Antimelanoma CTL recognizes peptides derived from an ORF transcribed from the antisense strand of the 3' untranslated region of TRIT1.

Noncoding regions of the genome play an important role in tumorigenesis of cancer. Using expression cloning, we have identified a cytotoxic T lymphocyte (CTL)-defined antigen that recognizes a protein sequence derived from an open reading frame transcribed from the reverse strand in the 3' untranslated region of tRNA isopentenyltransferase 1 (TRIT1). A peptide derived from this open reading frame (ORF) sequence and predicted to bind to HLA-B57, sensitized HLA-B57(+) tumor cells to lysis by CTL793. The peptide also induced a CTL response in peripheral blood mononuclear cells (PBMC) of patient 793 and in two other melanoma patients. The CTL lysed peptide-pulsed HLA-B57(+) target cells and melanoma cells with endogenous antigen expression. The recognition of this antigen is not limited to HLA-B57-restricted CTLs. An HLA-A2 peptide derived from the ORF was able to induce CTLs in PBMC of 2 HLA-A2(+) patients. This study describes for the first time a CTL-defined melanoma antigen that is derived from an ORF on the reverse strand of the putative tumor suppressor gene TRIT1. This antigen has potential use as a vaccine or its ability to induce CTLs in vitro could be used as a predictive biomarker.

Concurrent MEK2 mutation and BRAF amplification confer resistance to BRAF and MEK inhibitors in melanoma.

Although BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it.

There is a world beyond protein mutations: the role of non-coding RNAs in melanomagenesis.

Until recently, the general perception has been that mutations in protein-coding genes are responsible for tumorigenesis. With the discovery of (V600E)BRAF in about 50% of cutaneous melanomas, there was an increased effort to find additional mutations. However, mutations characterized in melanoma to date cannot account for the development of all melanomas. With the discovery of microRNAs as important players in melanomagenesis, protein mutations are no longer considered the sole drivers of tumors. Recent research findings have expanded the view for tumor initiation and progression to additional non-coding RNAs. The data suggest that tumorigenesis is likely an interplay between mutated proteins and deregulation of non-coding RNAs in the cell with an additional role of the tumor environment. With the exception of microRNAs, our knowledge of the role of non-coding RNAs in melanoma is in its infancy. Using few examples, we will summarize some of the roles of non-coding RNAs in tumorigenesis. Thus, there is a whole world beyond protein-coding sequences and microRNAs, which can cause melanoma.

Nucleophosmin is recognized by a cytotoxic T cell line derived from a rectal carcinoma patient.

Immunotherapy of colorectal carcinoma (CRC) has great promise as the presence of T lymphocytes in CRC tissues in situ is correlated with reduced recurrence and increased survival. Thus, identification of the antigens recognized by T cells of CRC patients may permit development of vaccines with potential benefit for these patients. Using expression cloning, we identified the antigen, nucleophosmin (Npm), recognized by an HLA-A1 restricted cytotoxic T lymphocyte (CTL) line derived from the peripheral blood mononuclear cells (PBMC) of a rectal cancer patient. A decamer peptide derived from the Npm sequence sensitized peptide-pulsed HLA-A1 positive cells to lysis by the CTL line. The peptide also induced proliferative and cytotoxic T lymphocytes in the PBMC of 4 of 6 CRC patients, which lysed HLA-A1 positive peptide-pulsed target cells and CRC cells endogenously expressing Npm. Overexpression of Npm by tumors of various histological types, recognition of the antigen by T cells derived from different CRC patients and association of the antigen with poor prognostic outcome make it a promising target for immunotherapeutic intervention in cancer patients.

Shared MHC class II-dependent melanoma ribosomal protein L8 identified by phage display.

Antigens recognized by T helper (Th) cells in the context of MHC class II molecules have vaccine potential against cancer and infectious agents. We have described previously a melanoma patient's HLA-DR7-restricted Th cell clone recognizing an antigen, which is shared among melanoma and glioma cells derived from various patients. Here, this antigen was cloned using a novel antigen phage display approach. The antigen was identified as the ribosomal protein L8 (RPL8). A peptide of RPL8 significantly stimulated proliferation and/or cytokine expression of the Th cell clone and lymphocytes in four of nine HLA-DR7(+) melanoma patients but not in healthy volunteers. The RPL8 antigen may represent a relevant vaccine target for patients with melanoma, glioma, and breast carcinoma whose tumors express this protein.

