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The prognosis for patients with glioblastoma is grim. Ex vivo expanded tumor-associated antigen (TAA)-reactive T-cells from patients with glioma may represent a viable source for anticancer-directed cellular therapies. Immunohistochemistry was used to test the survivin (n = 40 samples) and NY-ESO-1 (n = 38 samples) protein expression in tumor specimens. T-cells from peripheral blood were stimulated with TAAs (synthetic peptides) in IL-2 and IL-7, or using a combination of IL-2, IL-15 and IL-21. CD4 + and CD8 + T-cells were tested for antigen-specific proliferation by flow cytometry, and IFN-γ production was tested by ELISA. Twenty-eight out of 38 cancer specimens exhibited NY-ESO-1 protein expression, 2/38 showed a strong universal (4+) NY-ESO-1 staining, and 9/40 cancer lesions exhibited a strong (4+) staining for survivin. We could detect antigen-specific IFN-γ responses in 25% blood samples for NY-ESO-1 and 30% for survivin. NY-ESO-1-expanded T-cells recognized naturally processed and presented epitopes. NY-ESO-1 or survivin expression in glioma represents viable targets for anticancer-directed T-cells for the biological therapy of patients with glioma.

CD4(+) Treg cells expressing the transcription factor FOXP3 (forkhead box P3) are abundant in tumor tissues and appear to hinder the induction of effective antitumor immunity. A substantial number of T cells, including Treg cells, in tumor tissues and peripheral blood express C-C chemokine receptor 4 (CCR4). Here we show that CCR4 was specifically expressed by a subset of terminally differentiated and most suppressive CD45RA(-)FOXP3(hi)CD4(+) Treg cells [designated effector Treg (eTreg) cells], but not by CD45RA(+)FOXP3(lo)CD4(+) naive Treg cells, in peripheral blood of healthy individuals and cancer patients. In melanoma tissues, CCR4(+) eTreg cells were predominant among tumor-infiltrating FOXP3(+) T cells and much higher in frequency compared with those in peripheral blood. With peripheral blood lymphocytes from healthy individuals and melanoma patients, ex vivo depletion of CCR4(+) T cells and subsequent in vitro stimulation of the depleted cell population with the cancer/testis antigen NY-ESO-1 efficiently induced NY-ESO-1-specific CD4(+) T cells. Nondepletion failed in the induction. The magnitude of the responses was comparable with total removal of FOXP3(+) Treg cells by CD25(+) T-cell depletion. CCR4(+) T-cell depletion also augmented in vitro induction of NY-ESO-1-specific CD8(+) T cells in melanoma patients. Furthermore, in vivo administration of anti-CCR4 mAb markedly reduced the eTreg-cell fraction and augmented NY-ESO-1-specific CD8(+) T-cell responses in an adult T-cell leukemia-lymphoma patient whose leukemic cells expressed NY-ESO-1. Collectively, these findings indicate that anti-CCR4 mAb treatment is instrumental for evoking and augmenting antitumor immunity in cancer patients by selectively depleting eTreg cells.

During cancer progression, malignant cells may evade immunosurveillance. However, evidence for immunological escape in humans is scarce. We report here the clinical course of a melanoma patient whose initial tumor was positive for the antigens NY-ESO-1, MAGE-C1, and Melan-A. Upon immunization with a recombinant vaccinia/fowlpox NY-ESO-1 construct, the patient experienced a mixed clinical response and spreading of the NY-ESO-1 epitopes in the CD4+ T cell compartment. After NY-ESO-1 protein + CpG immunization, the patient's anti-NY-ESO-1 IgG response increased. Over the following years, progressing lesions were resected and found to be NY-ESO-1-negative while being positive for MAGE-C1, Melan-A, and MHC-I. The fatal, inoperable brain metastasis was analyzed after his death and also proved to be NY-ESO-1-negative, while being positive for MAGE-C1 and Melan-A, as well as MHC-I. We propose that cancer control and cancer escape in this patient were governed by NY-ESO-1-specific immunological pressure. Our findings provide evidence for the existence of immunoediting and immunoescape in this cancer patient.

Purpose: Mixed bacterial vaccine (MBV, Coley's toxins) is a historical, vaguely defined preparation of heat-inactivated Streptococcus pyogenes and Serratia marcescens used as nonspecific immunotherapy in the treatment of cancer. The mechanism of action is suspected to have an immunologic basis, yet it is poorly defined up to now. We developed a new, biochemically well defined and current good manufacturing practice-compliant MBV preparation, which has been investigated in patients with NY-ESO-1 expressing cancers., Experimental Design: Patients received MBV subcutaneously at a starting dose of 250 EU (endotoxin units) twice a week. The MBV dose was escalated in each patient until a body temperature of 38°C to 39.5°C was induced or up to the maximum dose of 547.000 EU. Changes in serum cytokine levels were determined and immune responses to NY-ESO-1 were evaluated. Tumor response was assessed according to RECIST., Results: Twelve patients were enrolled and 11 of them developed fever after the administration of MBV. Ten of 12 patients showed a consistent increase in serum IL-6 levels with the highest levels coinciding with the highest body temperature. A subgroup of patients showed increasing levels of TNF-α, IFN-γ, and IL1-β. A patient with metastatic bladder cancer showed a partial tumor response strongly correlated with MBV-induced fever and highly elevated levels of several cytokines., Conclusions: MBV at fever-inducing dose levels can lead to a massive induction of immunoregulatory cytokines that may be involved in inducing tumor regressions. We propose to further explore the role of MBV as a potent immune modulator at higher dose levels and in conjunction with antigen-specific cancer vaccines.

