: 4 months, 12 months); the median time to local progression after IRE was 12 months (95% CI: 8 months, 16 months). The median OS was 11 months from IRE (95% CI: 9 months, 13 months) and 17 months from diagnosis (95% CI: 10 months, 24 months). There were 12 minor complications (grade I or II) and 11 major complications (nine grade III, two grade IV) in 10 patients. There were no deaths within 90 days after IRE. Conclusion Percutaneous IRE for locally advanced pancreatic cancer is generally well tolerated, although major adverse events can occur. Preliminary survival data are encouraging and support the setup of larger phase II and III clinical trials to assess the efficacy of IRE plus chemotherapy in the neoadjuvant and adjuvant or second-line setting compared with more widely adopted regimens such as chemotherapy and/or radiation therapy. (©) RSNA, 2016 Online supplemental material is available for this article.

Item does not contain fulltext Immune checkpoint blockade enhances antitumor responses, but can also lead to severe immune-related adverse events (IRAE). To avoid unnecessary exposure to these potentially hazardous agents, it is important to identify biomarkers that correlate with clinical activity and can be used to select patients that will benefit from immune checkpoint blockade. To understand the consequences of CTLA-4 blockade and identify biomarkers for clinical efficacy and/or survival, an exploratory T cell monitoring study was performed in a phase I/II dose escalation/expansion trial (n = 28) of combined Prostate GVAX/ipilimumab immunotherapy. Phenotypic T cell monitoring in peripheral blood before and after Prostate GVAX/ipilimumab treatment revealed striking differences between patients who benefited from therapy and patients that did not. Treatment-induced rises in absolute lymphocyte counts, CD4(+) T cell differentiation, and CD4(+) and CD8(+) T cell activation were all associated with clinical benefit. Moreover, significantly prolonged overall survival (OS) was observed for patients with high pre-treatment frequencies of CD4(+)CTLA-4(+), CD4(+)PD-1(+), or differentiated (i.e., non-naive) CD8(+) T cells or low pre-treatment frequencies of differentiated CD4(+) or regulatory T cells. Unsupervised clustering of these immune biomarkers revealed cancer-related expression of CTLA-4(+) in CD4(+) T cells to be a dominant predictor for survival after Prostate GVAX/ipilimumab therapy and to thus provide a putative and much-needed biomarker for patient selection prior to therapeutic CTLA4 blockade.

Item does not contain fulltext BACKGROUND: The granulocyte-macrophage colony-stimulating factor-transduced allogeneic prostate cancer cells vaccine (GVAX) has antitumour activity against prostate cancer; preclinical studies have shown potent synergy when combined with ipilimumab, an antibody that blocks cytotoxic T-lymphocyte antigen 4. We aimed to assess the safety of combined treatment with GVAX and ipilimumab in patients with metastatic castration-resistant prostate cancer (mCRPC). METHODS: We did an open-labelled, single-centre, dose-escalation study of ipilimumab concurrent with a fixed dose of GVAX, with a subsequent expansion phase, both at the VU University Medical Centre (Amsterdam, Netherlands). Eligible patients had documented mCRPC and had not been previously treated with chemotherapy. All patients received a 5x10(8) cell priming dose of GVAX intradermally on day 1 with subsequent intradermal injections of 3x10(8) cells every 2 weeks for 24 weeks. The vaccinations were combined with intravenous ipilimumab every 4 weeks. We enrolled patients in cohorts of three; each cohort received an escalating dose of ipilimumab at 0.3, 1.0, 3.0, or 5.0 mg/kg. Our primary endpoint was safety. This study is registered with ClinicalTrials.gov, number NCT01510288. FINDINGS: We enrolled 12 patients into our dose-escalation cohort. We did not record any severe immune-related adverse events at the first two dose levels. At the 3.0 mg/kg dose level, one patient had grade 2 and two patients grade 3 hypophysitis; at the 5.0 mg/kg dose level, two patients had grade 3 hypophysitis and one patient developed grade 4 sarcoid alveolitis (a dose-limiting toxic effect). Due to observed clinical activity and toxic events, we decided to expand the 3.0 mg/kg dose level, rather than enrol a further three patients at the 5.0 mg/kg level. 16 patients were enrolled in the expansion cohort, two of whom developed grade 2 hypophysitis, three colitis (one grade 1 and two grade 2), and one grade 3 hepatitis--all immune-related adverse events. The most common adverse events noted in all 28 patients were injection-site reactions (grade 1-2 events seen in all patients), fatigue (grade 1-2 in 20 patients, grade 3 in two), and pyrexia (grade 1-2 in 15 patients, grade 3 in one). 50% or greater declines in prostate-specific antigen from baseline was recorded in seven patients (25%); all had received 3.0 mg/kg or 5.0 mg/kg ipilimumab. INTERPRETATION: GVAX combined with 3.0 mg/kg ipilimumab is tolerable and safe for patients with mCRPC. Further research on the combined treatment of patients with mCRPC with vaccination and ipilimumab is warranted. FUNDING: Cell Genesys Inc, Prostate Cancer Foundation, Dutch Cancer Society (KWF-VU 2006-3697), and Foundation Stichting VUmc Cancer Center Amsterdam.

We have been studying the re-activation of tumor-associated antigen (TAA)-specific CD8 + T cells in sentinel lymph nodes (SLN) of melanoma patients upon intradermal administration of the CpG-B oligodeoxynucleotide PF-3512676. To facilitate functional testing of T cells from small SLN samples, high-efficiency polyclonal T cell expansion is required. In this study, SLN cells were expanded via classic methodologies with plate- or bead-bound anti-CD3/CD28 antibodies and with the K562/CD32/4-1BBL artificial APC system (K32/4-1BBL aAPC) and analyzed for responsiveness to common recall or TAA-derived peptides. K32/4-1BBL-expanded T cell populations contained significantly more effector/memory CD8 + T cells. Moreover, recall and melanoma antigen-specific CD8 + T cells were more frequently detected in K32/4-1BBL-expanded samples as compared with anti-CD3/CD28-expanded samples. We conclude that K32/4-1BBL aAPC are superior to anti-CD3/CD28 antibodies for the expansion of in vivo -primed specific CD8 + T cells and that their use facilitates the sensitive monitoring of functional anti-tumor T cell immunity in SLN.

Dendritic cells (DC) are increasingly applied as a cellular adjuvant in immunotherapy of cancer. Two major myeloid DC subsets are recognized: interstitial DC (IDC) that infiltrate connective tissues and Langerhans cells (LC) that line epithelial surfaces. Yet, functional differences between IDC and LC remain to be defined. We recently showed that the CD34+ acute myeloid leukemia cell line MUTZ-3 supports differentiation of both DC-SIGN+ IDC and Langerin-positive Birbeck granule-expressing LC. By comparative functional characterization of MUTZ-3 IDC and MUTZ-3 LC, we aimed to elucidate the relative abilities of these two DC subsets to induce a specific T cell response and reveal the more suitable candidate for use as a clinical vehicle of tumor vaccines. Although mature LC and IDC displayed comparable lymph node-homing potential, mature LC showed higher allogeneic T cell stimulatory capacity. Nevertheless, IDC supported the induction of tumor Ag-specific CD8+ T cells at an overall higher efficiency. This might be related to the observed inability of LC to release T cell stimulatory cytokines such as IL-12p70, IL-23, and IL-15. Although this inability did not result in a detectable deviation in the cytokine expression profile of primed T cells, transduction with IL-12p70 significantly improved priming efficiency of LC, and ensured a functional equivalence with IDC in this regard. In conclusion, except for the inability of LC to release distinct type 1 T cell stimulatory cytokines, in vitro function of LC and IDC suggests comparable abilities of both subsets for the in vivo induction of antitumor T cells.