Colon carcinoma cells induce CXCL11-dependent migration of CXCR3-expressing cytotoxic T lymphocytes in organotypic culture.

Adoptive immunotherapy of cancer patients with cytolytic T lymphocytes (CTL) has been hampered by the inability of the CTL to home into tumors in vivo. Chemokines can attract T lymphocytes to the tumor site, as demonstrated in animal models, but the role of chemokines in T-lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the role of chemokines and their receptors in the migration of a colon carcinoma (CC) patient's CTL toward autologous tumor cells has been studied in a novel three-dimensional organotypic CC culture. CTL migration was mediated by chemokine receptor CXCR3 expressed by the CTL and CXCL11 chemokine secreted by the tumor cells. Excess CXCL11 or antibodies to CXCL11 or CXCR3 inhibited migration of CTL to tumor cells. T cell and tumor cell analyses for CXCR3 and CXCL11 expression, respectively, in ten additional CC samples, may suggest their involvement in other CC patients. Our studies, together with previous studies indicating angiostatic activity of CXCL11, suggest that CXCL11 may be useful as an immunotherapeutic agent for cancer patients when transduced into tumor cells or fused to tumor antigen-specific Ab.

High-grade glioma formation results from postnatal pten loss or mutant epidermal growth factor receptor expression in a transgenic mouse glioma model.

High-grade gliomas are devastating brain tumors associated with a mean survival of <50 weeks. Two of the most common genetic changes observed in these tumors are overexpression/mutation of the epidermal growth factor receptor (EGFR) vIII and loss of PTEN/MMAC1 expression. To determine whether somatically acquired EGFRvIII expression or Pten loss accelerates high-grade glioma development, we used a previously characterized RasB8 glioma-prone mouse strain, in which these specific genetic changes were focally introduced at 4 weeks of age. We show that both postnatal EGFRvIII expression and Pten inactivation in RasB8 mice potentiate high-grade glioma development. Moreover, we observe a concordant loss of Pten and EGFR overexpression in nearly all high-grade gliomas induced by either EGFRvIII introduction or Pten inactivation. This novel preclinical model of high-grade glioma will be useful in evaluating brain tumor therapies targeted to the pathways specifically dysregulated by EGFR expression or Pten loss.

Human leukocyte antigen-A2-restricted CTL responses to mutated BRAF peptides in melanoma patients.

Mutated BRAF (BRAF(V600E)) is a potential immunotherapeutic target for melanoma because of its tumor specificity and expression in the majority of these lesions derived from different patients. BRAF(V600E) is expressed intracellularly and not on the cell surface, therefore providing a target for T cells but not B cells. Demonstration of patients' T cell responses to BRAF(V600E) would suggest the feasibility of active specific immunotherapy targeting the mutation in these patients. In the present study, BRAF(V600E) peptides with putative binding sites for human leukocyte antigen (HLA)-A2 were used to stimulate T lymphocytes of HLA-A2-positive melanoma patients. Four of five patients with BRAF(V600E)-positive lesions showed lymphoproliferative responses to BRAF(V600E) peptide stimulation. These responses were specific for the mutated epitope and HLA-A2 was restricted in three patients. Lymphocytes from these three patients were cytotoxic against HLA-A2-matched BRAF(V600E)-positive melanoma cells. None of the four patients with BRAF(V600E)-negative lesions and none of five healthy donors had lymphoproliferative responses specific for the mutated epitope. The high prevalence (approximately 50%) of HLA-A2 among melanoma patients renders HLA-A2-restricted BRAF(V600E) peptides attractive candidate vaccines for these patients.

Migration of cytotoxic T lymphocytes toward melanoma cells in three-dimensional organotypic culture is dependent on CCL2 and CCR4.