Recombinant poxviruses (vaccinia and fowlpox) expressing tumor-associated antigens are currently being evaluated in clinical trials as cancer vaccines to induce tumor-specific immune responses that will improve clinical outcome. To test whether a diversified prime and boost regimen targeting NY-ESO-1 will result in clinical benefit, we conducted two parallel phase II clinical trials of recombinant vaccinia-NY-ESO-1 (rV-NY-ESO-1), followed by booster vaccinations with recombinant fowlpox-NY-ESO-1 (rF-NY-ESO-1) in 25 melanoma and 22 epithelial ovarian cancer (EOC) patients with advanced disease who were at high risk for recurrence/progression. Integrated NY-ESO-1-specific antibody and CD4(+) and CD8(+) T cells were induced in a high proportion of melanoma and EOC patients. In melanoma patients, objective response rate [complete and partial response (CR+PR)] was 14%, mixed response was 5%, and disease stabilization was 52%, amounting to a clinical benefit rate (CBR) of 72% in melanoma patients. The median PFS in the melanoma patients was 9 mo (range, 0-84 mo) and the median OS was 48 mo (range, 3-106 mo). In EOC patients, the median PFS was 21 mo (95% CI, 16-29 mo), and median OS was 48 mo (CI, not estimable). CD8(+) T cells derived from vaccinated patients were shown to lyse NY-ESO-1-expressing tumor targets. These data provide preliminary evidence of clinically meaningful benefit for diversified prime and boost recombinant pox-viral-based vaccines in melanoma and ovarian cancer and support further evaluation of this approach in these patient populations.

Purpose: NY-ESO-1, one of the most immunogenic tumor antigens, is expressed in 15% to 25% of metastatic prostate cancers. The immunological and clinical effects of vaccination with recombinant NY-ESO-1 protein combined with CpG as adjuvant were evaluated., Experimental Design: In a phase I clinical study, patients with advanced prostate cancer were vaccinated with recombinant NY-ESO-1 protein (100 μg) mixed with CpG 7909 (2.5 mg) every 3 weeks intradermally for 4 doses. Objectives of the study were the safety of the vaccine and changes of specific humoral and cellular immunological responses to NY-ESO-1 in relation to detectable NY-ESO-1 expression in the individual tumor., Results: All 12 baseline sero-negative patients developed high-titer NY-ESO-1 antibody responses. B-cell epitope mapping identified NY-ESO-1 p91-110 to be recognized most frequently by vaccine-induced antibodies. Two patients developed significant antibody titers against the adjuvant CpG. NY-ESO-1-specific CD4+ and/or CD8+ T-cell responses were induced in 9 patients (69%). Five of these 9 patients did not express NY-ESO-1 in the autologous tumor. Postvaccine CD8+ T-cell clones recognized and lyzed HLA-matched tumor cell lines in an antigen-specific manner., Conclusion: Our data provide clear evidence for the capacity of NY-ESO-1 protein/CpG vaccine to induce integrated antigen-specific immune responses in vivo and to efficiently prime CD8+ T-cell responses in NY-ESO-1 antigen-negative patients. Our results may also support further clinical vaccination protocols with NY-ESO-1 protein not only focused on the treatment of existing cancer, but also to prevent further development of NY-ESO-1 positive cancers in vivo., (©2010 AACR.)

Azanucleoside DNA-hypomethylating agents have remarkable clinical activity in myelodysplastic syndromes and acute myeloid leukemia (AML), particularly at low, non-cytotoxic doses favoring hypomethylation over cytotoxicity. Cancer/testis antigens (CTAs) encoding immunogenic proteins are not expressed in almost all normal tissues and many tumor types, but are consistently derepressed by epigenetically active agents in various cancer cell lines. Since the expression of CTA genes is usually very low or absent in myeloid leukemias, we treated various AML cell lines with 5-aza-2'-deoxycytidine (DAC) and quantified mRNA expression of the CTAs NY-ESO-1, MAGEA1, MAGEA3 and MAGEB2. Consistent time- and dose-dependent reactivation of all 4 CTA genes was observed, with maximum mRNA levels 72-144h after treatment start. As determined by RNA microarray analyses, numerous other CTA genes (all located on the X-chromosome) were also derepressed in a time-dependent fashion by DAC. NY-ESO-1 derepression was confirmed at the protein level. By Elispot and chromium release assays we showed that the de novo expressed NY-ESO-1 protein was naturally processed and presented in a time- and dose-dependent fashion up to 8 days after the start of DAC treatment, and converted the cell lines susceptible to antigen-specific recognition by CD8+ T-cell clones. In conclusion, NY-ESO-1 and numerous other CTAs localized on the X-chromosome are readily and transiently derepressed in AML cell lines treated with DAC. The susceptibility of DAC-treated AML cell lines to antigen-specific T-cell recognition has clear implications for future clinical trials combining DAC and specific immunotherapy in AML., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Peptide-based vaccines have led to the induction of antigen-specific CD8(+) T-cell responses in patients with NY-ESO-1 positive cancers. However, vaccine-induced T-cell responses did not generally correlate with improved survival. Therefore, we tested whether a synthetic CpG 7909 ODN (deoxycytidyl-deoxyguanosin oligodeoxy-nucleotides) mixed with NY-ESO-1 peptide p157-165 and incomplete Freund's adjuvants (Montanide(R) ISA-51) led to enhanced NY-ESO-1 antigen-specific CD8(+) immune responses in patients with NY-ESO-1 or LAGE-1 expressing tumors. Of 14 HLA-A2+ patients enrolled in the study, 5 patients withdrew prematurely because of progressive disease and 9 patients completed 1 cycle of immunization. Nine of 14 patients developed measurable and sustained antigen-specific CD8(+) T-cell responses: Four had detectable CD8+ T-cells against NY-ESO-1 after only 2 vaccinations, whereas 5 patients showed a late-onset but durable induction of NY-ESO-1 p157-165 specific T-cell response during continued vaccination after 4 months. In 6 patients, vaccine-induced antigen-specific T-cells became detectable ex vivo and reached frequencies of up to 0.16 % of all circulating CD8(+) T-cells. Postvaccine T-cell clones were shown to recognize and lyse NY-ESO-1 expressing tumor cell lines in vitro. In 6 of 9 patients developing NY-ESO-1-specific immune responses, a favorable clinical outcome with overall survival times of 43+, 42+, 42+, 39+, 36+ and 27+ months, respectively, was observed.