CD1d-restricted natural killer T (NKT) cells are involved in the regulation of various immune responses, and have been shown to inhibit viral replication in animal hepatitis models when activated by the glycolipid alpha-galactosylceramide (alpha-GalCer, KRN7000). Previous studies have indicated that alpha-GalCer-induced activation of the immune system requires both CD1d expression by antigen-presenting cells as well as (normal) numbers of NKT cells. Discrepancies exist over circulating numbers of human invariant Valpha24+Vbeta11+ NKT cells during hepatitis C virus (HCV) infection. Here, by cross-sectional analysis and longitudinal analysis of patients undergoing effective combination antiviral therapy, we demonstrate that circulating Valpha24+Vbeta11+ NKT cell numbers are not decreased during active HCV infection. Importantly, as we also show that CD1d is expressed at comparable levels by peripheral blood monocytes and CD1c+ myeloid dendritic cells (DC) of healthy individuals and HCV-infected patients, these data indicate that all ingredients for evaluating the antiviral effects of the Valpha24+Vbeta11+ NKT cell ligand alpha-GalCer in HCV-infected patients are present.

Purpose: Immunotherapy holds promise as a new strategy for the eradication of residual cells in acute myeloid leukaemia (AML). Leukaemic antigen presenting cells (APCs) combining optimal antigen presentation and tumour antigenicity could be used as potent T cell activators. For clinical purposes it is desirable to culture APCs under serum-free conditions. Therefore, we compared morphological, immunophenotypical and functional outcome of the serum-free culture of AML-APCs to their serum-enriched culture. Methods: AML blasts (n=19) were cultured in the presence of either a cytokine mix or calcium ionophore (CI) for 14 and 2 days, respectively, in FCS-containing medium (FCS), StemSpan serum-free medium (SP) and CellGro serum-free medium (CG). After culture relative yields were calculated and immunophenotypic analysis of APC markers was performed. The mixed leukocyte reaction (MLR) was used to determine T cell stimulating capacity. Results: Serum-free culture of AML-APCs resulted in comparable morphology, relative yields and immunophenotype to serum-enriched culture. By comparing both serum-free media we observed a trend towards a more mature phenotype of CI-cultured AML-APCs in SP. MLR showed that serum-free cultured cells have equal T cell stimulatory capacity in comparison with serum-enriched culture. Conclusion: These data show that the serum-free culture of AML-APCs is feasible and that these APCs are comparable to serum-enriched cultured AML-APCs with regard to morphological, immunophenotypical and functional characteristics. These AML-APCs are suitable for the development of active specific immunisation protocols which meet the criteria for good clinical practise (GCP).

The ability of acute myeloid leukaemia (AML) cells to acquire dendritic cell (DC)-like characteristics in vitro with a rapid culture method based either on the phorbol ester PMA or calcium ionophores has been studied in comparison to conventional AML-DC cultures with the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-α), interleukin-3 (IL-3), SCF, FLT3-L and IL-4. In all AML patients, antigen-presenting cells (APC) could be generated from leukaemic cells in 2 days by incubation with PMA or calcium ionophore (A23187 or ionomycin) in the presence as well as in the absence of IL-4. In 30 out of 36 patients APC could be generated after 2 weeks of culture in cytokine-enriched medium. AML-APC cultured with PMA or calcium ionophores immunophenotypically and functionally were at a more mature stage than those cultured in cytokine-enriched medium. The most mature APC were generated by calcium ionophore A23187 plus IL-4, as evidenced by the higher expression of CD40, CD80, CD86 and HLA-DR. Autologous T cell mediated cytotoxicity towards AML blast cells in vitro was observed in 2 cases tested. The persistence of cytogenetic abnormalities confirmed the leukaemic origin of the AML-APC. The generation of AML-APC was possible from freshly isolated as well as cryopreserved material. Our data show that generation of sufficient AML-APC by A23187 plus IL-4 is feasible, for vaccination purposes, in approximately 70% of AML specimens, offering a time-saving and cost-effective approach in preparing anti-leukaemia vaccines.

P-glycoprotein (Pgp) and vaults are associated with multidrug resistance in tumor cells, but their physiological functions are not yet clear. Pgp, the prototypical transmembrane transporter molecule, may also facilitate the migration of skin dendritic cells (DC). Vaults—ribonucleoprotein cell organelles, frequently overexpressed in Pgp-negative drug-resistant tumor cells—have also been associated with intracellular transport processes. Given the pivotal role of DC in dealing with exposure to potentially harmful substances, the present study was set out to examine the expression of Pgp and vaults during differentiation and maturation of DC. DC were obtained from different sources, including blood-derived monocytes, CD34+ mononuclear cells, and chronic myeloid leukemia cells. Whereas flow cytometric and immunocytochemical analyses showed slightly augmented levels of Pgp, up-regulation of vault expression during DC culturing was strong, readily confirmed by Western blotting, and independent of the source of DC. In further exploring the functional significance of vault expression, it was found that supplementing DC cultures with polyclonal or mAbs against the major vault protein led to lower viabilities of LPS- or TNF-α-matured monocytes-DC. Moreover, expression of critical differentiation, maturation, and costimulatory molecules, including CD1a and CD83, was reduced and their capacity to induce Ag-specific T cell proliferative and IFN-γ release responses was impaired. These data point to a role for vaults in both DC survival and functioning as APC.

de Bruin, R. C. G., A. G. M. Stam, A. Vangone, P. M. P. van Bergen en Henegouwen, H. M. W. Verheul, Z. Sebestyen, J. Kuball, A. M. J. J. Bonvin, T. D. de Gruijl, and H. J. van der Vliet. 2017. Prevention of Vγ9Vδ2 T cell activation by a Vγ9Vδ2 TCR nanobody. J. Immunol . 198: [308–317][1].

Background: Currently there is no standard adjuvant treatment following surgical resection of metastatic melanoma. We investigated whether surgery followed by autologous tumor cell-BCG vaccination was beneficial for malignant melanoma patients. In this study we focus on the prognostic value of DTH response following vaccination therapy. Patients and methods: Eighty-one patients with AJCC stage III and IV melanoma were selected. Whenever feasible, radical metastasectomy was performed. ASI was initiated by the administration of three weekly intra-cutaneous vaccinations with l07 irradiated autologous tumor cells, starting four weeks after surgery. Depending on the size of DTH response to the first three injections, subsequent vaccinations were planned. The first two vaccines also contained l07 BCG organisms as an immune stimulatory adjuvant. Results: Induration as well as erythema correlated strongly with survival (P < 0.0001 and P = 0.0004). After radical metastasectomy in stage III melanoma patients a five-year survival of 48% was observed. In stage IV disease, a five-year survival of 34% was seen, after radical surgery had been performed. When macroscopic disease was present at start of vaccination treatment, no clinical responses occurred. Apart from transient skin ulceration at the site of BCG-containing vaccinations, no serious side effects were observed. Conclusions: This study shows that large-scale preparation of autologous melanoma cell vaccines is feasible, while vaccination results in DTH responses that correlate significantly with survival. ASI seemed to be beneficial in stage III and stage IV melanoma when given in the adjuvant setting, while causing only very mild side effects.

