Spontaneous immunity against Bcl-xL in cancer patients.

It is well-established that peptide epitopes derived from human tumor-associated Ags can be recognized by CTL in the context of the MHC molecule. However, the vast majority of Ags described are not vital for survival and growth of the tumor cells, and immunoselection of Ag-loss variants during immunotherapy has been demonstrated in several cases. Malfunctions in death pathways observed in human cancers are often due to overexpression of antiapoptotic proteins in the Bcl-2 protein family, i.e., Bcl-2, Mcl-1, and Bcl-xL. These antiapoptotic proteins are implicated in cancer development, tumor progression, and drug resistance. The general overexpression of the antiapoptotic members of the Bcl-2 family in cancer and the fact that down-regulation or loss of expression of these proteins as a means of immune escape would impair sustained tumor growth makes them very attractive targets for anticancer immunotherapy. Recently, we identified spontaneous T cell responses against Bcl-2- and Mcl-1-derived peptides in patients suffering from cancers of different origin. In this study, we demonstrate that Bcl-xL is a target for T cell recognition in cancer patients. Thus, we describe spontaneous HLA-A2-restricted cytotoxic T cell responses against peptide epitopes derived from Bcl-xL by means of ELISPOT and flow cytometry stainings, whereas no responses were detected against any of the Bcl-xL epitopes in any healthy controls. Moreover, Bcl-xL-specific T cells are cytotoxic against HLA-matched cancer cells of different origin. Thus, cellular immune responses against apoptosis inhibitors like the Bcl-2 family proteins appear to represent a general feature in cancer.

Immunogenicity of Bcl-2 in patients with cancer.

B-cell lymphoma 2 (Bcl-2) is a pivotal regulator of apoptotic cell death and it is overexpressed in many cancers. Consequently, the Bcl-2 protein is an attractive target for drug design, and Bcl-2-specific antisense oligonucleotides or small-molecule Bcl-2 inhibitors have shown broad anticancer activities in preclinical models and are currently in several clinical trials. The clinical application of immunotherapy against cancer is rapidly moving forward in multiple areas, including the adoptive transfer of anti-tumor-reactive T cells and the use of "therapeutic" vaccines. The overexpression of Bcl-2 in cancer and the fact that immune escape by down-regulation or loss of expression of this protein would impair sustained tumor growth makes Bcl-2 a very attractive target for anticancer immunotherapy. Herein, we describe spontaneous T-cell reactivity against Bcl-2 in peripheral blood from patients suffering from unrelated tumor types (ie, pancreatic cancer, breast cancer, acute myeloid leukemia [AML], and chronic lymphocytic leukemia [CLL]). Additionally, we show that these Bcl-2-reactive T cells are indeed peptide-specific, cytotoxic effector cells. Thus, Bcl-2 may serve as an important and widely applicable target for anticancer immunotherapeutic strategies (eg, in the combination with conventional radiotherapy and chemotherapy).

Spontaneous T-cell responses against peptides derived from the Taxol resistance-associated gene-3 (TRAG-3) protein in cancer patients.

Expression of the cancer-testis antigen Taxol resistance-associated gene-3 (TRAG-3) protein is associated with acquired paclitaxel (Taxol) resistance, and is expressed in various cancer types; e.g., breast cancer, leukemia, and melanoma. Thus, TRAG-3 represents an attractive target for immunotherapy of cancer. To identify HLA-A*02.01-restricted epitopes from TRAG-3, we screened cancer patients for spontaneous cytotoxic T-cell responses against TRAG-3-derived peptides. The TRAG-3 protein sequence was screened for 9mer and 10mer peptides possessing HLA-A*02.01-binding motifs. Of 12 potential binders, 9 peptides were indeed capable of binding to the HLA-A*02.01 molecule, with binding affinities ranging from strong to weak binders. Subsequently, lymphocytes from cancer patients (9 breast cancer patients, 12 melanoma patients, and 13 patients with hematopoietic malignancies) were analyzed for spontaneous reactivity against the panel of peptides by ELISpot assay. Spontaneous immune responses were detected against 8 epitope candidates in 7 of 9 breast cancer patients, 7 of 12 melanoma patients, and 5 of 13 patients with hematopoietic malignancies. In several cases, TRAG-3-specific CTL responses were scattered over several epitopes. Hence, no immunodominance of any single peptide was observed. Furthermore, single-peptide responses were detected in 2 of 12 healthy HLA-A2(+) donors, but no responses were detectable in 9 HLA-A2(-) healthy donors or 4 HLA-A2(-) melanoma patients. The identified HLA-A*02.01-restricted TRAG-3-derived epitopes are targets for spontaneous immune responses in breast cancer, hematopoietic cancer, and melanoma patients. Hence, these epitopes represent potential target structures for future therapeutic vaccinations against cancer, possibly appropriate for strategies that combine vaccination and chemotherapy; i.e., paclitaxel treatment.