Studies in experimental animal models have demonstrated that chemokines produced by tumor cells attract chemokine receptor-positive T lymphocytes into the tumor area. However, in cancer patients, the role of chemokines in T lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the migration of a melanoma patient's CTL toward autologous tumor cells has been studied in a novel three-dimensional organotypic melanoma culture. In this model, CTL migrated toward tumor cells, resulting in tumor cell apoptosis. CTL migration was mediated by the CC chemokine receptor (CCR)4 expressed by the CTL and the CC chemokine ligand (CCL)2 secreted by the tumor cells, as evidenced by blockage of CTL migration by CCL2 or antibodies to CCL2 or CCR4. These results were confirmed in a Transwell migration assay in which the CTL actively migrated toward isolated CCL2 and migration was inhibited by anti-CCR4 antibody. These studies, together with previous studies in mice indicating regression of CCL2-transduced tumor cells, suggest that CCL2 may be useful as an immunotherapeutic agent for cancer patients.

Serum antibodies to EpCAM in healthy donors but not ulcerative colitis patients.

PURPOSE: The gastrointestinal carcinoma-associated antigen epithelial cell adhesion molecule (EpCAM) has been a target for passive and active immunotherapy of gastrointestinal carcinoma patients. The antigen is expressed by both tumor and normal tissues. The immunogenicity of EpCAM in colorectal cancer patients has been described previously. The purpose of this study was to evaluate humoral and cellular immune responses of healthy individuals and ulcerative colitis patients to EpCAM and to relate immune responses to colonic tissue expression of EpCAM. METHODS: An inhibition radioimmunoassay was used to detect anti-EpCAM serum antibodies. Anti-EpCAM antibodies of a healthy donor were expressed by phages and sequenced. (3)H-thymidine incorporation assay was used for detection of lymphoproliferative responses to stimulation with EpCAM. EpCAM tissue expression was determined by immunohistochemistry. RESULTS: We detected anti-EpCAM serum antibodies in 4 of 10, and EpCAM-specific lymphoproliferation responses in 1 of 10 healthy volunteers. The majority of anti-EpCAM antibodies derived from a healthy donor were germline-encoded. In contrast, none of the 23 patients with ulcerative colitis showed serum antibodies to EpCAM (P=0.005). Antigen expression was greatly reduced and altered in ulcerative colitis patients, whereas colon from healthy individuals and uninvolved colon of colorectal cancer patients expressed high levels of EpCAM. CONCLUSION: The results of these studies suggest an association between EpCAM antibody production and colonic EpCAM expression in healthy individuals and patients with ulcerative colitis. Decreased and altered colonic EpCAM expression in ulcerative colitis patients may be related to the disease induction, based on the previously demonstrated adhesion function of this molecule. Healthy individuals with anti-EpCAM immune responses and high risk for developing colorectal carcinoma are prime candidates for prophylactic immunization against EpCAM.

CXC chemokine ligand 12 (stromal cell-derived factor 1 alpha) and CXCR4-dependent migration of CTLs toward melanoma cells in organotypic culture.

Studies in experimental animal models have demonstrated that chemokines produced by tumor cells attract chemokine receptor-positive T lymphocytes into the tumor area, which may lead to tumor growth inhibition in vitro and in vivo. However, in cancer patients, the role of chemokines in T lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the role of chemokines and their receptors in the migration of a melanoma patient's CTL toward autologous tumor cells has been studied in a novel organotypic melanoma culture, consisting of a bottom layer of collagen type I with embedded fibroblasts followed successively by a tumor cell layer, collagen/fibroblast separating layer, and, finally, a top layer of collagen with embedded fibroblasts and T cells. In this model, CTL migrated from the top layer through the separating layer toward tumor cells, resulting in tumor cell apoptosis. CTL migration was mediated by chemokine receptor CXCR4 expressed by the CTL and CXCL12 (stromal cell-derived factor 1alpha) secreted by tumor cells, as evidenced by blockage of CTL migration by Abs to CXCL12 or CXCR4, high concentrations of CXCL12 or small molecule CXCR4 antagonist. These studies, together with studies in mice indicating regression of CXCL12-transduced tumor cells, followed by regression of nontransduced challenge tumor cells, suggest that CXCL12 may be useful as an immunotherapeutic agent for cancer patients, when transduced into tumor cells, or fused to anti-tumor Ag Ab or tumor Ag.

Immune responses of breast cancer patients to mutated epidermal growth factor receptor (EGF-RvIII, Delta EGF-R, and de2-7 EGF-R).