Active immunotherapy may prevent the relapse of acute myeloid leukemia (AML) by inducing leukemia-specific T cells. Here, we investigated whether Wilms' tumor 1 (WT1) and preferentially expressed antigen in melanoma (PRAME)-specific T cells could be induced upon the priming of healthy donor- and AML patient-derived T cells with HLA-A2-matched, peptide-loaded allogeneic dendritic cells. AML-reactive, tetramer (Tm)-binding and interferon-producing, cytotoxic T lymphocytes specific for PRAME could readily be isolated from healthy individuals and maintained in culture. In this setting, priming efficacy was significantly higher for PRAME than for WT1. The priming of T cells from patient-derived material proved to be near-to-impossible: No leukemia-associated antigen (LAA)-specific T cell could be primed in 4 patients that had recently achieved a complete response (CR), and in only 1 out of 3 patients exhibiting a sustained CR we did observe WT1-specific T cells, though with a low frequency. These findings suggest that the functionality and/or repertoire of T cells differ in healthy subjects and AML patients in CR, and may have repercussions for the implementation of active vaccination approaches against AML.

The toxicity of high-dose recombinant interleukin-2 (rIL-2) treatment limits its use in tumour therapies. This paper describes in vitro studies of whether a single, peak rIL-2 dose, followed by low maintenance doses, could enhance the cytotoxic potential of peripheral blood mononuclear cells (PBMC) without inducing a significant sustained release of secondary cytokines, known to contribute to undesirable side-effects of therapy. Pre-pulsing of PBMC with high-dose rIL-2 (16,000 IU/ml for 30 min), followed by low-dose (5 IU/ml) maintenance culturing, was found to induce persistent augmentation of cytotoxic activity towards natural-killer(NK)-sensitive and -insensitive tumour targets, as well as increased T-cell-mediated target cell killing. Under these conditions the level of killing was as high as that achieved by higher maintenance doses (20-100 IU/ml). Although not reflected by overexpression of cell surface markers, enhanced activation of cytotoxic capacities by high-dose pre-pulsing remained clearly apparent for at least 12 days of culture. Increased secondary cytokine production (tumour necrosis factor, interleukin-6 and interferon gamma) was only evident during the first 24-72 h after pulsing, and not at later stages of culturing at the low maintenance dose of 5 IU rIL-2/ml. These results may warrant a human phase-1 B study to investigate the in vivo effect of high-dose prepulsing, followed by low-dose maintenance.

While systemic immune checkpoint blockade is unsurpassed in the induction of clinically effective T cell immunity in advanced-stage melanoma patients, the risk of autoimmune-related side effects precludes its application in earlier stages. In a Phase-1 dose escalation study we have studied the safety, feasibility and immunological effects of intradermal delivery of a single low dose of anti-CTLA-4/tremelimumab at the primary tumor excision site of patients with clinically Stage I-II melanoma. Dose escalation (4x3 cohorts of 2, 5, 10 and 20 mg) has been concluded with one additional patient enrolled in a 20 mg expansion cohort. Breslow thickness ranged from 0.9 to 6 mm (mean 1.98 mm). Examination of the sentinel lymph node (SLN) revealed (micro-)metastases in five of the patients. The tremelimub administration was found to be safe without apparent side effects except for one case of localized vitiligo at the highest (20 mg) dose level. Of note, potent immunological effects were observed in equal measure at all dose levels. In all patients a decrease in systemic rates of activated Tregs (aTregs, defined as CD4+CD25hiFoxP3hi T cells) was observed after 7 days of treatment (p Specific T cell reactivity against long peptides derived from the melanoma antigens NY-ESO-1 and MART-1 was tested after in vitro re-stimulation in an IFNγ Elispot assay. In 7/13 patients evidence was found of tremelimumab-induced increases in systemic T cell rates, which remained elevated up to 12 weeks post-treatment. Remarkably, 5/13 patients showed pre-existent NY-ESO-1 reactivity, which in all cases had decreased by day 7, and in 3/5 patients had increased over baseline levels by day 21. T cell reactivity to either NY-ESO-1 or MART-1 was observed in 4/4 tested SLN. Of note, both NY-ESO-1 and MART-1 reactive T cells were detected in the SLN of the patient experiencing an isolated incidence of vitiligo after receiving 20 mg tremelimumab. In conclusion, local administration of a single low dose of anti-CTLA-4/tremelimumab has proven safe and effective in terms of attenuating aTreg levels in peripheral blood and boosting systemic antitumor immunity. As such it may offer an attractive, and up to now sorely lacking, adjuvant treatment option for early-stage melanoma patients at risk for tumor recurrence. Citation Format: Anita G.M. Stam, Kim M. van Pul, Bas D. Koster, Dafni Chondronasiou, Sinéad M. Lougheed, M. Petrousjka van den Tol, Karin Jooss, Ronald J.C.L.M. Vuylsteke, Alfons J.M. van den Eertwegh, Tanja D. de Gruijl. Single low-dose administration of anti-CTLA-4/tremelimumab at the tumor excision site boosts systemic antitumor immunity in early-stage melanoma patients: Results from a Phase-1 study. [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 B138.

Tumor-derived vascular endothelial growth factor (VEGF) has previously been identified as a causative factor in the disturbed differentiation of myeloid dendritic cells (DC) in advanced cancer patients. Here, we investigated the potential of vascular endothelial growth factor receptor (VEGFR) tyrosine kinase (TK) inhibition to overcome this defective DC differentiation. To this end, peripheral blood DC (PBDC) precursor and subset frequencies were measured in 13 patients with advanced cancer before and after treatment with AZD2171, a TK inhibitor (TKI) of VEGFR, coadministered with gefitinib, and an epidermal growth factor receptor (EGFR) TKI. Of note, not only myeloid DC but also plasmacytoid DC frequencies were significantly reduced in the blood of the cancer patients prior to treatment, as compared to healthy controls. Moreover, besides an accumulated population of immature myeloid cells (ImC), a population of myeloid suppressor cells (MSC) was significantly increased. Upon systemic VEGFR TK inhibition, DC frequencies did not increase, whereas the rate of circulating MSC showed a slight, but not significant, decrease. In conclusion, TK inhibition of VEGFR with AZD2171 does not restore the defective PBDC differentiation observed in advanced cancer patients.

Vaccination with autologous leukaemia-derived dendritic cells (DC) presents an adjuvant treatment option for chronic myeloid leukaemia (CML). Here, we show that high-efficiency CD40-targeted adenoviral gene transfer of GM-CSF to CML-derived DC induces long-lived maturation in the absence of exogenous cytokines and may thus ensure protracted stimulation of CML-specific T cells upon vaccination.

