T-cell response against human malignant melanoma.

The present Thesis is introduced with a discussion of reverse transcription polymerase chain reaction (RT-PCR) based analysis of TCRBV regions, including the description of a panel of primers. Using this primer panel, we analysed for TCR clonality in progressive versus regressive parts of partially regressive primary melanoma lesions (Paper 1). The main conclusion drawn from this study is that clonotypic T cells were present in both regressive and progressive parts of the same lesions. However, sequencing of “non clonal” BV-regions demonstrated that most of them were not polyclonal by nature but oligoclonal e.g. that in some cases all transcripts were of the same length or only a limited number of BJ regions were used. This indicated that some T-cell clones were left undetected by direct sequencing. To facilitate full and detailed analysis of T-cell clonotypes in the infiltrates we aimed at establishing a suitable method for the detection of clonally expanded T cells. Denaturing gradient gel electrophoresis (DGGE) was chosen because this method is highly sensitive and excludes laborious steps that are obligatory in almost all other methods dealing with the detection of TCR clonality (Paper 2). We applied the DGGE based method to analyse for in situ T-cell clonality in 6 subcutaneous melanoma lesions from two patients. The results demonstrated two important characteristics of in situ TIL in melanoma. First, the infiltrate constitutes an exceedingly high number of different T-cell clones, and second, the T-cell response appears to be executed by local T cells that do not enter the periphery (Paper 3). The final study deals with a comparative analysis of in situ T cells and T cells propagated in vitro, demonstrating that standard in vitro culture conditions do not support the growth of in vivo expanded T-cell clones (Paper 4)

Characterization of Ex Vivo Expanded Tumor Infiltrating Lymphocytes from Patients with Malignant Melanoma for Clinical Application

Clinical trials of adoptive transfer of autologous tumor infiltrating lymphocytes (TILs) to patients with advanced malignant melanoma have shown remarkable results with objective clinical responses in 50% of the treated patients. In order to initiate a clinical trial in melanoma, we have established a method for expanding TILs to clinical relevant quantities in two steps with in 8 weeks. Further characterization of expanded TILs revealed an oligoclonal composition of T-cells with an effector memory like phenotype. When autologous tumor was available, TILs showed specific activity in all patients tested. TIL cultures contained specificity towards tumor cells as well as peptides derived from tumor-associated antigens (TAAs) during expansion procedures

The Melanoma Inhibitor of Apoptosis Protein: A Target for Spontaneous Cytotoxic T Cell Responses

The identification of tumor antigens which expression is essential for the survival of tumor cells is a new avenue to prevent antigen loss variants emerging due to immunoselection, particularly during immune therapy. The melanoma inhibitor of apoptosis protein, ML-IAP (also named livin) counteracts apoptosis induced by death receptors, hypooxgenic conditions, or chemotherapeutic agents. Thus, elevated expression of ML-IAP renders melanoma cells resistant to apoptotic stimuli and thereby potentially contributes to the oncogenic phenotype. Here, we demonstrate that T cells in a large proportion of melanoma patients infiltrating the tumor or circulating in the peripheral blood specifically recognize ML-IAP-derived peptides. Interestingly, the responses against the peptide epitope ML-IAP280–289 were not restricted to melanoma patients but present among peripheral blood T cells in a few healthy controls. In situ peptide/HLA-A2 multimer staining, however, confirmed the infiltration of ML-IAP-reactive cells into the tumor microenvironment. Moreover, ML-IAP-reactive T cells isolated by magnetic beads coated with peptide/HLA-A2 complexes were cytotoxic against HLA-matched melanoma cells. In conclusion, out data strongly indicate ML-IAP as a suitable target for immunologic intervention

Exercise Oncology and Immuno-Oncology; A (Future) Dynamic Duo

Recent advances in clinical oncology is based on exploiting the capacity of the immune system to combat cancer: immuno-oncology. Thus, immunotherapy of cancer is now used to treat a variety of malignant diseases. A striking feature is that even patients with late-stage disease may experience curative responses. However, most patients still succumb to disease, and do not benefit from treatment. Exercise has gained attention in clinical oncology and has been used for many years to improve quality of life, as well as to counteract chemotherapy-related complications. However, more recently, exercise has garnered interest, largely due to data from animal studies suggesting a striking therapeutic effect in preclinical cancer models; an effect largely mediated by the immune system. In humans, physical activity is associated with a lower risk for a variety of malignancies, and some data suggest a positive clinical effect for cancer patients. Exercise leads to mobilization of cells of the immune system, resulting in redistribution to different body compartments, and in preclinical models, exercise has been shown to lead to immunological changes in the tumor microenvironment. This suggests that exercise and immunotherapy could have a synergistic effect if combined

HLA-A24 and survivin: possibilities in therapeutic vaccination against cancer

Recently, it was described that an HLA-A24 restricted peptide derived from the survivin splice variant survivin-2B can be recognized by CD8(+) cytotoxic T-cells. The identification of an HLA-A24 epitope is critical for survivin-based immunotherapy as HLA-24 is the most frequent HLA allele in Asia. Consequently, this survivin-2B epitope is already a target in a clinical study in patients with advanced or recurrent colorectal cancer expressing survivin. However, the splice variant survivin-2B has been described to be pro-apoptotic, and is only expressed at low levels in most malignant tissues. Furthermore, survivin-2B expression are significantly decreased in later tumor stages and inversely correlated with tumor differentiation and invasion. Consequently, survivin is a more general vaccination candidate than the splice variant survivin-2B. Here, we on the basis of spontaneous immune responses in HLA-A24+ cancer patients describes that a HLA-A24-restricted survivin epitopes does indeed exist. Consequently, this epitope is an attractive target for the ongoing survivin-based peptide immunotherapy against cancer

Therapeutic Cancer Vaccines in Combination with Conventional Therapy

The clinical efficacy of most therapeutic vaccines against cancer has not yet met its promise. Data are emerging that strongly support the notion that combining immunotherapy with conventional therapies, for example, radiation and chemotherapy may improve efficacy. In particular combination with chemotherapy may lead to improved clinical efficacy by clearing suppressor cells, reboot of the immune system, by rendering tumor cells more susceptible to immune mediated killing, or by activation of cells of the immune system. In addition, a range of tumor antigens have been characterized to allow targeting of proteins coupled to intrinsic properties of cancer cells. For example, proteins associated with drug resistance can be targeted, and form ideal target structures for use in combination with chemotherapy for killing of surviving drug resistant cancer cells. Proteins associated with the malignant phenotype can be targeted to specifically target cancer cells, but proteins targeted by immunotherapy may also simultaneously target cancer cells as well as suppressive cells in the tumor stroma