Andreas Albers

Antitumor Activity of Human Papillomavirus Type 16 E7–Specific T Cells against Virally Infected Squamous Cell Carcinoma of the Head and Neck

Human papillomavirus (HPV)-associated squamous cell carcinoma of the head and neck (SCCHN) seems to be a suitable target for cancer vaccination. HPV-encoded oncogenic proteins, such as E7, are promising tumor-specific antigens and are obligatory for tumor growth. Because few immunologic studies have analyzed the endogenous HPV-specific immune response in this subset of SCCHN patients, we studied T-cell frequencies against HPV-16 E7(11-20) or E7(86-93) in tumor-bearing, human leukocyte antigen (HLA)-A*0201+ SCCHN patients, whose tumors were either HPV-16+ or HPV-16-. In HPV-16+ SCCHN patients, frequencies of T cells against either peptide were significantly elevated (P < 0.005) compared with HPV-16- patients or healthy volunteers. Tetramer+ T cells showed evidence of terminally differentiated phenotype (CD45RA+CCR7-) and an elevated level of CD107a staining for degranulation. Despite detectable expression of the restricting HLA class I allele, HLA-A*0201-E7(11-20)- or HLA-A*0201-E7(86-93)-specific CTL obtained by in vitro stimulation of healthy donor peripheral blood mononuclear cells only recognize a naturally HPV-16-transformed, HLA-A*0201+ SCCHN cell line after pretreatment with IFN-gamma. This cell line had little or no expression of LMP2, TAP1, and tapasin, critical components of the HLA class I antigen-processing machinery, which were up-regulated by IFN-gamma treatment. Immunohistochemistry of HPV-16+ SCCHN tumors showed that these antigen-processing machinery components are down-regulated in tumors in vivo compared with adjacent normal squamous epithelium. Thus, immunity to HPV-16 E7 is associated with the presence of HPV-16 infection and presentation of E7-derived peptides on SCCHN cells, which show evidence of immune escape. These findings support further development of E7-specific immunotherapy and strategies for up-regulation of antigen-processing machinery components in HPV-associated SCCHN.

Immune responses to p53 in patients with cancer: enrichment in tetramer+ p53 peptide-specific T cells and regulatory T cells at tumor sites.

Objective: A majority of human cancers, including head and neck cancer (HNC), “overexpress” p53. Although T cells specific for wild-type (wt) sequence p53 peptides are detectable in the peripheral blood of patients with HNC, it is unknown whether such T cells accumulate in tumor-involved tissues. Also, the localization of “regulatory” T cells (Treg) to tumor sites in HNC has not been investigated to date. Methods: Tumor infiltrating lymphocytes (TIL), tumor-involved or non-involved lymph node lymphocytes (LNL) and peripheral blood mononuclear cells (PBMC) were obtained from 24 HLA-A2.1+ patients with HNC. Using tetramers and four-color flow cytometry, the frequency of Treg and CD3+CD8+ T cells specific for wt p53 epitopes as well as their functional attributes were determined. Results: The CD3+CD8+ tetramer+ cell frequency was significantly higher (P<0.001) in TIL than autologous PBMC as was the percentage of CD4+CD25+ T cells (P<0.003). TIL were enriched in FOXp3+, GITR+ and CTLA-4+ Treg. CD8+ TIL had low Ζ expression and produced little IFN-γ after ex vivo stimulation relative to autologous PBMC or PBMC from NC. Conclusions: Anti-wt p53 epitope-specific T cells and Treg preferentially localize to tumor sites in patients with HNC. However, despite enrichment in tumor peptide-specific T cells, the effector cell population (CD3+CD8+) in TIL or PBMC was unresponsive to activation in the tumor microenvironment enriched in Treg.

