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.

Vaccination against Oncoproteins of HPV16 for Noninvasive Vulvar/Vaginal Lesions: Lesion Clearance Is Related to the Strength of the T-Cell Response.

PURPOSE: Therapeutic vaccination with human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides (SLP) is effective against HPV16-induced high-grade vulvar and vaginal intraepithelial neoplasia (VIN/VaIN). However, clinical nonresponders displayed weak CD8(+) T-cell reactivity. Here, we studied if imiquimod applied at the vaccine site could improve CD8(+) T-cell reactivity, clinical efficacy, and safety of HPV16-SLP (ISA101). EXPERIMENTAL DESIGN: A multicenter open-label, randomized controlled trial was conducted in patients with HPV16(+) high-grade VIN/VaIN. Patients received ISA101 vaccination with or without application of 5% imiquimod at the vaccine site. The primary objective was the induction of a directly ex vivo detectable HPV16-specific CD8(+) T-cell response. The secondary objectives were clinical responses (lesion size, histology, and virology) and their relation with the strength of vaccination-induced immune responses. RESULTS: Forty-three patients were assigned to either ISA101 with imiquimod (n = 21) or ISA101 only (n = 22). Imiquimod did not improve the outcomes of vaccination. However, vaccine-induced clinical responses were observed in 18 of 34 (53%; 95% CI, 35.1-70.2) patients at 3 months and in 15 of 29 (52%; 95% CI, 32.5-70.6) patients, 8 of whom displayed a complete histologic response, at 12 months after the last vaccination. All patients displayed vaccine-induced T-cell responses, which were significantly stronger in patients with complete responses. Importantly, viral clearance occurred in all but one of the patients with complete histologic clearance. CONCLUSIONS: This new study confirms that clinical efficacy of ISA101 vaccination is related to the strength of vaccine-induced HPV16-specific T-cell immunity and is an effective therapy for HPV16-induced high-grade VIN/VaIN. Clin Cancer Res; 22(10); 2342-50. ©2016 AACRSee related commentary by Karaki et al., p. 2317.

Antigen-specific immunotherapy in ovarian cancer and p53 as tumor antigen.

Immunotherapy for ovarian cancer is one of the new treatment strategies currently investigated in epithelial ovarian cancer. This review discusses the results of different immunization strategies, identifies possible drawbacks in study design and provides potential solutions for augmentation of clinical efficacy. A potential target for cancer immunotherapy is p53, as approximately 50% of ovarian cancer cells carry p53 mutations. Therefore we review the immunological and clinical responses observed in ovarian cancer patients vaccinated with p53 targeting vaccines in particular. In most studies antigen-specific vaccine-induced immunological responses were observed. Unfortunately, no clinical responses with significant reduction of tumor-burden have been reported. Based on the currently available results we emphasize the necessity of multimodality treatment of ovarian cancer, combining classical cytoreductive surgery, (neo) adjuvant chemotherapy, immunotherapy and/or targeted therapy.

Long-term clinical and immunological effects of p53-SLP® vaccine in patients with ovarian cancer.

Vaccine-induced p53-specific immune responses were previously reported to be associated with improved response to secondary chemotherapy in patients with small cell lung cancer. We investigated long-term clinical and immunological effects of the p53-synthetic long peptide (p53-SLP®) vaccine in patients with recurrent ovarian cancer. Twenty patients were immunized with the p53-SLP® vaccine between July 2006 and August 2007. Follow-up information on patients was obtained. Clinical responses to secondary chemotherapy after p53-SLP® immunizations were determined by computerized tomography and/or tumor marker levels (CA125). Disease-specific survival was compared to a matched historical control group. Immune responses were analyzed by flow cytometry, proliferation assay, interferon gamma (IFN-γ) ELISPOT and/or cytokine bead array. Lymphocytes cultured from skin biopsy were analyzed by flow cytometry and proliferation assay. Of 20 patients treated with the p53-SLP® vaccine, 17 were subsequently treated with chemotherapy. Eight of these patients volunteered another blood sample. No differences in clinical response rates to secondary chemotherapy or disease-specific survival were observed between immunized patients and historical controls (p = 0.925, resp. p = 0.601). p53-specific proliferative responses were observed in 5/8 patients and IFN-γ production in 2/7 patients. Lymphocytes cultured from a prior injection site showing inflammation during chemotherapy did not recognize p53-SLP®. Thus, treatment with the p53-SLP® vaccine does not affect responses to secondary chemotherapy or survival, although p53-specific T-cells do survive chemotherapy.

Potentiation of a p53-SLP vaccine by cyclophosphamide in ovarian cancer: a single-arm phase II study.