Vaccination of chronic myeloid leukemia patients with autologous in vitro cultured leukemic dendritic cells

In a phase-I dose escalation trial in patients (N = 28) with CRPC we showed that GVAX and ipilimumab had an acceptable safety profile (Lancet Oncology 2012). We observed tumor responses and prolonged survival. Patients had CRPC and were chemotherapy-naive. They received bi-weekly GVAX for a 24 week period combined with monthly intravenous administrations of ipilimumab. Each cohort of 3 patients received an escalating dose of ipilimumab at 0·3, 1·0, 3·0 or 5·0 mg/kg. In an expansion cohort 16 patients were treated with GVAX and 3·0 mg/kg ipilimumab. Since the teratment can be accompanied by side-effects (colitis, hypophysitis), we looked for lymphoid and myeloid markers as well as antibody formation. We observed a significantly prolonged OS for patients with high pre-treatment frequencies of CD4 + CTLA-4 + , CD4 + PD-1 + , or differentiated CD8+ T cells, or low pre-treatment frequencies of differentiated CD4+ T cells or CD4 + CD25hiFoxP3+ regulatory T cells. In contrast, increased frequencies of granulocytic Myeloid-Derived Suppressor Cells and high pre-treatment frequencies of monocytic CD14 + HLA-DRlo/- MDSC were associated with reduced OS. Antibody formation against PSMA, NRP2, and PNPO was associated with prolonged survival and especially when multiple antibodies were detected. Conclusions Together these data provide an immune profile to predict clinical outcome; both pre-treatment CD4+ T-cells as well as antibody formation was associated with prolonged survival. These potentially biomarkers for patient selection should be validated in patients treated with ipilimumab. Disclosure W.R. Gerritsen: member of advisory board of BMS, K. Hege: former employee of Cell Genesys, N. Sacks: former employee of Cell Genesys, I. Lowy: former employee of Medarex, T. Harding: former emloyee of Cell Genesys, A. van den Eertwegh: member advisory board BMS, T. De Gruijl: educational grant from Cell Genesys. All other authors have declared no conflicts of interest.

We have been studying the safety and clinical efficacy of the Prostate GVAX vaccine (consisting of two allogeneic GM-CSF secreting tumor cell lines: LNCaP and PC3) and the CTLA-4 blocking antibody ipilimumab in prostate cancer patients in a phase I/II dose escalation/expansion trial. To assess immune reactivity against the GVAX vaccine, immune infiltrate analysis of the vaccination sites was performed. Punch biopsies of the vaccination site were taken 48 hours after the 1st, 5th, and 11th injections and analyzed by immunohistochemical staining. Injection of the GVAX vaccine evoked striking local reactions that were characterized by local erythema, induration and swelling in all patients. Analysis of the vaccination site biopsies revealed that the GVAX/ipilimumab combination resulted in an inflammatory response in the skin with intradermal infiltrates of CD1a+ dendritic cells (DC), DC-SIGN+/CD68+ immature DC/macrophages, CD14+ monocyte-like cells, eosinophils, and of CD4+, CD8+, and Granzyme-B+ T lymphocytes. Strikingly, CD56+ NK cells were completely absent. The inflammatory response significantly increased over the course of treatment and resulted in accelerated clearance of the vaccine in vivo. Moreover, analysis revealed that the PC3 vaccine was more immunogenic, and induced a more potent anti-vaccine T cell response. The latter was confirmed in an in vitro cytotoxicity assay. Upon multiple vaccinations T cell reactivity to PC3 was significantly enhanced in the PC3 but not in the LNCaP vaccination sites, whereas reactivity to LNCaP was not enhanced in either vaccination site. We conclude that the GVAX/ipilimumab combination elicits a local immune infiltrate that increases over time, consistent with an induced CD8+ effector T cell mediated cytolytic response, only in vaccination sites of the PC3 component of the vaccine. Citation Format: Saskia J. Santegoets, Leonieke JJ van Mens, Jeroen AM Beliën, Anita GM Stam, Sinead M. Lougheed, Rik J. Scheper, Shabnam Samiei, Helen Gall, Karin Jooss, Jean-Marie Cuillerot, Natalie Sacks, Kirsten Hege, Winald R. Gerritsen, Alfons JM van den Eertwegh, Tanja D. de Gruijl. Inflammatory responses with infiltrating DC and functional cytotoxic T lymphocytes in the dermal vaccination sites of GVAX/ipilimumab treated prostate cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 496. doi:10.1158/1538-7445.AM2013-496

The effects of a GM-CSF-secreting allogeneic prostate cancer vaccine (Prostate GVAX) and the anti-CTLA4 antibody ipilimumab were investigated in a Phase I dose escalation/expansion trial of patients with metastatic castration resistant prostate cancer (van den Eertwegh et al, Lancet Oncology, 13: 509-17 2012). The GVAX and ipilimumab combination treatment led to PSA declines of more than 50% (Partial Response, PR) in 5 of 28 patients, PSA stabilizations (Stable Disease, SD) in 12 of 28 patients and disease progression (PD) in 11 of 28 patients. Regressing bone and lymph node metastases were observed in 2/5 PR patients. As an indication of tumor antigen-specific responsiveness we analyzed serologic and T cell responses against NY-ESO-1 before, during and after treatment. NY-ESO-1 seroreactivity (determined by ELISA) was observed in seven of 28 patients (25%). Five patients were seropositive at baseline and 2 became seropositive following treatment; no significant correlations were found with clinical benefit or survival. NY-ESO-1 T cell reactivity was determined in 19 patients by IFNγ ELISPOT after stimulation with a peptide pool of overlapping 20-mer peptides covering the immunodominant region aa121-180. NY-ESO-1-specific T cell responses were detected in 13 of 19 patients (68%) at any time before or during treatment. Increases in the frequency of NY-ESO-1 specific T cells following treatment were only observed in five of these (26%). A remarkably high number of nine out of 19 patients (47%) already exhibited NY-ESO-1-specific T cell reactivity before treatment. Interestingly, three of four (75%) PR patients displayed pre-existing frequencies of NY-ESO-1 T cells, whereas these cells were only detected in six of 15 SD/PD (40%) patients. Moreover, patients with pre-existing NY-ESO-1 T cell reactivity demonstrated a significantly prolonged overall survival (p=0.044, log-rank test). These data suggest that baseline NY-ESO-1 T cell reactivity may have predictive value for the clinical outcome of prostate GVAX and/or ipilimumab treatment. Citation Format: Anita GM Stam, Saskia JAM Santegoets, Alfons JM van den Eertwegh, Rik J. Scheper, Sinéad M. Lougheed, Helen Gall, Karin Jooss, Natalie Sacks, Thomas Harding, Kristen Hege, Israel Lowy, Sacha Gnjatic, Jedd D. Wolchok, Winald R. Gerritsen, Tanja D. de Gruijl. Pre-existing NY-ESO-1-specific T cell reactivity is associated with improved clinical outcome in castration resistant prostate cancer patients treated with Prostate GVAX and ipilimumab. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1229. doi:10.1158/1538-7445.AM2013-1229