A major objective of peptide vaccination is the induction of tumor-reactive CD8+ T-cells. We have shown that HLA-A2 positive cancer patients frequently develop an antigen-specific CD8+ T-cell response after vaccination with NY-ESO-1 peptides p157-165/p157-167. These T-cells are highly reactive with the peptides used for vaccination, but only rarely recognize HLA-matched, NY-ESO-1 expressing tumor cell lines. To address the apparent lack of tumor recognition of vaccine-induced CD8+ T-cell responses, we used autologous tumor cells for in vitro stimulation and expansion of pre- and postvaccine CD8+ T-cells. In contrast to standard presensitization methods with peptide-pulsed antigen-presenting cells, mixed lymphocyte tumor culture favored the selective expansion of low-frequency tumor-reactive T-cells. In four patients, we were able to demonstrate that antigen-specific and tumor-reactive T-cells are detectable and are indeed elicited as a result of NY-ESO-1 peptide vaccination. Further analyses of postvaccine antigen-specific T-cells at a clonal level show that vaccine-induced antigen-specific T-cells are heterogeneous in functional activity. These results suggest that the methods of immunomonitoring are critical to identify the proportion of tumor-reactive T-cells within the population of vaccine-induced antigen-specific effector cells. Our results show that immunization with NY-ESO-1 peptides leads to strong tumor-reactive CD8+ T-cell responses. Our findings suggest that approaches to peptide vaccination may be improved to induce higher numbers of antigen-specific T-cells and to selectively increase the proportion of CD8+ T-cells that have the capacity to recognize and eliminate tumor cells., (Copyright (c) 2007 Wiley-Liss, Inc.)

NY-ESO-1 is a cancer-testis antigen and an attractive target for immunotherapy in patients with different malignancies. Here we report the results of a phase I clinical study of intensive course NY-ESO-1 peptide vaccination, evaluating the safety, immunogenicity and clinical response in HLA-A2 positive patients with NY-ESO-1 expressing cancers. Of 20 patients enrolled in the trial, 14 completed at least 2 cycles of immunization and were evaluable for clinical and immunological response. Five of these evaluable patients were treated in cohort 1 (baseline seropositive) and 9 patients were treated in cohort 2 (baseline seronegative). During vaccination, NY-ESO-1-specific CD8+ T-cells were induced in 3 of 9 baseline seronegative patients. In patients with pre-existing antigen-specific CD8+ T-cells, their number increased or remained stable. In contrast to previous immunization protocols with less intensive immunization schedules, we observed a rapid induction of high magnitude NY-ESO-1 peptide-specific T-cell responses detectable already on day 15-22 of immunization. A specific immune response of high magnitude and early onset may be more effective in eliminating minimal residual disease in adjuvant treatment situations and in preventing tumor progression due to immune escape mechanisms.

Bacterial vectors may offer many advantages over other antigen delivery systems for cancer vaccines. We engineered a Salmonella typhimurium vaccine strain to deliver the NY-ESO-1 tumor antigen (S. typhimurium-NY-ESO-1) through a type III protein secretion system. The S. typhimurium-NY-ESO-1 construct elicited NY-ESO-1-specific CD8+ and CD4+ T cells from peripheral blood lymphocytes of cancer patients in vitro. Oral administration of S. typhimurium-NY-ESO-1 to mice resulted in the regression of established NY-ESO-1-expressing tumors. Intratumoral inoculation of S. typhimurium-NY-ESO-1 to NY-ESO-1-negative tumors resulted in delivery of antigen in vivo and led to tumor regression in the presence of preexisting NY-ESO-1-specific CD8+ T cells. Specific T cell responses against at least 2 unrelated tumor antigens not contained in the vaccine were observed, demonstrating epitope spreading. We propose that antigen delivery through the S. typhimurium type III secretion system is a promising novel strategy for cancer vaccine development.

Presentation of intracellular tumor-associated Ags (TAAs) in the context of HLA class I molecules offers unique cancer-specific cell surface markers for the identification and targeting of tumor cells. For most peptide Ags, the levels of and variations in cell surface presentation remain unknown, yet these parameters are of crucial importance when considering specific TAAs as targets for anticancer therapy. Here we use a soluble TCR with picomolar affinity for the HLA-A2-restricted 157-165 epitope of the NY-ESO-1 and LAGE-1 TAAs to investigate presentation of this immunodominant epitope on the surface of a variety of cancer cells. By single molecule fluorescence microscopy, we directly visualize HLA-peptide presentation for the first time, demonstrating that NY-ESO-1/LAGE-1-positive tumor cells present 10-50 NY-ESO-1/LAGE-1(157-165) epitopes per cell.