Mutated epidermal growth factor receptor (EGF-RvIII, DeltaEGF-R, and de2-7 EGF-R) is the result of an 801-bp deletion within the extracellular domain of wild-type EGF-R and is expressed by breast carcinomas, but not by normal breast tissues. EGF-RvIII is expressed both on the surface and in the cytoplasm of tumor cells. Thus, EGF-RvIII is a potential tumor-specific target for both Abs and T cells. However, it is not known whether breast cancer patients can raise immune responses to EGF-RvIII expressed by their tumors. The demonstration of EGF-RvIII-specific immune responses in patients would suggest that immunization of patients with EGF-RvIII vaccines is feasible, because these vaccines may boost a pre-existing immune response. We have evaluated humoral and cellular immune responses to EGF-RvIII in 16 breast cancer patients and three healthy donors. Seven of 16 patients developed EGF-RvIII-specific Abs that bound to isolated EGF-RvIII protein or the protein expressed by EGF-RvIII-transfected mouse fibroblasts. The Abs that bound to EGF-RvIII did not bind to wild-type EGF-R, and anti-EGF-RvIII Abs were not found in the sera of healthy donors. Three patients had EGF-RvIII peptide-specific lymphoproliferative responses, and two of these patients also had humoral immune responses. Humoral and cellular immune responses correlated with EGF-RvIII expression by patients' tumors in most cases. These studies demonstrate that breast cancer patients specifically recognize EGF-RvIII with an overall immune response rate of 50%, suggesting that patients may benefit from vaccination against EGF-RvIII, boosting pre-existing immune responses.

Recombinant CD63/ME491/neuroglandular/NKI/C-3 antigen inhibits growth of established tumors in transgenic mice.

Attempts to vaccinate against tumors can be hindered by the induction of immunological tolerance to the target Ag as a result of Ag expression on normal tissues. In this study, we find that transgenic mice expressing the melanoma-associated Ag CD63/ME491/neuroglandular/NKI/C-3 on their normal tissues do, in fact, exhibit immunological tolerance to the Ag, recapitulating the conditions in cancer patients. In these mice, growth of murine melanoma cells expressing the Ag after gene transfer was inhibited by immunization with Ag-expressing recombinant vaccinia virus combined with IL-2, but not by immunization with the protein alone, anti-idiotypic Abs, or irradiated tumor cells. The effect of the recombinant virus was demonstrated both for nonestablished and established tumors. Infiltration with both CD4(+) and CD8(+) T lymphocytes was significantly more extensive in tumors from experimental mice than in tumors from control mice. MHC class I-positive, but not class I-negative, tumors were inhibited by the vaccine, suggesting that MHC class I-restricted T lymphocytes play a role in the antitumor effects. Abs did not appear to be involved in the vaccine effects. CD63 was immunogenic in 2 of 13 melanoma patients, pointing to the potential of this Ag, combined with IL-2, as a vaccine for melanoma patients.

Induction of cellular immunity by anti-idiotypic antibodies mimicking GD2 ganglioside.

Gangliosides are potentially useful targets for tumor destruction by antibodies. However, the role of gangliosides in T cell-mediated immunity to tumors is unclear. We produced three murine monoclonal anti-idiotypic antibodies (Ab2) against a monoclonal antibody (Ab1) that binds strongly to melanoma-associated GD2 ganglioside and weakly to GD3 ganglioside. All three Ab2 induced anti-anti-idiotypic antibodies (Ab3) with Ab1-like binding specificity to tumor cells and antigen in rabbits. The Ab3 specifically bound to GD2(+) tumor cells and isolated GD2, and shared idiotopes with the Ab1. Two of the three Ab2 induced GD2-specific delayed-type hypersensitivity responses in BALB/c and C57BL/6 mice, but not in C57BL/6/CD4(-/-) mice. Peripheral blood mononuclear cells (PBMC) from a melanoma patient proliferated specifically in response to in vitro stimulation with Ab2. Proliferation was accompanied by Th1-type cytokine production. Our studies demonstrate the induction of ganglioside-specific T cell-dependent immunity by Ab2 in mice. These T cells showed specific reactivity to ganglioside expressed by tumor cells.

A CD4+, HLA-DR7-restricted T-helper lymphocyte clone recognizes an antigen shared by human malignant melanoma and glioma.