Contains fulltext : 118572.pdf (Publisher’s version ) (Open Access) BACKGROUND: Adoptive cell transfer of tumor infiltrating lymphocytes has shown clinical efficacy in the treatment of melanoma and is now also being explored in other tumor types. Generation of sufficient numbers of effector T cells requires extensive ex vivo expansion, often at the cost of T cell differentiation and potency. For the past 20 years, IL-2 has been the key cytokine applied in the expansion of TIL for ACT. However, the use of IL-2 has also led to collateral expansion of regulatory T cells (Tregs) and progressive T cell differentiation, factors known to limit in vivo persistence and activity of transferred TIL. The use of alternative T cell growth factors is therefore warranted. Here, we have compared the effects of IL-2, -15 and -21 cytokines on the expansion and activation of TIL from single-cell suspensions of non-small cell lung cancer, ovarian cancer and melanoma. METHODS: We applied the K562-based artificial APC (aAPC) platform for the direct and rapid expansion of tumor infiltrating lymphocytes isolated from primary cancer specimens. These aAPC were engineered to express the Fc-gamma receptor CD32 (for anti-CD3 antibody binding), the co-stimulatory molecule 4-1BBL, and to secrete either IL-2, IL-15 or IL-21 cytokine. RESULTS: Although IL-2 aAPC induced the greatest overall TIL expansion, IL-21 aAPC induced superior expansion of CD8+ T cells with a CD27+CD28+ "young" phenotype and superior functional cytotoxic effector characteristics, without collateral expansion of Tregs. CONCLUSION: Our data rationalize the clinical application of IL-21-secreting aAPC as a standardized cell-based platform in the expansion of "young" effector TIL for ACT.

2562 Background: In a phase-I dose escalation trial in patients with castration-resistant prostate cancer we showed that GVAX and ipilimumab had an acceptable safety profile. Moreover, we observed tumor responses and prolonged survival as compared to the Halabi predicted overall survival (OS). However, ipilimumab can also lead to severe immune-related adverse events. To avoid unnecessary exposure to this risk, it is essential to identify biomarkers that correlate with clinical activity. Methods: Patients had castration-resistant prostate cancer and were chemotherapy-naïve. They received bi-weekly GVAX for a 24 week period combined with monthly intravenous administrations of ipilimumab. Each cohort of 3 patients received an escalating dose of ipilimumab at 0·3, 1·0, 3·0 or 5·0 mg/kg. In an expansion cohort 16 patients were treated with GVAX and 3·0 mg/kg ipilimumab. Flowcytometric monitoring of lymphoid and myeloid subsets in blood were performed. Results: We observed a significantly prolonged OS for patients with high pre-treatment frequencies of CD4+CTLA-4+, CD4+PD-1+, or differentiated CD8+ T cells, or low pre-treatment frequencies of differentiated CD4+ T cells or CD4+CD25hiFoxP3+ regulatory T cells. In contrast, increased frequencies of granulocytic Myeloid-Derived Suppressor Cells (MDSC) and high pre-treatment frequencies of monocytic CD14+HLA-DRlo/- MDSC were associated with reduced OS. Treatment-induced CD4+ T cell differentiation and CD4+ and CD8+ T cell activation was associated with clinical benefit. Moreover, treatment-induced activation of CD1c+ conventional Dendritic Cells (cDC) and 6-sulfo LacNAc+ inflammatory DC were associated with significantly prolonged OS. Conclusions: Together these data provide an immune profile to predict clinical outcome. Importantly, cluster analysis revealed pre-treatment, CRPC-associated expression of CTLA-4+ by CD4+ T cells to be a dominant predictor for OS after GVAX/ipilimumab. This potentially biomarker for patient selection should be validated in patients treated with ipilimumab.

Blockade of the CTLA-4 immune checkpoint can enhance anti-tumor responses and prolong survival, but it can also lead to the development of severe and potentially life-threatening immune-related adverse events (IRAE). To avoid unnecessary exposure to this risk, it is essential to identify biomarkers that correlate with clinical activity and can be used to recognize and select patients that will benefit from immune checkpoint blockade. We therefore performed extensive immune monitoring in a phase I/II dose escalation/expansion trial of combined Prostate GVAX (a GM-CSF-secreting allogeneic prostate cancer vaccine) and antiCTLA-4/ipilimumab immunotherapy in patients with Castration Resistant Prostate Cancer (CRPC). Here we report on the effects of the treatment on circulating myeloid cells and the identification of potential myeloid-related biomarkers. The GVAX/ipilimumab combination was clinically active with PSA declines of more than 50% in 5, and PSA stabilizations in 12 of 28 patients. Regressing bone and lymph node metastasis were observed in 2/5 PR patients. Flowcytometric monitoring of myeloid subsets in peripheral blood before and after Prostate GVAX/ipilimumab treatment revealed some striking differences between patients who benefited from therapy and patients who did not. Significant treatment-induced decreases of conventional and plasmacytoid Dendritic Cell subsets (cDC and pDC, respectively) were observed, which were paralleled by increased DC activation and recruitment to the vaccination sites. Treatment-induced activation of BDCA1/CD1c+ cDC and 6-sulfo LacNAc+ inflammatory DC was associated with significantly prolonged over-all survival (OS). In contrast, increased frequencies of CD11b+CD14−CD15+ granulocytic myeloid-derived suppressor cells (MDSC) and high pre-treatment levels of CD14+HLA-DRlo/− monocytic MDSC were associated with reduced OS. Together with similar analyses of T cell subsets, these studies have yielded an immune profile with predictive value for clinical outcome. The profile is characterized by pre- or on-treatment activation of immune effector subsets and low frequencies of regulatory/suppressive subsets. It may thus provide a potentially useful tool for patient selection and should be validated as such in other patient groups treated by antiCTLA-4 blockade. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5381. doi:1538-7445.AM2012-5381

Serum samples from 217 cancer patients participating in phase I/II clinical trials were analysed for the development of anti-interleukin-2 (IL-2) antibodies. Patients received recombinant human IL-2 (rIL-2) by continuous intravenous infusion (c.i.v.; n = 86) or by subcutaneous (s.c.) injections (n = 131). Both patient groups developed anti-rIL-2 antibodies as detected by ELISA with similar frequencies and titres: 52% (median titre, 23) and 47% (median titre, 24), respectively. Using an IL-2-dependent T-cell proliferation assay, sera from 5 c.i.v.-treated patients (6%) and 13 s.c.-treated patients (10%) exhibited neutralising activity. Immunoabsorption studies with rIL-2-coated beads, demonstrated that in 8 of 15 patients with neutralising sera, the neutralising activity was correlated with specific anti-rIL-2 immunoglobulin. All 8 patients had received at least two cycles of rIL-2 by s.c. injections. Specific IL-2 neutralising activity affected both recombinant and natural IL-2 in all 8 patients. Development of anti-rIL-2 antibodies, irrespective of whether these exhibited neutralising activity or not, did not affect the frequency or duration of clinical responses.