NY-ESO-1 is one of the most immunogenic cancer antigens eliciting strong humoral and cellular immune responses in patients with NY-ESO-1-expressing malignancies. Since CD4+ T cells play a critical role in generating and maintaining antigen-specific cellular and humoral immune responses, we searched for new NY-ESO-1 epitopes presented by MHC class II molecules. CD4+ T cells of patients with NY-ESO-1-expressing cancer were presensitized with 18-mer overlapping synthetic peptides spanning the entire sequence of NY-ESO-1. Two partly overlapping NY-ESO-1 epitopes p49-66 and p55-72 were identified as targets for NY-ESO-1-specific CD4+ T cells. Peptide-specific CD4+ T-cell clones were generated by repetitive stimulation with NY-ESO-1 p49-66 and p55-72. Further experiments confirmed distinct specificities for the CD4+ T-cell clones indicating that at least 2 different CD4+ T-cell epitopes are located in the region p49-72 of the NY-ESO-1 sequence. Using a set of partially histocompatible EBV-B cell lines and MHC class II-specific antibodies, we found that both CD4+ T-cell epitopes were presented in the context of HLA-DQ B1 03011(DQ7). Natural processing and presentation of these epitopes was demonstrated by recognition of an HLA-DQ B1 03011- and NY-ESO-1-expressing lymphoma cell line and by recognition of dendritic cells (DC) exogenously loaded with NY-ESO-1 protein or infected with recombinant NY-ESO-1 adenoviral constructs. The specific production of IFN-gamma and TNF-alpha suggests that the NY-ESO-1-specific CD4+ T-cell clones belong to the Th1 subtype. The characterization of the new HLA-DQ B1 03011-restricted NY-ESO-1 peptides broadens the repertoire of epitopes that can be used to monitor NY-ESO-1-specific spontaneous and vaccine-induced T-cell responses in cancer patients., (Copyright 2005 Wiley-Liss, Inc.)

In the 9 years since its discovery, cancer-testis antigen NY-ESO-1 has made one of the fastest transitions from molecular, cellular, and immunological description to vaccine and immunotherapy candidate, already tested in various formulations in more than 30 clinical trials worldwide. Its main characteristic resides in its capacity to elicit spontaneous antibody and T-cell responses in a proportion of cancer patients. An overview of immunological findings and immunotherapeutic approaches with NY-ESO-1, as well the role of regulation in NY-ESO-1 immunogenicity, is presented here.

NY-ESO-1 is one of the most immunogenic cancer antigens known to date, eliciting spontaneous immune responses in approximately 50% of patients with NY-ESO-1+ cancers. Spontaneous CD4+ and CD8+ T cell responses were found in patients with detectable NY-ESO-1 serum antibody, indicating an integrated type of immune response induced by NY-ESO-1+ malignancies. A close association between spontaneous NY-ESO-1 immunity and the HLA-DP4 allele was suggested in a recent study. To address these results, we assessed the NY-ESO-1 antibody and HLA-DP4 status of 102 patients with NY-ESO-1+ malignancies. However, no correlation between HLA-DP4 and NY-ESO-1 immunity was found. To explore the role of HLA-DP4-restricted CD4+ T cells in cancer immunity, we established HLA-DP4- restricted NY-ESO-1-specific CD4+ T cell clones by limiting dilution and repeated stimulation with NY-ESO-1 peptide p157-170 from NY-ESO-1 seropositive patients. A subset of CD4+ T cell clones was reactive with naturally processed NY-ESO-1 presented by autologous DCs that were pulsed with recombinant NY-ESO-1 protein, lysates of NY-ESO-1-expressing tumor cell lines, or transduced with recombinant NY-ESO-1 viral constructs in ELISPOT assays. Three different CD4+ T cell clones were used to mediate the specific lysis of allogeneic HLA-DP4+ Epstein-Barr virus-transformed B cells (EBV-B) pulsed with NY-ESO-1 p157-170. The Th1 phenotype and effector functions of the CD4+ T cell clones described here provide an important rationale for the activation of antigen-specific CD4+ T cells along with CD8+ T cells in cancer vaccination strategies.

The NY-ESO-1 antigen is expressed in a significant proportion of patients with epithelial ovarian cancer (EOC) and appears to be an ideal target for immunotherapy. In order to elucidate the nature of the HLA-DPB1*0401/0402 (DP4+)-restricted CD4+ immune response in patients with NY-ESO-1-expressing EOC, peripheral blood CD4+ T cells from HLA-DP4+ patients were stimulated with the NY-ESO-1 epitope 157-170 and tested for the release of type 1 (IFN-gamma) and type 2 (IL-5) cytokines in enzyme-linked immunospot assays. Of 14 DP4+ EOC patients who tested seronegative for NY-ESO-1, 3 patients had a detectable CD4+ T cell response to NY-ESO-1 epitope 157-170 by IFN-gamma ELISPOT assay. Six of 10 DP4+ EOC patients with serum antibodies to NY-ESO-1 had CD4+ T cell responses to NY-ESO-1 epitope 157-170 by IFN-gamma assay. Six patients had mixed Th1/Th2 CD4+ T cell responses to NY-ESO-1 epitope 157-170 regardless of their antibody response to NY-ESO-1. Four EOC patients had Th1 cells expressing IFN-gamma, but not IL-5. This suggests that the NY-ESO-1 epitope 157-170 stimulates both Th1 and Th2 type CD4+ T cell responses in EOC patients. These data suggest the NY-ESO-1 epitope 157-170 has a key role in the induction of cellular and humoral immune responses against NY-ESO-1-expressing EOC tumors. Our study supports the relevance of cancer vaccine trials with the NY-ESO-1 epitope 157-170 in HLA-DP4+ EOC patients with NY-ESO-1-expressing tumors and strategies to improve Th1-dominated tumor-reactive CD4+ T cell bias.