CD4(+) Th cells that are restricted by MHC class II molecules play an important role in the induction of antitumor immune responses. We have established a stable CD4(+) Th cell clone (Th35-1A) from the PBMCs of a patient with primary cutaneous melanoma. The Th cell clone is noncytolytic and proliferates specifically in the presence of irradiated autologous melanoma cells or autologous EBV-transformed B cells pulsed with melanoma tumor cell lysates. Th35-1A produces IFN-gamma (a Th1-type cytokine) after autologous tumor cell stimulation, and its proliferative reactivity is HLA class II-restricted. Th cells showed helper activity for PWM responses of PBMCs. Using a panel of HLA class II-matched and unmatched EBV-B cells as APCs and allogeneic melanoma tumor cell lysate as stimulant, DR7 was delineated as the HLA class II restriction element used by the Th cell clone. In agreement with these results, transfection of an allogeneic melanoma cell line with HLA-DR7 isolated from autologous EBV-B cells rendered the cell line stimulatory for Th35-1A cells. Specificity studies using autologous EBV-B cells (EBV-B35) pulsed with a panel of allogeneic tumor cell lysates of various tissue origins indicated that the Th cell clone recognizes an antigen shared by melanoma and glioma cells. The availability of the Th cell clone may lead to the development of new therapies against melanoma, using adoptive Th cell transfer and/or active immunization with a shared Th cell antigen.

Inhibition of cytolytic T lymphocyte proliferation by autologous CD4+/CD25+ regulatory T cells in a colorectal carcinoma patient is mediated by transforming growth factor-beta.

Cancer patients often develop CTLs that lyse autologous tumor cells in culture. However, tumors can progress in vivo despite the presence of CTLs. Various mechanisms have been reported to down-modulate CTL functions. In this study, the role of CD4+/CD25+ regulatory T cells in CTL induction and proliferation of established CTLs was investigated in a patient with CRC. CD4+ cytotoxic and regulatory T-cell lines were derived from the peripheral blood mononuclear cells of the same patient in mixed-lymphocyte tumor culture. The cytotoxic T-cell line and a clonal derivative specifically lysed the autologous tumor cells but not the B lymphocytes. Only HLA-A1-matched allogeneic CRC cells were lysed by the CTL clone. The clone produced IFN-gamma and TNF-alpha. The regulatory CD4+/CD25+ T-cell line was tumor cell-dependent in its growth but did not lyse autologous tumor cells. This T-cell line suppressed pokeweed mitogen responses of allogeneic lymphocytes, proliferative activity of the established, autologous CTLs, and induction of CTLs in autologous, freshly isolated peripheral blood mononuclear cells. The immunosuppressive effect of the CD4+/CD25+ regulatory T cells was mediated by transforming growth factor-beta and did not require cell-to-cell contact. Thus, although CRC patients can develop specific CTLs against their tumors, the development of regulatory T cells may allow the escape of tumor cells from immune surveillance by the CTLs in vivo.

A comprehensive study of Candida-specific antibodies in the saliva of human immunodeficiency virus-positive individuals with oropharyngeal candidiasis.

Oropharyngeal candidiasis (OPC) is a common oral opportunistic infection among human immunodeficiency virus (HIV)-positive individuals. Although most cases of OPC correlate with reduced systemic levels of CD4(+) T cells, the role of humoral immunity in protection against mucosal candidiasis, including OPC, remains questionable. In the present study, a comprehensive analysis of saliva from 33 HIV-negative and 68 HIV-positive individuals, stratified by OPC status and peripheral CD4(+) cell count, was conducted to measure levels of total and Candida-specific immunoglobulin A(IgA) and IgG antibodies, including subclasses and secretory IgA. Despite changes in total immunoglobulin levels, when levels of Candida-specific antibodies were normalized to total protein or total immunoglobulin of the corresponding isotype, no distinct differences in IgG (including subclasses), IgA (including subclasses), or secretory IgA levels were seen, regardless of HIV status, OPC status, or CD4(+) cell count. These data suggest that when a complete repertoire of antibodies is evaluated, with appropriate normalization of data, there is no evidence of appreciable changes in levels of Candida-specific antibodies in saliva that would account for the prevalence of OPC among HIV-positive individuals.