TRAIL, Fasl and a blocking anti-DR5 antibody augment paclitaxel-induced apoptosis in human non-small-cell lung cancer

Lung carcinoma is one of the most frequent causes of malignancy‐related mortality in the world. Paclitaxel (PA) is an antineoplastic agent used in the treatment of non‐small‐cell lung cancer (NSCLC) and possesses a single‐agent response rate approaching 25%. PA kills tumor cells by inducing both cellular necrosis and apoptosis. Fas and Trail receptors (DR4 and DR5) are TNF family members and act as death signal transduction proteins in the apoptosis cascade. Despite the importance of PA in lung cancer treatment, the function of Fas, DR4 and DR5 in PA‐induced apoptosis, as well as the effect of their respective ligands FasL and TRAIL alone or in combination with PA, remains poorly understood. We show here that 10 μM PA induces a significant 10‐ to 57‐fold increase in primary lung cancer cell apoptosis and is associated with 20–215% increases in caspase‐3 activity in various NSCLC cell types. All the lung cancer cells express Fas, FasL, DR4 and DR5; however PA did not significantly modify their levels. We provide here the first time evidence that TRAIL is a potent inducer of apoptosis in multiple NSCLC cell lines. Noticeably, CH11, the Fas receptor cross‐linking and the antagonistic anti‐DR5 antibody enhance considerably the spontaneous apoptotic rate in 3 out of 5 cell types. The combination treatments, FasL+PA, TRAIL+PA or PA+anti‐DR5 antibody, greatly enhance PA‐apoptotic effect in most cell lines. These data suggest that the use of new combination treatment with PA and ligands targeting Fas or TRAIL receptors would be particularly efficacious.

Human tumor-derived genomic DNA transduced into a recipient cell induces tumor-specific immune responses ex vivo

This article describes a DNA-based vaccination strategy evaluated ex vivo with human cells. The vaccine was prepared by transferring tumor-derived genomic DNA to PCI-13 cells, a highly immunogenic tumor cell line (“recipient cell”), which had been genetically modified to secrete IL-2 (PCI-13/IL-2). PCI-13 cells expressed class I MHC determinants (HLA-A2) shared with the tumor from which the DNA was obtained as well as allogeneic determinants. DNA from a gp100+ melanoma cell line was transduced into gp100− PCI-13/IL-2 cells (PCI-13/IL-2/DNA). A T cell line specific for the gp100 epitope responded to PCI-13/IL-2/DNA cells by IFN-γ-secretion measured in enzyme-linked immunospot assays. The T cell line also recognized the gp100 epitope presented by dendritic cells that ingested PCI-13/IL-2/DNA cells, which had been induced by UVB irradiation to undergo apoptosis. After up-take and processing of apoptotic PCI-13/IL-2/DNA cells, the dendritic cells primed normal peripheral blood lymphocytes to generate effector T cells specific for the tumor donating the DNA. The results indicate that tumor epitopes encoded in such DNA are expressed in recipient cells and can induce tumor-specific T cells. The findings support translation of this vaccination strategy to a phase I trial in patients with cancer.

Function but not phenotype of melanoma peptide-specific CD8(+) T cells correlate with survival in a multiepitope peptide vaccine trial (ECOG 1696).

ECOG 1696 was a Phase II multi‐center trial testing vaccination with melanoma peptides, gp100, MART‐1 and tyrosinase delivered alone, with GM‐CSF, IFN‐α2b or both cytokines to HLA‐A2+ patients with metastatic melanoma. Here, the frequency of circulating CD8+tetramer+ (tet+) T cells and maturation stages of responding T cells were serially monitored and compared with baseline values in a subset of patients (n = 37) from this trial. Multiparameter flow cytometry was used to measure the frequency of CD8+ T cells specific for gp100, MART‐1, tyrosinase and influenza (FLU) peptides. Expression of CD45RA/CCR7 on CD8+tet+ T cells and CD25, CD27, CD28 on all circulating T cells was determined. Vaccine‐induced changes in the CD8+tet+ T cell frequency and phenotype were compared with results of IFN‐γ ELISPOT assays and with clinical responses. The frequency of CD8+tet+ T cells in the circulation was increased for the melanoma peptides (p < 0.03–0.0001) but not for FLU (p < 0.9). Only gp100‐ and MART‐1‐specific T cells differentiated to CD45RA+CCR7‐ effector/memory T cells. In contrast to the IFN‐γ ELISPOT frequency, previously correlated with overall survival (Kirkwood et al., Clin Cancer Res 2009;15:1443‐51), neither the frequency nor differentiation stage of CD8+tet+ T cells correlated with clinical responses. Delivery of GM‐CSF and/or IFN‐α2b had no effects on the frequency or differentiation of CD8+tet+, CD8+ or CD4+ T cells. Phenotypic analyses of CD8+tet+ T cells did not correlate with clinical responses to the vaccine, indicating that functional assessments of peptide‐specific T cells are preferable for monitoring of anti‐tumor vaccines.