Immunogenicity of constitutively active V599EBRaf.

Activating BRAF somatic missense mutations within the kinase domain are present in 60-66% of melanomas. The vast majority of these represent a single substitution of glutamate for valine (V599E). Here, we demonstrate spontaneous HLA-B*2705-restricted cytotoxic T-cell responses against an epitope derived from (V599E)BRaf. These T-cell responses were mutation specific as the corresponding epitope derived from wild-type BRaf was not recognized. The loss of the (V599E)BRAF genotype during progression from primary to metastatic melanoma in patients with (V599E)BRaf specific T-cell responses suggests an active immune selection of nonmutated melanoma clones by the tumor-bearing host.

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.

Identification of novel survivin-derived CTL epitopes.

The identification of tumor antigens, which are essential for the survival of tumor cells is a new avenue to prevent antigen loss variants emerging due to immunoselection, particularly during immune therapy. In the search for such immunogenic tumor antigens, we recently identified spontaneous cytotoxic lymphocyte (CTL) responses against the inhibitor of apoptosis protein survivin. Thus, we identified two HLA-A2-restricted, survivin-derived CTL epitopes, which both were targets for spontaneous CTL responses in melanoma, breast cancer, and CLL. Here, we extend these data and describe the characterization of novel HLA-A1-, HLA-A2-, HLA-A3-, and HLA-A11-restricted survivin epitopes on the basis of spontaneous CTL responses in cancer patients. These epitopes significantly increase the number of patients eligible for immunotherapy based on survivin derived peptides. Additionally, the collective targeting of several restriction elements is likely to decrease the risk of immune escape by HLA-allele loss.

HLA-B35-restricted immune responses against survivin in cancer patients.

Two HLA-A2 restricted epitopes have recently been identified from the broadly expressed tumor antigen survivin, and several vaccination trials in cancer patients based on these survivin-derived peptides have been initiated. Consequently, there is a crucial need for the identification of survivin epitopes restricted to other HLA-molecules in order to extend the proportion of patients that can enter these ongoing clinical trials. In the present study, we characterized 2 survivin-derived epitopes, which are restricted to HLA-B35. Specific T-cell reactivity against these survivin-derived epitopes was found in the peripheral blood from patients with different B-cell malignancies and melanoma. Substitution of the C-terminal anchor residue of the survivin-derived peptides improved the recognition by tumor-infiltrating lymphocytes from melanoma patients. Furthermore, we demonstrated spontaneous cytotoxic T-cell responses to survivin in a primary melanoma lesion. The characterization of these epitopes allows more patients can be included in the ongoing peptide-based survivin vaccination trials against cancer.

Dynamic changes of specific T cell responses to melanoma correlate with IL-2 administration.

Interleukin 2 (IL-2) is a promising immunotherapeutic agent for the treatment of metastatic melanoma and renal cell carcinoma. Systemic administration of high dose IL-2 produces objective responses in up to 25% of melanoma patients, and a low but significant proportion of these patients experience durable responses. Nevertheless, the cells and molecules responsible for induction of tumor regression over the course of IL-2 treatment remain unknown. New strategies in tumor immunotherapy have evolved over the past decade as a consequence of significant progress in the field, in particular with respect to the characterization of peptide epitopes derived from tumor associated antigens, and the role of antigen presenting cells in the initiation of cellular immune responses. Alongside with these factual as well as conceptual advances, new methods have been developed to monitor and characterize anti-tumor T cell responses in cancer patients. Application of these tools to dissect anti-tumor responses has demonstrated that various immune therapeutic approaches can induce powerful systemic anti-tumor cytotoxic T lymphocyte (CTL) responses. However, only limited efforts have been made to use present days tool to analyze anti-tumor immune responses in patients treated with IL-2 based immunotherapy. We have examined CTL responses against known tumor antigens in melanoma patients over the course of IL-2 based immunotherapy (electrochemotherapy). Surprisingly, anti-tumor CTL responses significantly declined upon initiation of therapy, but reappeared when IL-2 administration was paused. Molecular analyses of the clonotypic composition of responding T cells demonstrated that new clones emerged over the course of treatment, and that tumor-specific T cells that had left the peripheral blood could subsequently be detected at the tumor site. These data provide new insight into the biological actions of IL-2 and highlight the difficulties associated with the monitoring of anti-tumor immune responses. This underlines the importance of frequent sampling of blood and tumor biopsies to be analyzed with a combination of state of the art technologies in order to gain detailed information on the interactions between cancer cells and cells of the immune system.