Complete and long-lasting clinical responses in immune checkpoint inhibitor-resistant, metastasized melanoma treated with adoptive T cell transfer combined with DC vaccination.

Development of T cell-directed immune checkpoint inhibitors (ICI) has revolutionized metastatic melanoma (MM) therapy, but <50% of treated patients experience durable responses. This phase I trial (NCT01946373) investigates the safety/feasibility of tumor-infiltrating lymphocyte (TIL) adoptive cell therapy (ACT) combined with dendritic cell (DC) vaccination in MM patients progressing on ICI. An initial cohort (5 patients) received TIL therapy alone to evaluate safety and allow for optimization of TIL expansion protocols. A second cohort (first-in-man, 5 patients) received TIL combined with autologous tumor lysate-loaded DC vaccination. All patients received cyclophosphamide/fludarabine preconditioning prior to, and intravenous (i.v.) IL-2 after, TIL transfer. The DC vaccine was given as five intradermal injections after TIL and IL-2 administration. [18F]-FDG PET/CT radiology was performed to evaluate clinical response, according to RECIST 1.1 (on the CT part). Immunological monitoring was performed by flow cytometry and T-cell receptor (TCR) sequencing. In the safety/optimization cohort, all patients had a mixed response or stable disease, but none durable. In the combination cohort, two patients experienced complete responses (CR) that are still ongoing (>36 and >18 months, respectively). In addition, two patients had partial responses (PR), one still ongoing (>42 months) with only a small bone-lesion remaining, and one of short duration (<4 months). One patient died early during treatment and did not receive DC. Long-lasting persistency of the injected TILs was demonstrated in blood. In summary, we report clinical responses by TIL therapy combined with DC vaccination in 4 out of 4 treated MM patients who previously failed ICI.

Lenalidomide as immune adjuvant to a dendritic cell vaccine in chronic lymphocytic leukemia patients.

OBJECTIVES: We previously showed that immunization with ex vivo- generated autologous dendritic cells loaded with apoptotic tumor cells (Apo-DC) potentiated tumor-specific immunity in chronic lymphocytic leukemia (CLL) patients. Here, we evaluated safety and immunogenicity of Apo-DC in combination with lenalidomide, granulocyte-macrophage colony-stimulating factor (GM-CSF), and low-dose cyclophosphamide (CTX). METHODS: Ten previously untreated patients with slowly progressing CLL received 5 Apo-DC vaccinations and lenalidomide orally for 24 weeks either alone (cohort I, n = 5) or together with subcutaneous GM-CSF and intravenous CTX (cohort II, n = 5). Tumor-specific T-cell responses were measured by proliferation and IFN-γ ELISPOT assays. Immune monitoring was performed by flow cytometry. RESULTS: Dose-limiting toxicity was observed in 3/10 patients, 2 in cohort I and one in cohort II. One patient developed autoimmune hemolytic anemia and another grade 4 thrombocytopenia. Vaccine-induced immune responses were seen in 5/5 and 4/5 patients in cohort I and II, respectively. The expression of immune checkpoints on T cells did not change significantly. CONCLUSIONS: Lenalidomide alone or in combination with GM-CSF and low-dose CTX as immune adjuvant to the Apo-DC vaccine elicited tumor-specific T-cell responses in CLL patients. However, unexpected toxicity was observed and caution is suggested in further exploring this drug as immune adjuvant in CLL.

Intratumorally injected pro-inflammatory allogeneic dendritic cells as immune enhancers: a first-in-human study in unfavourable risk patients with metastatic renal cell carcinoma.