The effects of Prostate GVAX and the anti-CTLA4 antibody Ipilimumab were investigated in a Phase I dose escalation/expansion trial of patients with castration resistant prostate cancer. Results showed that the GVAX/Ipilimumab combination was clinically active with PSA declines of more than 50% (Partial Response, PR) in 6 and PSA stabilizations (Stable Disease, SD) in 11 of 28 patients. Immune response monitoring was performed to identify changes that might predict or correlate with clinical efficacy. Upon administration of high Ipilimumab dosages (3 or 5 mg/kg), significant increases in frequencies of activated CD4+ and/or CD8+ T cells were observed by HLA-DR, PD-1, FoxP3 and ICOS expression. Monitoring over the course of treatment, revealed these markers to be differentially associatied with survival, as were levels of effector/memory T cells or Tregs. Early HLA-DR upregulation appeared useful as a marker for response prediction, since it was observed to significant levels in PR or SD, but not in PD patients. In addition, GVAX/Ipilimumab administration was found to induce Th2/Th17 cytokine profiles, as determined ex vivo by intracellular staining of peripheral T cells. Significantly increased levels of IL-4 in both CD4+ and CD8+ T cells were observed in patients with PR or SD, but not in patients with PD and, importantly, profound up-regulation of CD4+IL-5+ T cell frequencies was associated with improved overall survival (p=0.03). Similarly, significantly increased Th17 rates were only observed in patients with PR and SD (p As an indication of tumor-specific responsiveness, IgG antibody responses against 11 (prostate) tumor-associated antigens were determined by western blot and ELISA. Increased seroreactivity to prostate-specific membrane antigen (PSMA), pyridoxamine 5′-phosphate oxidase (PNPO) and/or Neuropilin-2 (NRP2) was significantly correlated with improved overall survival (p Our data indicate that changes in frequencies of activated memory/effector T cells and Tregs, Th2 and Th17 rates and seroconversion to multiple tumor antigens may define a predictive immune profile for patients with potential benefit from Prostate GVAX and/or anti-CTLA4 immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5512. doi:10.1158/1538-7445.AM2011-5512

The effects of a GM-CSF-secreting allogeneic prostate cancer vaccine (Prostate GVAX) and the anti-CTLA4 antibody Ipilimumab were investigated in a Phase I dose escalation/expansion trial of patients with metastatic, hormone-refractory prostate cancer (mHRPC). Patients received a 500 million cell GVAX priming dose on day 1 followed by 12 bi-weekly intradermal administrations of 300 million cells, while Ipilimumab was administered every 4 wks from day 1 for a total of 6 times. Initially, 12 patients were enrolled in cohorts of 3 and each cohort was assigned an escalating dose of Ipilimumab at 0.3, 1, 3 or 5 mg/kg. 16 additional patients were enrolled in the expansion cohort of 3 mg/kg Ipilimumab. Results showed that the GVAX and Ipilimumab combination was clinically active in mHRPC patients. PSA declines of more than 50% (Partial Response, PR) were observed in 5 of 22 patients in the 3–5 mg/kg Ipilimumab dose cohorts, and were associated with Autoimmune Breakthrough Events (ABE), including Grade 2 or 3 hypophysitis and Grade 3 alveolitis. PSA stabilizations (Stable Disease, SD) were observed in 1/3 patients in lower (0.3 and 1 mg/kg), and in 7 of 22 in the higher (3–5 mg/kg) dose levels. Moreover, regressing bone and lymph node metastases were observed in 2/5 PR patients. Immune response monitoring was performed to identify changes that might predict or correlate with clinical efficacy. Most notably, pronounced and significant increases in frequencies of activated and effector CD4+ and CD8+ T cells were observed by HLA-DR and ICOS expression upon administration of high (3 and 5 mg/kg) Ipilimumab doses; a relation to clinical behaviour was only observed for HLA-DR with earlier and more pronounced increases in patients with PR or SD. As an indication of tumor-specific responsiveness HLA-Tetramer (Tm) and seroreactivity to NY-ESO and PSMA were tested. For NY-ESO, therapy-induced increased seroreactivity (by Western Blot or ELISA) was observed in 6/28 patients. Of these 6 patients, three could be tested for Tm reactivity and in two (1SD, 1 Progressive Disease, PD) increased NY-ESO157 rates were found concomitant with increasing serum Ab levels, whereas no Tm reactivity was detected in 8 patients without seroconversions. PSMA seroconversions were observed in a total of 12/28 patients (and, of note, in 4/5 PR), but no Tm positivity at any time was found in a total of 15 patients tested. In the 3–5 mg/kg dose levels (n=22), PSMA seroconversion was associated with increased overall survival (P=0.062). In addition, combined GVAX/Ipilimumab administration was found to induce Type-2/Th17 profiles, as determined ex vivo by intracellular staining of peripheral T cells. Significantly increased levels of IL-4 in both CD4+ and CD8+ T cells were observed in patients with PR or SD (P In summary, our analyses so far reveal a relationship between clinical efficacy, PSMA seroconversion, and generalized T cell activation accompanied by Th2 and Th17 skewing. Together these data point to a mechanism of action whereby combined Prostate GVAX and anti-CTLA4 immunotherapy can induce both Th2/humoral and Th17/cell-mediated immune responses, resulting in tumor destruction and collateral autoimmunity. Supported by the Prostate Cancer Foundation and the Dutch Cancer Society (KWF-VU 2006-3697) Citation Information: Clin Cancer Res 2010;16(7 Suppl):B20

The effects of a GM-CSF-secreting allogeneic prostate cancer vaccine (Prostate GVAX) and the anti-CTLA4 antibody Ipilimumab were investigated in a Phase I dose escalation/expansion trial of patients with metastatic, hormone-refractory prostate cancer (mHRPC). All patients received a 500 million cell priming dose of GVAX on day 1 followed by bi-weekly intradermal treatments of 300 million cells over a 24-week period. Ipilimumab was administered every 4 wks from day 1 during the same period. Initially, 12 patients were enrolled in cohorts of 3 and each cohort was assigned an escalating dose of Ipilimumab at 0.3, 1, 3 or 5 mg/kg. 16 additional patients were enrolled in the expansion cohort of 3 mg/kg Ipilimumab. Results showed that the GVAX and Ipilimumab combination was active in mHRPC patients. PSA declines of more than 50% (Partial Response, PR) were observed in 5 of 22 patients in the 3-5 mg/kg Ipilimumab doses, and were associated with Autoimmune Breakthrough Events (ABE), including Grade 2 or 3 hypophysitis and Grade 3 alveolitis. PSA stabilizations (Stable Disease, SD) were observed in 1/3 pts in lower (0.3 and 1 mg/kg), and in 7 of 22 in the higher (3-5 mg/kg) dose levels. In this study, cytokine profiles of peripheral blood-derived T cells were determined ex vivo and after in vitro stimulation with PMA and ionomycin by intracellular staining for IL-2, −4, −5, −10, TNF-α, IFN-γ and IL-17A. Of these cytokines, only IL-4 was detectable ex vivo in CD4+ and CD8+ T cells; post-treatment increases were detectable in the higher (3-5 mg/kg) but not in the lower (0.3-1 mg/kg) Ipilimumab dose levels. After PMA/ionomycin stimulation, no consistent changes were observed in the frequencies of IFN-γ, IL-2 and TNF-α-producing type-1 T cells compared to pre-treatment values. In contrast, significantly increased rates of IL-4 and IL-5 producing CD4+ and CD8+ T cells were found upon treatment (visit 1 versus visit 5) at higher (3-5 mg/kg) Ipilimumab dose levels. Overall these data clearly demonstrate a shift in favor of a Type-2 cytokine profile due to the GVAX/Ipilimumab administration. The frequencies of IL-17A producing CD4+ T (Th17) cells increased over the course of treatment in 5 of 18 patients. Of note, in 3 (PR) out of 5 of these patients this increase of the Th17 subset coincided with onset of hypophysitis and/or adrenal insufficiencies, as well as the onset of PSA decline. The two patients with increased Th17 rates without these pituitary or adrenal-related ABE showed SD. In summary, our results show that combined GVAX/Ipilimumab administration gives rise to Type-2/Th17 cytokine profiles in mHRPC patients, but that only increases in Th17 show a relation to ABE and PSA responses. 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 2934.