NY-ESO-1 is one of the most immunogenic proteins described in human cancers, based on its capacity to elicit simultaneous antibody and CD8+ T cell responses in vivo. Although HLA class II restricted epitopes from NY-ESO-1 have been identified, no broad survey has yet established the status of natural CD4+ T cell responses in cancer patients in relation to CD8+ and antibody responses. We used a recently developed general strategy for monitoring CD4+ responses that overcomes the need for prior knowledge of epitope or HLA restriction to analyze a series of 31 cancer patients and healthy donors for the presence of CD4+ T cells to NY-ESO-1, and related this response to NY-ESO-1 expression in tumor cells and serum antibodies to NY-ESO-1. None of the 18 patients that tested seronegative for NY-ESO-1 had detectable CD4+ T cell responses. On the contrary, 11 of 13 cancer patients with serum antibodies to NY-ESO-1 had polyclonal CD4+ T cell responses directed against various known and previously undescribed NY-ESO-1 epitopes. NY-ESO-1 peptide 80-109 was the most immunogenic, with 10 of 11 patients responding to this peptide. We show here that 12-mer determinants from NY-ESO-1 eliciting a CD4+ T cell response were peptide 87-98 with promiscuous HLA class II presentation, peptide 108-119 restricted by HLA-DP4, and peptides 121-132 and 145-156, both shorter epitopes from previously described HLA-DR4 peptides, also presented by HLA-DR7. This study represents the next step in compiling a comprehensive picture of the adaptive immune response to NY-ESO-1, and provides a general strategy for analyzing the CD4+ T cell response to other tumor antigens eliciting a humoral immune response.

The serological analysis of antigens by recombinant expression cloning (SEREX) has identified a multitude of new tumor antigens in many different tumor entities. These antigens can be grouped into different classes according to their specificities, with cancer/testis antigens appearing to be the most attractive candidates for vaccine development. The observation that CD8 and CD4 T-cell responses against cancer/testis antigens such as NY-ESO-1 correlate with the presence of specific antibodies demonstrates the importance of serological monitoring patients participating in vaccine trials. However, all serological assays available (Western blot, phage display and ELISA) are hampered by the fact that the protein cannot be analyzed in its natural conformation. We have thus developed a yeast display system where the antigen is expressed on the yeast surface (RAYS), allowing for a more natural folding of the protein. To validate this approach we displayed the A33 colorectal cancer antigen on the yeast cell surface and demonstrated specific binding by an A33 monoclonal antibody recognizing a conformation-dependent epitope on the A33 antigen. We then compared RAYS with the more commonly used ELISA and Western blot serological monitoring methods by analyzing 50 sera from cancer patients with known NY-ESO-1 antibody status and 10 sera from patients with unknown SSX2 antibody status in a blind fashion. RAYS appears at least equivalent to both ELISA and Western blotting for the monitoring of antibodies against NY-ESO-1 as regards specificity and sensitivity, while antibodies against SSX2 were detected more frequently by RAYS than by ELISA or phage display.

NY-ESO-1, a germ cell Ag often detected in tumor tissues, frequently elicits Ab and CD8(+) T cell responses in cancer patients. Overlapping long peptides spanning the NY-ESO-1 sequence have been used to map HLA class I-restricted epitopes recognized by NY-ESO-1-specific CD8(+) T lymphocytes. To address the antigenicity of long peptides, we analyzed two synthetic 30-mer peptides from NY-ESO-1, polypeptides 80-109 and 145-174, for their capacity to be processed by APCs and to stimulate CD8(+) T cells. By incubating APCs with polypeptides at different temperatures or in the presence of protease inhibitors, we found that NY-ESO-1 polypeptides were rapidly internalized by B cells, T2 cells, or PBLs and submitted to cellular proteolytic action to yield nonamer epitopes presented by HLA class I. Polypeptides were also immunogenic in vitro and stimulated the expansion of CD8(+) T cells against naturally processed NY-ESO-1 epitopes in the context of three different HLA class I alleles. Polypeptides can thus serve as exogenous Ags that are cross-presented on HLA class I without requiring the action of professional APCs. These findings support innovative vaccination strategies using NY-ESO-1 polypeptides that would circumvent current limitations of HLA class I peptide vaccination, i.e., HLA eligibility criteria and knowledge of epitope, while allowing for facilitated immunogenicity in the presence of helper epitopes.

NY-ESO-1 is one of the most immunogenic cancer antigens known to date, inducing humoral and cellular immune responses in a high proportion of patients with advanced NY-ESO-1-expressing cancers. The assessment of spontaneous and vaccine-induced CD8+ T cell responses has been limited to a small number of known NY-ESO-1 epitopes presented by MHC class I alleles. Recently, a new method to monitor NY-ESO-1-specific CD8+ T cell responses was introduced that does not depend on the individual MHC class I status and on predefined peptide epitopes. Antigen-presenting cells transduced with recombinant adenoviral vectors encoding NY-ESO-1 were used to stimulate CD8+ selected NY-ESO-1-specific T cells. Effector cells were tested for recognition of autologous B cell targets transfected with NY-ESO-1 using a recombinant vaccinia virus construct. Using a modified approach we identified the NY-ESO-1 p94-102 peptide as being recognized by CD8+ T cells in the context of HLA- B51. NY-ESO-1 p94-102 specific CD8+ T cells recognized naturally processed NY-ESO-1 presented by HLA-B51+ monocyte-derived dendritic and tumor cells. Transfection of target cells with NY-ESO-1 combined with different HLA class I alleles confirmed that the NY-ESO-1 peptide was naturally processed and recognized by HLA-B51-restricted CD8+ T cell lines and clones. Therefore, NY-ESO-1 p94-102 is a new candidate peptide antigen for cancer immunotherapy and for the monitoring of spontaneous and vaccine-induced NY-ESO-1-specific T cell responses in HLA- B51+ patients with NY-ESO-1 expressing malignancies.