BACKGROUND: Accumulating pre-clinical data indicate that the efficient induction of antigen-specific cytotoxic CD8+ T cells characterizing viral infections is caused by cross-priming where initially infected DCs produce an unique set of inflammatory factors that recruit and activate non-infected bystander DCs. Our DC-based immunotherapy concept is guided by such bystander view and accordingly, we have developed a cellular adjuvant consisting of pre-activated allogeneic DCs producing high levels of DC-recruiting and DC-activating factors. This concept doesn't require MHC-compatibility between injected cells and the patient and therefore introduces the possibility of using pre-produced and freeze-stored DCs from healthy blood donors as an off- the-shelf immune enhancer. The use of MHC-incompatible allogeneic DCs will further induce a local rejection process at the injection site that is expected to further enhance recruitment and maturation of endogenous bystander DCs. METHODS: Twelve intermediate and poor risk patients with newly diagnosed metastatic renal cell carcinoma (mRCC) where included in a phase I/II study. Pro-inflammatory allogeneic DCs were produced from a leukapheresis product collected from one healthy blood donor and subsequently deep-frozen. A dose of 5-20 × 106 DCs (INTUVAX) was injected into the renal tumor twice with 2 weeks interval before planned nephrectomy and subsequent standard of care. RESULTS: No INTUVAX-related severe adverse events were observed. A massive infiltration of CD8+ T cells was found in 5 out of 12 removed kidney tumors. No objective tumor response was observed and 6 out of 11 evaluable patients have subsequently received additional treatment with standard tyrosine kinase inhibitors (TKI). Three of these 6 patients experienced an objective tumor response including one sunitinib-treated patient who responded with a complete and durable regression of 4 brain metastases. Median overall survival (mOS) is still not reached (currently 42.5 months) but has already passed historical mOS in patients with unfavourable risk mRCC on standard TKI therapy. CONCLUSIONS: Our findings indicate that intratumoral administration of proinflammatory allogeneic DCs induces an anti-tumor immune response that may prolong survival in unfavourable risk mRCC-patients given subsequent standard of care. A randomized, multi-center, phase II mRCC trial (MERECA) with INTUVAX in conjuction with sunitinib has been initiated. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT01525017.

Enhanced stimulation of human tumor-specific T cells by dendritic cells matured in the presence of interferon-γ and multiple toll-like receptor agonists.

Dendritic cell (DC) vaccines have been demonstrated to elicit immunological responses in numerous cancer immunotherapy trials. However, long-lasting clinical effects are infrequent. We therefore sought to establish a protocol to generate DC with greater immunostimulatory capacity. Immature DC were generated from healthy donor monocytes by culturing in the presence of IL-4 and GM-CSF and were further differentiated into mature DC by the addition of cocktails containing different cytokines and toll-like receptor (TLR) agonists. Overall, addition of IFNγ and the TLR7/8 agonist R848 during maturation was essential for the production of high levels of IL-12p70 which was further augmented by adding the TLR3 agonist poly I:C. In addition, the DC matured with IFNγ, R848, and poly I:C also induced upregulation of several other pro-inflammatory and Th1-skewing cytokines/chemokines, co-stimulatory receptors, and the chemokine receptor CCR7. For most cytokines and chemokines the production was even further potentiated by addition of the TLR4 agonist LPS. Concurrently, upregulation of the anti-inflammatory cytokine IL-10 was modest. Most importantly, DC matured with IFNγ, R848, and poly I:C had the ability to activate IFNγ production in allogeneic T cells and this was further enhanced by adding LPS to the cocktail. Furthermore, epitope-specific stimulation of TCR-transduced T cells by peptide- or whole tumor lysate-loaded DC was efficiently stimulated only by DC matured in the full maturation cocktail containing IFNγ and the three TLR ligands R848, poly I:C, and LPS. We suggest that this cocktail is used for future clinical trials of anti-cancer DC vaccines.

Immunotherapy in atypical teratoid-rhabdoid tumors: Data from a survey of the HGG-Immuno Group.