The Wild-Type Sequence (wt) p53 25–35 Peptide Induces HLA-DR7 and HLA-DR11-Restricted CD4 + Th Cells Capable of Enhancing the Ex Vivo Expansion and Function of Anti-wt p53 264–272 Peptide CD8 + T Cells

Tumor peptide-based vaccines are more effective when they include tumor-specific Th cell-defined as well as CTL-defined peptides. Presently, two overlapping wild-type sequences (wt) p53 helper peptides, p53108–122 and p53110–124, have been identified as HLA-DR1- and/or HLA-DR4-restricted epitopes. These HLA-DR alleles are expressed by ∼35% of subjects with cancer. To identify Th cell-defined wt p53 peptides suitable for use on the remaining subject population, a dendritic cell (DC)-based coculture system was developed. CD4+ T cells isolated from PBMC obtained from HLA-DR4− normal donors were stimulated ex vivo with autologous DC transfected with wt p53 or mutant p53 cDNA. Reactivity of T cells was tested in ELISPOT IFN-γ assays against DC pulsed individually with a panel of algorithm-predicted, multiple HLA-DR-binding wt p53 peptides. The wt p5325–35 peptide was identified as capable of inducing and being recognized by CD4+ T cells in association, at a minimum, with HLA-DR7 and -DR11 molecules, each of which is expressed by ∼15% of the population. In addition, the presence of anti-p5325–35 CD4+ Th cells was shown to enhance the in vitro generation/expansion of HLA-A2-restricted, anti-wt p53264–272 CD8+ T cells, which from one donor were initially “nonresponsive” to the wt p53264–272 peptide. The wt p5325–35 peptide has attributes of a naturally presented Th cell-defined peptide, which could be incorporated into antitumor vaccines applicable to a broader population of subjects for whom a wt p53 helper peptide is presently unavailable, as well as used for monitoring anti-p53 Th cell activity in cancer subjects receiving p53-based immunotherapy.

Alterations in the T-Cell Receptor Variable β Gene–Restricted Profile of CD8+ T Lymphocytes in the Peripheral Circulation of Patients with Squamous Cell Carcinoma of the Head and Neck

Purpose: Apoptosis of activated CD8+ T cells is often seen in tumor-infiltrating lymphocytes and circulating peripheral blood mononuclear cells (PBMC) in patients with squamous cell carcinoma of the head and neck (SCCHN). We investigated whether T-cell receptor (TCR) variable β chain (Vβ)–restricted T cells were more sensitive to apoptosis than non–TCR Vβ-restricted T cells. Experimental Design: Flow cytometry analysis with anti-TCR Vβ antibodies was used to define expansions and contractions of Vβ-restricted T cells in patients with SCCHN relative to normal donors. This staining was combined with Annexin V binding to indicate early T-cell apoptosis. Results: The TCR Vβ profiles of CD3+ T cells in tumor-infiltrating lymphocytes and PBMCs of patients with SCCHN were altered relative to controls, with one to five expansions and numerous contractions of TCR Vβ-restricted T cells detected. These types of alterations were significantly greater in CD8+ than CD4+ T cells. Enhanced Annexin V binding to CD8+ T cells was evident in PBMCs obtained from all patients, with 3 of 13 showing preferential targeting for apoptosis of TCR Vβ-restricted T cells. Conclusions: TCR Vβ profiles of CD8+ T cells were altered in patients with SCCHN relative to normal controls. This may reflect increased apoptosis of expanded or contracted CD8+ T cells, which define the TCR Vβ profile of antigen-responsive T-cell populations in patients with cancer.