NY-ESO-1 is a germ cell antigen aberrantly expressed in different tumor types that elicits strong humoral and cellular immune responses in cancer patients. Monitoring spontaneous CD8(+) T cell responses against NY-ESO-1 peptides 157-165 (S9C) and 157-167 (S11L) in a series of HLA-A2(+) cancer patients showed that these two peptides had overlapping antigenic profiles and were equally immunogenic. However, discrepancies between S9C and S11L reactivities were observed upon vaccination with both peptides to generate or boost T cell responses to NY-ESO-1 in cancer patients. We here analyze the fine specificity of these responses and describe an HLA-A2-restricted epitope, NY-ESO-1 peptide 159-167 (L9L), which is strongly recognized by CD8(+) T cells as a result of peptide vaccination of cancer patients. Responses to L9L were stimulated by S11L and appeared early in the course of vaccination, independently of S9C responses. However, L9L-specific CD8(+) T cells failed to recognize tumor cells naturally expressing NY-ESO-1 or B lymphoblastoid cells transduced with NY-ESO-1. Processing of L9L could be rescued after IFN-gamma treatment of tumor cells or by dendritic cells pulsed with NY-ESO-1 protein/antibody immune complexes. The present results demonstrate a dual specificity within peptide S11L, with S9C as the natural antigenic tumor epitope, and L9L as a cryptic epitope with dominant immunogenicity upon vaccination that diverts the immune response from tumor recognition. These unanticipated findings raise questions about the use of S11L in the clinic and emphasize the importance of analyzing the fine specificity of vaccine-induced T cell responses in patients as a basis for constructing effective cancer vaccines.

NY-ESO-1 is one of the most immunogenic tumor antigens known to date. Spontaneous humoral and cellular immune responses against NY-ESO-1 are detected in a substantial proportion of patients with NY-ESO-1 positive cancers. NY-ESO-1 serum antibody is dependent on the presence of NY-ESO-1+ cancer cells, and antibody titers correlate with the clinical development of the disease. NY-ESO-1 serum antibody is associated with detectable NY-ESO-1-specific CD8+ T cell reactivity. High titers of NY-ESO-1 serum antibodies are found in patients with advanced NY-ESO-1+ malignancies. Urine samples of seropositive patients with normal kidney function were tested for NY-ESO-1 antibody by Western blotting and enzyme-linked immunosorbent assay (ELISA). Antibodies to NY-ESO-1 were found in the urine of patients whose NY-ESO-1 serum antibody titers were 1:10,000 or higher by Western blotting. In patients with weak (positive at 1:250, negative at 1:1,000) or no reactivity, urine antibody was not detectable. No urine NY-ESO-1 antibody was found in patients without detectable NY-ESO-1 serum antibody. Our results show that urine analysis for NY-ESO-1 antibody identifies patients with strong NY-ESO-1 immunity. Urine antibody detection may also be of value in the monitoring of spontaneous and vaccine-induced immunity against other defined tumor antigens.

Tumour associated antigens recognised by cellular or humoral effectors of the immune system are potential targets for antigen specific cancer immunotherapy. Different categories of cancer antigens have been identified that induce cytotoxic T lymphocyte (CTL) responses in vitro and in vivo, namely: (1) "cancer testis" (CT) antigens, expressed in different tumours and normal testis, (2) melanocyte differentiation antigens, (3) point mutations of normal genes, (4) self antigens that are overexpressed in malignant tissues, and (5) viral antigens. Clinical studies with peptides and proteins derived from these antigens have been initiated to study the efficacy of inducing specific CTL responses in vivo. Immunological and clinical parameters for the assessment of antigen specific immune responses have been defined-delayed type hypersensitivity (DTH), CTL, autoimmmune, and tumour regression responses. Specific DTH and CTL responses and tumour regression have been observed after the intradermal administration of tumour associated peptides alone. Peptide specific immune reactions were enhanced after using granulocyte macrophage stimulating factor (GM-CSF) as a systemic adjuvant by increasing the frequency of dermal antigen presenting Langerhans cells. Complete tumour regression has been observed in the context of measurable peptide specific CTL. However, in single cases with disease progression after an initial tumour response, either a loss of single antigens targeted by CTL or of the presenting major histocompatibility complex (MHC) class I allele was detected, pointing towards immunisation induced immune escape. Cytokines to modulate antigen and MHC class I expression in vivo are being evaluated to prevent immunoselection. Recently, a new CT antigen, NY-ESO-1, has been identified on the basis of spontaneous antibody responses to tumour associated antigens. NY-ESO-1 appears to be one of the most immunogenic antigens known to date, with spontaneous immune responses observed in 50% of patients with NY-ESO-1 expressing cancers. Clinical studies have been initiated to evaluate the immunogenicity of different NY-ESO-1 constructs to induce both humoral and cellular immune responses in vivo.