The effects of a GM-CSF-secreting allogeneic prostate cancer vaccine (Prostate GVAX) and the anti-CTLA4 antibody Ipilimumab were investigated in a Phase I dose escalation/expansion trial of patients with metastatic, hormone-refractory prostate cancer (mHRPC). Patients received a 500 million cell GVAX priming dose on day 1 followed by 12 bi-weekly intradermal administrations of 300 million cells, while Ipilimumab was administered every 4 wks from day 1 for a total of 6 times. Initially, 12 patients were enrolled in cohorts of 3 and each cohort was assigned an escalating dose of Ipilimumab at 0.3, 1, 3 or 5 mg/kg. 16 additional patients were enrolled in the expansion cohort of 3 mg/kg Ipilimumab. Results showed that the GVAX and Ipilimumab combination was clinically active in mHRPC patients. PSA declines of more than 50% (Partial Response, PR) were observed in 5 of 22 patients in the 3-5 mg/kg Ipilimumab doses and PSA stabilizations (Stable Disease, SD) were observed in 1/3 pts in lower (0.3 and 1 mg/kg), and in 7/22 in the higher (3-5 mg/kg) dose levels. Phenotypic and functional (anti-tumor) effector T cell activation was monitored to identify changes that might predict or correlate with clinical efficacy. Most notably, pronounced and significant increases in frequencies of activated and effector CD4+ and CD8+ T cells were observed by HLA-DR and ICOS expression upon administration of high (3 and 5 mg/kg) but not of low (0.3 and 1 mg/kg) Ipilimumab doses. We also observed a significant increase of CTLA4 and PD-1 expression on CD4+ T cells compared to pre-treatment values in the 3 mg/kg Ipilimumab-administered patients. The latter might be indicative of the induction of concomitant negative feedback signaling. As an indiciation of Tumor-Associated Antigen (TAA) specific responsiveness we tested HLA-Tetramer (Tm) reactivity to NY-ESO and PSMA in HLA-A2 or -A3 positive patients. For NY-ESO, GVAX/Ipi-induced increased seroreactivity (by Western Blot or ELISA) was observed in 6/28 patients. Of these 6 patients, three could be tested for Tm reactivity and in two (1SD, 1 Progressive Disease, PD) increased NY-ESO157 rates were found concomitant with increasing serum Ab levels, whereas no Tm reactivity was detected in 8 patients without seroconversions. Whereas PSMA seroconversions were observed in a total of 12/28 patients (and of note, in 4/5 PR), no Tm positivity at any time was found in a total of 15 patients tested. Further functional Treg and CTL analyses are ongoing. So far, only early HLA-DR upregulation on CD4+ or CD8+ T cells (visit 1 versus visit 3) showed any value for response prediction, since it was observed to significant levels in patients with PR or SD, but not in patients with PD. 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 5588.

Abstract 4139 Background Therapeutic vaccination with dendritic cells (DCs) is regarded as an option for the treatment of minimal residual disease in acute myeloid leukemia (AML). In this respect, defined epitope sequences derived from leukemia-associated antigens (LAA) are an attractive source of antigen. WT1 and PRAME are both LAA that are over-expressed in various hematological malignancies and that share a potential for the induction of cellular immune responses. Expression rates in AML of each of these LAA are high, ranging from 60 to 90%. To evaluate the feasibility of using these LAA in DC based vaccine formulations in AML, we investigated the relative cytotoxic T lymphocyte (CTL) priming efficiencies in response to WT and PRAME derived HLA-A2 binding epitopes in healthy donors (HD) and leucapheresis (LF) material or peripheral blood (PB) samples of AML patients in first complete remission (CR). Methods CD8+ cells were isolated from peripheral blood of HLA-A2+ HD and from G-CSF stimulated HLA-A2+ patients and stimulated with peptide-loaded DCs. DCs were derived from the human HLA-A2 positive AML cell line MUTZ-3 (MUTZ3-DCs) (Santegoets, et al. Cancer Immunol Immunother. 2006). The peptides used were WT1 (126-134) and PRAME (100-108). Irradiated CD8-cells from the same donor were also added as helper cells. CD8+ cells were plated at one million per well in 8 to 30 wells per donor or patient. CD8+ cells were weekly restimulated with peptide-loaded MUTZ3-DCs. HLA-tetramer positivity (Tm+) for the WT1 and PRAME epitopes was weekly screened by flow cytometry. Wells were scored positive if more than 0.02% Tm+, viable CTLs were present for at least 2 weeks. Functionality of Tm+ cells was studied by analysis of IFN-gamma production after stimulation with the JY cell line loaded with relevant peptides or with an irrelevant control peptide. PRAME Tm+ CTLs were isolated by flow cytometric sorting and a CTL clone was derived by limiting dilution. Recognition of HLA-A2 and PRAME positive tumor cell lines and patient samples by the CTL clone was tested in a direct cytotoxicity assay and ELISPOT assay. Results On the whole, frequencies of the induced peptide-specific CTLs were low as shown in the Table below. Priming efficiencies were highest for PRAME. Furthermore, both PRAME and WT1 specific T cells derived from HD produced IFN-gamma in an epitope specific fashion, confirming functionality of the primed T cells. Most notably, no PRAME specific CTLs could be cultured from the patients' blood, whereas a PRAME specific CTL clone, cultured from sorted Tm+ CTLs from HD, recognized and lysed HLA-A2 PRAME positive tumor cell lines and patient-derived AML blasts Conclusions LAA-specific CD8+ T cell frequencies were generally low. Most consistently successful priming results were obtained for PRAME in HD and functional CTL were derived thereof indicating that this peptide may be an attractive candidate for vaccination strategies. However, priming of patient samples was unsuccessful, implying possible anergy induction or clonal deletion. As a consequence, active immunotherapy with PRAME-loaded DC may require combined therapy with matched allogeneic donor lymphocyte Infusion (DLI). Disclosures: No relevant conflicts of interest to declare.