Spontaneous apoptosis of tumor-specific tetramer+ CD8+ T lymphocytes in the peripheral circulation of patients with head and neck cancer.

In cancer, tumor escape from the host immune system includes apoptosis of circulating CD3+CD8+ effector T lymphocytes. Here, we compare sensitivity to apoptosis of virus‐ with tumor‐specific circulating CD8+ T cells in patients with head and neck cancer.

Additive Effects of TRAIL and Paclitaxel on Cancer Cells: Implications for Advances in Cancer Therapy

In cancer therapy outgrowth of chemoresistant tumor cells is the most important factor that ultimately determines-apart from immediate adverse effects during treatment-the life span and prognosis of cancer patients. Despite many advances in cancer treatment and the integration of supportive medications, including new and better drugs for pain management, antiemesis, infection, and reconstitution of the hematopoietic system, both toxic effects and the development of resistance in response to the treatment remain a major problem. New treatment regimens have to be developed to target cancer more specifically using multiple cellular pathways. This will reduce toxic effects as well as the development of chemoresistance. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is the ligand for death receptors that belong to the TNF death receptor family. TRAIL triggers apoptosis in vitro in various cancer cell types. The antitumor drug, Paclitaxel (PA) was shown to increase the survival of patients with cancer. In in vitro experiments, PA also induces apoptosis in cancer cells. Together, PA and TRAIL lead to tumor regression in in vivo therapy and induce apoptosis through the interaction of TNF family death receptors, caspase activation, and?or cytochrome c release from mitochondria. PA and TRAIL complement each other using two distinct pathways that trigger apoptosis in addition to the anti-microtubule effect of PA. The combination of TRAIL and PA suppresses tumor growth that is otherwise resistant to treatment with either PA or TRAIL alone, by improving proapoptotic effects of the drugs. This observation support the use of the PA and TRAIL in future clinical trials.

Impact of p53-based immunization on primary chemically-induced tumors.

In mice as well as humans, cytotoxic T lymphocytes (CTL) specific for wild‐type‐sequence (wt) p53 peptides have been shown to react against a wide range of tumors, but not normal cells. As such, they are attractive candidates for developing broadly applicable cancer vaccines. Of particular interest is the potential of using p53‐based vaccines in high‐risk individuals to prevent cancer. Methylcholanthrene, an immunosuppressive polycyclic hydrocarbon carcinogen implicated as a causative agent in human cancers, has long been used to induce murine tumors with a high incidence of genetic alterations and sensitivity to wt p53‐specific CTL. To analyze the potential of p53‐based vaccines on primary tumors, we evaluated the efficacy of DNA and dendritic cell vaccines targeting wt p53 peptides given to methylcholanthrene‐treated mice in the protection or therapy settings. The results indicate that the efficacy of these vaccines relative to reducing tumor incidence were severely compromised by vaccine‐induced tumor escape. As compared to tumors induced in non‐immunized mice, a higher incidence of epitope‐loss tumors was detected in tumors from the immunized mice. The increase in tumor escape arose as a consequence of either increased frequencies of mutations within/flanking p53 epitope‐coding regions or downregulation of expression of the major histocompatibility complex Class I molecules that present these epitopes for T cell recognition These findings are consistent with current views of immunoselection occurring in patients receiving tumor peptide‐based immunotherapy, and impact on the design and implementation of p53‐based vaccines, in particular, those aimed at treating individuals at high risk for developing cancer.