The purpose of the current phase II single-arm clinical trial was to evaluate whether pretreatment with low-dose cyclophosphamide improves immunogenicity of a p53-synthetic long peptide (SLP) vaccine in patients with recurrent ovarian cancer. Patients with ovarian cancer with elevated serum levels of CA-125 after primary treatment were immunized four times with the p53-SLP vaccine. Each immunization was preceded by administration of 300 mg/m2 intravenous cyclophosphamide as a means to affect regulatory T cells (Tregs). Vaccine-induced p53-specific interferon-gamma (IFN-γ)-producing T cells evaluated by IFN-γ ELISPOT were observed in 90% (9/10) and 87.5% (7/8) of evaluable patients after two and four immunizations, respectively. Proliferative p53-specific T cells, observed in 80.0% (8/10) and 62.5% (5/8) of patients, produced both T-helper 1 and T-helper-2 cytokines. Cyclophosphamide induced neither a quantitative reduction of Tregs determined by CD4+ FoxP3+ T cell levels nor a demonstrable qualitative difference in Treg function tested in vitro. Nonetheless, the number of vaccine-induced p53-specific IFN-γ-producing T cells was higher in our study compared to a study in which a similar patient group was treated with p53-SLP monotherapy (p≤0.012). Furthermore, the strong reduction in the number of circulating p53-specific T cells observed previously after four immunizations was currently absent. Stable disease was observed in 20.0% (2/10) of patients, and the remainder of patients (80.0%) showed clinical, biochemical and/or radiographic evidence of progressive disease. The outcome of this phase II trial warrants new studies on the use of low-dose cyclophosphamide to potentiate the immunogenicity of the p53-SLP vaccine or other antitumor vaccines.

Tumor-infiltrating cytotoxic T lymphocytes as independent prognostic factor in epithelial ovarian cancer with wilms tumor protein 1 overexpression.

Immune response characterization at the primary tumor site enables the design of therapeutic vaccination strategies with higher efficacy in epithelial ovarian cancer (EOC). In this study, we related Wilms tumor protein 1 (WT1) overexpression, a well-established immunotherapeutic target, to clinicopathological characteristics, immunological parameters, and survival in primary EOC. WT1 overexpression was evaluated in primary EOC tissue of 270 patients by immunohistochemistry on tissue microarrays (TMAs). Clinicopathological characteristics, follow-up, and data on infiltration of CD8⁺ cytotoxic T lymphocytes (CTLs), FoxP3⁺ regulatory T lymphocytes (Tregs), major histocompatibility complex (MHC) class I, and II molecule expression, were derived from a previously published dataset. WT1 overexpression was defined as positive immunostaining for WT1. WT1 overexpression, present in 56.3% of EOC, was associated with infiltration of Tregs [odds ratio (OR), 2.7; 95% confidence interval (95% CI), 1.6-4.7; P<0.001] and up-regulation of MHC class II (OR, 2.2; 95% CI, 1.2-4.1; P=0.014). Advanced stage (OR, 4.0; 95% CI, 1.9-8.6; P<0.001) and serous histology (OR, 6.7; 95% CI, 3.2-13.6; P<0.001) were independent predictors of WT1 overexpressing EOC. High number of CTL was an independent prognostic factor for progression-free survival (hazard ratio, 0.5; 95% CI, 0.3-0.8; P=0.006) in WT1 overexpressing EOC. As WT1 overexpressing EOC is associated with CTL and Treg infiltration next to MHC class II up-regulation, future clinical trials should evaluate the combination of therapeutic WT1 vaccines with strategies depleting Tregs and/or up-regulating MHC class I, in an attempt to enhance clinical efficacy in EOC patients.

Potential target antigens for a universal vaccine in epithelial ovarian cancer.

The prognosis of epithelial ovarian cancer (EOC), the primary cause of death from gynaecological malignancies, has only modestly improved over the last decades. Immunotherapeutic treatment using a cocktail of antigens has been proposed as a "universal" vaccine strategy. We determined the expression of tumor antigens in the context of MHC class I expression in 270 primary tumor samples using tissue microarray. Expression of tumor antigens p53, SP17, survivin, WT1, and NY-ESO-1 was observed in 120 (48.0%), 173 (68.9%), 208 (90.0%), 129 (56.3%), and 27 (11.0%) of 270 tumor specimens, respectively. In 93.2% of EOC, at least one of the investigated tumor antigens was (over)expressed. Expression of MHC class I was observed in 78.1% of EOC. In 3 out 4 primary tumors, (over)expression of a tumor antigen combined with MHC class I was observed. These results indicate that a multiepitope vaccine, comprising these antigens, could serve as a universal therapeutic vaccine for the vast majority of ovarian cancer patients.