Background Therapeutic vaccination with dendritic cells (DCs) is regarded as an option for the treatment of minimal residual disease in acute myeloid leukemia (AML). In this respect, defined epitope sequences derived from Leukemia-Associated Antigens (LAA) are an attractive source of antigen. WT1, PRAME, hTERT, and survivin are all LAA that are expressed in various hematological malignancies and that share a potential for the induction of cellular immune responses. Expression rates in AML of each of these LAA are high, ranging from 60 to 90%. To evaluate the feasibility of using these LAA in DC based vaccine formulations in AML, we investigated the relative Cytotoxic T Lymphocyte (CTL) priming efficiencies in response to a panel of LAA-derived HLA-A2 binding epitopes. Methods CD8+ cells were isolated from peripheral blood of healthy donors (HD) and stimulated with peptide-loaded DCs. DCs were derived from the human AML cell line MUTZ-3 (MUTZ3-DCs).1 The peptides used were WT1 (126–134), PRAME (100–108), hTERT (540–548), and survivin (96–104). Irradiated CD8− cells from the same donor were also added. CD8+ cells were plated at one million per well in 8 to 30 wells. Weekly CD8+ cells were restimulated with peptide-loaded MUTZ3-DCs. HLA-tetramer positivity (Tm+) for WT1, PRAME, hTERT or survivin epitopes was weekly screened by flow cytometry. Wells were scored positive if more than 0.02% Tm+, viable CTLs were present for at least 2 weeks. Functionality of Tm+ cells was studied by analysis of IFN-gamma production after stimulation with the JY cell line loaded with relevant LAA peptides or with an irrelevant control peptide (BCR-ABL). Results Percentages of the induced peptide-specific CTLs were low as shown in the table below. Priming efficiencies were highest for PRAME. Furthermore, PRAME and WT1 specific CTLs produced IFN-gamma in an epitope specific fashion, confirming functionality of the primed T cells. Number of HD that were Tm+ after co-culture Number of wells that were Tm+ after co-culture Median % Tm+ cells per positive well (range) Number of restimulations at maximum of Tm+ PRAME 3/4 28/60 0.10% (0.02–1.07) 1–5 WT1 2/5 4/104 0.07% (0.03–0.09) 1–4 hTERT 1/2 1/24 0.25% 5 Survivin 1/1 2/18 0.17% (0.12–0.21) 8 Conclusions On the whole, the detected LAA-specific CD8+ T cell frequencies were low. Most consistently successful priming results were obtained for PRAME, indicating that this peptide may be an attractive candidate for inclusion in AML-specific DC-based vaccine regimens.

The effects of a combination of two allogeneic, GM-CSF-secreting prostate cancer cell lines (GVAX Immunotherapy for Prostate Cancer, Cell Genesys, Inc.) and escalating doses of the anti-CTLA4 antibody ipilimumab (MDX-010, Medarex, Inc.) are currently being evaluated in a Phase I trial of patients with metastatic, hormone-refractory prostate cancer (HRPC). The anticipated synergy of these two novel therapies may potentially lead to augmented T cell-mediated anti-tumor immunity via improved dendritic cell (DC) functions and blockade of inhibitory feedback loops in activated tumor-specific T cells. All patients received a 500 million cell prime dose of the cellular immunotherapy on day 1 followed by bi-weekly intradermal administrations of 300 million cells for a 24-week period. Ipilimumab was administered every 4 wks from day 1 during the same period. Patients were enrolled in cohorts of 3; each cohort was assigned an escalating dose of ipilimumab at 0.3, 1, 3 or 5 mg/kg. Results showed PSA declines of >50% in 5/6 patients at the two highest dose levels of anti-CTLA-4, as well as resolution of multiple lesions on bone scan in two patients, and resolution of abdominal lymph node disease by CT scan and improvement in bone pain in one patient each. T cell activation was monitored to identify changes that correlate with clinical efficacy. Preliminary DC and T cell data are encouraging. Pre-treatment frequencies of circulating myeloid DC (MDC) subsets were significantly reduced in the enrolled HRPC patients, as compared to age- and sex-matched healthy controls. A transient increase in MDC frequencies was observed at the lowest ipilimumab dose level (0.3 mg/kg), but not at the two highest dose levels. The latter may be explained by an observed massive recruitment of MDC to the immunotherapy injection sites. Evidence of transient activation of circulating MDC, concomitant increases of the activation marker HLA-DR on circulating T cells and increased frequencies of circulating memory/effector T cells was found during treatment at the higher ipilimumab dose levels. The transient activation of circulating MDC and T cells showed a reverse kinetics with serum PSA levels. Furthermore, intense skin reactions were observed in response to administration of the cellular immunotherapy, which is consistent with the observed increases in T cell infiltration and Granzyme B expression in injection site biopsies taken during the treatment course and is suggestive of early immune activation. To increase the chances of detecting tumor-specific T cells, both blood and injection sites are monitored for T cell reactivity. Functional regulatory T cell activity is tested in parallel. These on-going analyses are expected to yield valuable data concerning immune effects achieved by the GVAX/ipilimumab combination.

Background Cancer-related disturbances in myeloid lineage development, marked by high levels of myeloid-derived suppressor cells (MDSC) and impaired dendritic cell (DC) development, are associated with poor clinical outcome due to immune escape and therapy resistance. Redressing this balance may therefore be of clinical benefit. Here we investigated the effects of combined Prostate GVAX/ipilimumab immunotherapy on myeloid subsets in peripheral blood of castration-resistant prostate cancer (CRPC) patients as well as the putative predictive value of baseline and on-treatment myeloid parameters on clinical outcome. Methods Patients with CRPC (n = 28) received thirteen intradermal administrations of Prostate GVAX, consisting of two allogeneic GM-CSF-transduced and irradiated prostate cancer cell lines (LN-CaP and PC3) and six infusions of escalating doses of anti-CTLA4/ipilimumab. Frequencies and activation status of peripheral blood DC (PBDC) and MDSC were determined before, during and after treatment by flowcytometric analysis and related to clinical benefit. Results Significant treatment-induced activation of conventional and plasmacytoid DC subsets (cDC and pDC) was observed, which in the case of BDCA1/CD1c+ cDC1 and MDC8+/6-sulfoLacNAc+ inflammatory cDC3 was associated with significantly prolonged overall survival (OS), but also with the development of autoimmune-related adverse events. High pre-treatment levels of CD14+HLA-DR−monocytic MDSC (mMDSC) were associated with reduced OS. Unsupervised clustering of these myeloid biomarkers revealed particular survival advantage in a group of patients with high treatment-induced PBDC activation and low pretreatment frequencies of suppressive mMDSC in conjunction with our previously identified lymphoid biomarker of high pretreatment CD4+CTLA4+ T cell frequencies. Conclusions Our data demonstrate that DC and MDSC subsets are affected by prostate GVAX/ipilimumab therapy and that myeloid profiling may contribute to the identification of patients with possible clinical benefit of Prostate GVAX/ipilimumab treatment. Electronic supplementary material The online version of this article (doi:10.1186/s40425-014-0031-3) contains supplementary material, which is available to authorized users.

Meeting abstracts Immunotherapeutic approaches such as vaccination or immune checkpoint blockade have proven to be clinically active in prostate cancer, but only in fractions of treated patients; this calls for personalized application of these novel therapies based on predictive biomarkers. Our