The identification of tumor-associated antigens recognized by cellular or humoral effectors of the immune system has opened new perspectives for cancer immunotherapy. Different categories of cancer-associated antigens have been described as targets for CD8+ T cells in vitro and in vivo: (1) 'cancer-testis' (CT) antigens expressed in different tumors and normal testis; (2) melanocyte differentiation antigens; (3) point mutations of normal genes; (4) antigens that are overexpressed in malignant tissues, and (5) viral antigens. Clinical trials with antigenic peptides have been initiated to induce specific immunological responses in vivo. Immunological and clinical parameters for the assessment of peptide-specific reactions have been defined: DTH, CD8+ T cell, autoimmune and tumor regression responses. Preliminary results show that tumor-associated peptides alone elicit specific DTH and CD8+ T cell responses associated with tumor regression after intradermal vaccination. Granulocyte macrophage colony-stimulating factor has been shown to enhance peptide-specific immune reactions by amplification of dermal antigen-presenting dendritic cells. Complete tumor regressions have been observed after the induction of CD8+ T cell responses by peptide immunization. Based on these results, active immunotherapy with tumor-associated antigens may be a promising approach for patients in adjuvant treatment situations, who are at high risk for tumor recurrence. Recently, a strategy utilizing spontaneous antibody responses to tumor-associated antigens (SEREX) has led to the identification of a new CT antigen, NY-ESO-1. NY-ESO-1-specific spontaneous humoral and cellular immune responses were found in approximately 50% of patients with NY-ESO-1-positive tumors. Clinical studies have been initiated to evaluate the immunological effects of immunization with NY-ESO-1 peptides in cancer patients with detectable or absent immunity against NY-ESO-1.

Cancer-testis antigen NY-ESO-1 is one of the most immunogenic tumor antigens defined to date. Spontaneous humoral and CD8+ T-cell responses to NY-ESO-1 are detected in 40-50% of patients with advanced NY-ESO-1-expressing tumors. A clinical trial was initiated to study the immunological effects of intradermal vaccination with 3 HLA-A2-binding NY-ESO-1 peptides in 12 patients with metastatic NY-ESO-1-expressing cancers. Seven patients were NY-ESO-1 serum antibody negative, and five patients were NY-ESO-1 serum antibody positive at the outset of the study. Primary peptide-specific CD8+ T-cell reactions and delayed-type hypersensitivity responses were generated in four of seven NY-ESO-1 antibody-negative patients. Induction of a specific CD8+ T-cell response to NY-ESO-1 in immunized antibody-negative patients was associated with disease stabilization and objective regression of single metastases. NY-ESO-1 antibody-positive patients did not develop significant changes in baseline NY-ESO-1-specific T-cell reactivity. However, stabilization of disease and regression of individual metastases were observed in three of five immunized patients. These results demonstrate that primary NY-ESO-1-specific CD8+ T-cell responses can be induced by intradermal immunization with NY-ESO-1 peptides, and that immunization with NY-ESO-1 may have the potential to alter the natural course of NY-ESO-1-expressing tumors.

Expression of NY-ESO-1 in a high proportion of different human tumors makes this protein a very attractive vaccine target. NY-ESO-1 peptides, recognized by HLA-A2-restricted CTL, have recently been described. However, it remains unclear how efficiently tumors generate these epitopes, and whether peptide analogues can be used for optimal expansion and activation of NY-ESO-1-specific HLA-A2-restricted CTL. By generating unique CTL clones, we demonstrate that NY-ESO-1-positive tumor cells are efficiently killed by HLA-A2-restricted CTL specific for the peptide epitope NY-ESO-1 157-165. Presentation of this epitope is not affected by the presence or absence of the proteasome subunits low molecular proteins 2 and 7 and is not blocked by proteasome inhibitors, while it is impaired in the TAP-deficient cell line LBL 721.174. NY-ESO-1 157-165 peptide analogues were compared for their antigenicity and immunogenicity using PBL from melanoma patients. Three peptides, containing the carboxyl-terminal cysteine substituted for either valine, isoleucine, or leucine, were recognized at least 100 times more efficiently than the wild-type peptide by specific CTL. Peptide analogues were capable of stimulating the expansion of NY-ESO-1-specific CTL from PBL of melanoma patients much more efficiently than wild-type peptide. These findings define the processing requirements for the generation of the NY-ESO-1 157-165 epitope. Identification of highly antigenic NY-ESO-1 peptide analogues may be important for the development of vaccines capable of expanding NY-ESO-1-specific CTL in cancer patients.

NY-ESO-1, a member of the cancer-testis family of antigens, is expressed in a subset of a broad range of different human tumor types. Patients with advanced NY-ESO-1-expressing tumors frequently develop humoral immunity to NY-ESO-1, and three HLA A2-restricted peptides were defined previously as targets for cytotoxic CD8(+) T cells in a melanoma patient with NY-ESO-1 antibody. The objectives of the present study were (i) to develop enzyme-linked immunospot (ELISPOT) and tetramer assays to measure CD8(+) T cell responses to NY-ESO-1, (ii) to determine the frequency of CD8(+) T cell responses to NY-ESO-1 in a series of HLA-A2 patients with NY-ESO-1 expressing tumors, (iii) to determine the relation between CD8(+) T cell and humoral immune responses to NY-ESO-1, and (iv) to compare results of NY-ESO-1 ELISPOT assays performed independently in two laboratories with T cells from the same patients. NY-ESO-1 ELISPOT and tetramer assays with excellent sensitivity, specificity, and reproducibility have been developed and found to correlate with cytotoxicity assays. CD8(+) T cell responses to HLA-A2-restricted NY-ESO-1 peptides were detected in 10 of 11 patients with NY-ESO-1 antibody, but not in patients lacking antibody or in patients with NY-ESO-1-negative tumors. The results of ELISPOT assays were concordant in the two laboratories, providing the basis for standardized monitoring of T cell responses in patients receiving NY-ESO-1 vaccines.