BACKGROUND AIMS: Atypical rhabdoid/teratoid tumors (AT/RT) are the most common brain tumors in infants and associated with a dismal prognosis. Although intensification of first-line therapy has resulted in improvement of overall survival, novel treatment strategies are needed. Because immunotherapy has resulted in remarkable results in several adult tumor entities, incorporation of immunotherapy into AT/RT treatment offers a novel alternative. METHODS: We retrospectively analyzed data from 7 AT/RT patients from five countries treated within the HGG-Immuno Consortium. Two patients were ≤1 year and 4 patients were ≤2 years of age at diagnosis. All received immunotherapy with autologous, tumor-lysate-loaded dendritic cells (DCs) on a compassionate use basis using a schedule of three to four weekly DC vaccinations with up to 2 × 10(7) DCs per vaccine, followed by three lysate boosts each 1 month apart. RESULTS: Monocyte collections (median age at apheresis 31.5, range 20-143 months) and vaccinations were uneventful without any severe adverse event related to the vaccine, demonstrating feasibility and safety in this very young age group. Two children received immunotherapy during their primary and the remaining five during second- or third-line therapy. Three of seven patients survived long term with a follow-up of 143, 138 and 46 months, with at least two of them harboring somatic mutations. One long-term survivor was vaccinated during primary treatment and the other two after first or second relapse/progression. Two analyzed patients showed positive CD8(+) T-cell responses after vaccination. DISCUSSION: Our data demonstrate that anti-tumor immunotherapy with autologous DCs is feasible and safe in young children with ATRTs and that this approach warrants further investigation in controlled clinical trials.

Dendritic cell regulation of NK-cell responses involves lymphotoxin-α, IL-12, and TGF-ß.

Dendritic cell (DC) vaccines induce T-cell responses in cancer patients. However, there is a paucity of data regarding the role of DC vaccines in shaping natural killer (NK) cell responses. Here, we observe that NK cells are less activated following DC vaccination. In vitro, DC-mediated inhibition of NK cells did not require cell-to-cell contact, but required increased Signal transducer and activator of transcription 3 (STAT3) phosphorylation (pSTAT3) in DCs. When phosphorylation of STAT3 was inhibited in DCs, we found that DCs did not suppress NK cells, and observed an increase in the production of lymphotoxin-alpha (LTα) and interleukin-12 (IL-12) as well as reduced release of transforming growth factor beta (TGF-ß). The addition of recombinant LTα or IL-12 to the DC-NK-cell cocultures restored NK-cell activity, and neutralization of TGF-ß resulted in elevated production of LTα and IL-12 from DCs. Compared with LPS, DCs matured with a cocktail of R848, poly I:C, and IFN-γ showed reduced levels of pSTAT3 and higher levels of LTα and IL-12 and did not inhibit NK-cell activity. These results show that LTα, IL-12, and TGF-ß are involved in the cross-talk between NK cells and DCs. Our findings have important implications for the development of DC-based vaccination strategies to potentiate NK-cell responses in patients with cancer.

A phase I clinical trial combining dendritic cell vaccination with adoptive T cell transfer in patients with stage IV melanoma.

Adoptive transfer of in vitro-expanded tumor-infiltrating lymphocytes (TIL) has shown great clinical benefit in patients with malignant melanoma. TIL therapy itself has little side effects, but conditioning chemo- or radiotherapy and postinfusion interleukin 2 (IL-2) injections are associated with severe adverse advents. We reasoned that combining TIL infusion with dendritic cell (DC) vaccination could circumvent the need for conditioning and IL-2 support and thus represent a milder treatment approach. Eight patients with stage IV melanoma were enrolled in the MAT01 study, consisting of vaccination with autologous tumor-lysate-loaded DC, followed by TIL infusion. Six of eight patients were treated according to protocol, while one patient received only TIL and one only DC. Treatments were well tolerated with a single grade 3 adverse event. The small study size precludes analysis of clinical responses, though interestingly one patient showed a complete remission and two had stable disease. Analysis of the infusion products revealed that mature DC were generated in all cases. TIL after expansion were CD3+ T cells, dominated by effector memory CD8+ cytotoxic T cells. Analysis of the T cell receptor repertoire revealed presence of highly dominant clones in most infusion products, and many of these could be detected in the circulation for weeks after T cell transfer. Here, we report the first combination of DC vaccination and TIL infusion in malignant melanoma. This combined treatment was safe and feasible, though after evaluating both clinical and immunological parameters, we expect that administration of lymphodepleting chemotherapy and IL-2 will likely increase treatment efficacy.

Vaccination with dendritic cells loaded with tumor apoptotic bodies (Apo-DC) in patients with chronic lymphocytic leukemia: effects of various adjuvants and definition of immune response criteria.

We previously demonstrated that autologous dendritic cells that have endocytosed apoptotic bodies of chronic lymphocytic leukemia (CLL) cells (Apo-DC) can stimulate antileukemic T cell responses in vitro. In this phase I study, we vaccinated 15 asymptomatic CLL patients at five time points with Apo-DC administered intradermally either alone (cohort I), or in combination with subcutaneous granulocyte-macrophage-colony-stimulating-factor (GM-CSF) (cohort II) or with GM-CSF and intravenous low-dose cyclophosphamide (cohort III). Aim of the study was to evaluate the safety and immunogenicity of Apo-DC alone or in combination with GM-CSF and low-dose cyclophosphamide in CLL patients. All patients completed the vaccination schedule without dose-limiting toxicity. No objective clinical responses were seen. Vaccine-induced leukemia-specific immune responses were evaluated by IFN-γ ELISpot and proliferation assays over a 52 weeks observation period and immune response criteria were defined. According to these criteria, 10/15 patients were defined as immune responders. The frequency of immune-responding patients was higher in cohorts II (3/5) and III (5/5) than in cohort I (2/5). In order to further characterize the induced immune response, estimation of secreted cytokines and CD107-degranulation assay were performed. Clustering of T and CLL cells was observed in CD107-degranulation assay and visualized by confocal microscopy. Additionally, assessment of regulatory T cells (T(regs)) revealed their significantly lower frequencies in immune responders versus non-responders (P < 0.0001). Cyclophosphamide did not reduce T(regs) frequency. In conclusion, vaccination with Apo-DC + GM-CSF and cyclophosphamide was safe and elicited anti-CLL immune responses that correlated inversely with T(regs) levels. Lack of clinical responses highlights the necessity to develop more potent vaccine strategies in B cell malignancies.

Insulin and IGF-1 mediated inhibition of apoptosis in CHO cells grown in suspension in a protein-free medium.

When Chinese hamster ovary (CHO) cells were grown in suspension and deprived of serum, 40% of them became apoptotic after 72 hours, as determined by flow cytometry analysis of TUNEL-labelled cells. Cell viability, assessed by erythrocin B staining, decreased correspondingly. An increase in the total fraction of cells expressing interleukin converting enzyme (ICE; caspase 1), B-cell lymphoma 2 protein (Bcl-2,) and Bcl-2 associated x protein (Bax) was shown by antibody probing and subsequent flow cytometry. The p53 tumour suppressor gene product level remained low within the cell population. Insulin-like growth factor-1 (IGF-1) inhibited cell death in a concentration-dependent manner, and at 20 ng/ml, cell viability was maintained close to 100% and no apoptotic cells were detected. Also, insulin was shown to inhibit cell death - at 1.0 microg/ml, cell viability was 95%, whereas 10% of the cells stained for apoptosis. At the highest concentrations of IGF-1 and insulin, the expression of ICE, Bcl-2 and Bax was fully suppressed, whereas the p53 product level increased, despite still being detectable in a minority of cells. Under these conditions, IGF-1 may increase p53 expression to restrain abnormal cell proliferation. It is concluded that special attention should be paid to exposure and culture conditions that induce acquired susceptibility to a toxic insult, during the development and validation of cell-based assays.

Monocyte enriched apheresis for preparation of dendritic cells (DC) to be used in cellular therapy.

We describe that with one leukapheresis procedure it is feasible to obtain sufficient numbers of monocytes to be utilized in dendritic cell therapies. Twenty-two leukaphereses were performed on eight healthy volunteers and 13 cancer patients, using Cobe Spectra. An on-line sample was drawn as soon as a stable interface was established. The concentration of monocytes in the sample was used to calculate the volume to be collected to reach target numbers of monocytes. A recovery unit was used to calculate the efficacy of the leukaphereses and we demonstrate an efficacy for monocytes correlating with the amount of processed blood.