NY-ESO-1 is a member of the cancer-testis family of tumor antigens that elicits strong humoral and cellular immune responses in patients with NY-ESO-1-expressing cancers. Since CD4(+) T lymphocytes play a critical role in generating antigen-specific cytotoxic T lymphocyte and antibody responses, we searched for NY-ESO-1 epitopes presented by histocompatibility leukocyte antigen (HLA) class II molecules. Autologous monocyte-derived dendritic cells of cancer patients were incubated with recombinant NY-ESO-1 protein and used in enzyme-linked immunospot (ELISPOT) assays to detect NY-ESO-1-specific CD4(+) T lymphocyte responses. To identify possible epitopes presented by distinct HLA class II alleles, overlapping 18-mer peptides derived from NY-ESO-1 were synthetized and tested for recognition by CD4(+) T lymphocytes in autologous settings. We identified three NY-ESO-1-derived peptides presented by DRB4*0101-0103 and recognized by CD4(+) T lymphocytes of two melanoma patients sharing these HLA class II alleles. Specificity of recognition was confirmed by proliferation assays. The characterization of HLA class II-restricted epitopes will be useful for the assessment of spontaneous and vaccine-induced immune responses of cancer patients against defined tumor antigens. Further, the therapeutic efficacy of active immunization using antigenic HLA class I-restricted peptides may be improved by adding HLA class II-presented epitopes.

SEREX (serological analysis of recombinant tumor cDNA expression libraries) has been applied to several different tumor types and has led to the identification of a wide range of tumor antigens. In this study, a breast cancer library and a normal testicular library were analyzed using autologous and allogeneic breast cancer sera. Thirty genes were isolated, including 27 known genes and 3 previously unknown genes. Among the known genes, two cancer-testis (CT) antigens, NY-ESO-1 and SSX2, previously defined by SEREX analysis, were found. In addition, ING1, a candidate breast cancer suppressor gene, was isolated. This ING1 gene product was also recognized by 2 of 14 allogeneic sera from breast cancer patients but not 12 normal adult sera. Comparison of ING1 cDNA from normal and tumor tissues showed no mutation in the index breast cancer case and revealed the presence of at least three different mRNA transcripts with variable transcription initiation sites and exon usage. Tissue-specific expression of these transcripts was found in normal tissues and tumor cell line mRNAs. Furthermore, a novel gene, designated as ING2, sharing 76% nucleotide homology with ING1 was identified in the breast cancer cDNA library. The basis of the immunogenicity of ING1 and the biological role of ING1 and ING2 need further exploration.

Humoral immune responses against the "Cancer-Testis" (CT) antigen NY-ESO-1 are frequently observed in patients with NY-ESO-1 expressing tumors. This is in contrast to other known tumor antigens (TA) defined by antibody or cytotoxic T cell (CTL) reactivity, i.e., MAGE-1, MAGE-3, SSX2, Melan A, and tyrosinase. No NY-ESO-1 antibody has been detected in healthy controls and patients with NY-ESO-1 negative tumors. In this study, we have assessed the NY-ESO-1 serum antibody response in patients with NY-ESO-1 positive tumors of different histological types and stages using Western blotting and an ELISA. Of the 12 patients analyzed, 10 had demonstrable NY-ESO-1 antibodies at the start of the study. All patients were followed for changes in NY-ESO-1 antibody titers during the course of tumor treatment and clinical evolution. In 4 patients, an increase of NY-ESO-1 antibody titer was observed with progression of disease or extensive tumor necrosis under treatment. One patient showed a stable NY-ESO-1 antibody titer over 3 years along with gradual regression of a large tumor mass. In 5 patients, a decrease of NY-ESO-1 antibody was detected: in 1 patient after curative tumor resection, in 3 patients with partial regression of metastatic disease under chemo- and immunotherapy, and in another patient with a NY-ESO-1 negative tumor relapse. Our results indicate that the induction and maintenance of NY-ESO-1 antibody is dependent on the presence of NY-ESO-1 expressing tumors. Furthermore, changes in NY-ESO-1 antibody titers correlate with the evolution of NY-ESO-1 positive disease., (Copyright 1999 Wiley-Liss, Inc.)

Evidence is growing for both humoral and cellular immune recognition of human tumor antigens. Antibodies with specificity for antigens initially recognized by cytotoxic T lymphocytes (CTLs), e.g., MAGE and tyrosinase, have been detected in melanoma patient sera, and CTLs with specificity for NY-ESO-1, a cancer-testis (CT) antigen initially identified by autologous antibody, have recently been identified. To establish a screening system for the humoral response to autoimmunogenic tumor antigens, an enzyme-linked immunosorbent assay (ELISA) was developed using recombinant NY-ESO-1, MAGE-1, MAGE-3, SSX2, Melan-A, and tyrosinase proteins. A survey of sera from 234 cancer patients showed antibodies to NY-ESO-1 in 19 patients, to MAGE-1 in 3, to MAGE-3 in 2, and to SSX2 in 1 patient. No reactivity to these antigens was found in sera from 70 normal individuals. The frequency of NY-ESO-1 antibody was 9.4% in melanoma patients and 12.5% in ovarian cancer patients. Comparison of tumor NY-ESO-1 phenotype and NY-ESO-1 antibody response in 62 stage IV melanoma patients showed that all patients with NY-ESO-1(+) antibody had NY-ESO-1(+) tumors, and no patients with NY-ESO-1(-) tumors had NY-ESO-1 antibody. As the proportion of melanomas expressing NY-ESO-1 is 20-40% and only patients with NY-ESO-1(+) tumors have antibody, this would suggest that a high percentage of patients with NY-ESO-1(+) tumors develop an antibody response to NY-ESO-1.

A growing number of human tumor antigens have been described that can be recognized by cytotoxic T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class I-restricted fashion. Serological screening of cDNA expression libraries, SEREX, has recently been shown to provide another route for defining immunogenic human tumor antigens. The detection of antibody responses against known CTL-defined tumor antigens, e.g., MAGE-1 and tyrosinase, raised the question whether antibody and CTL responses against a defined tumor antigen can occur simultaneously in a single patient. In this paper, we report on a melanoma patient with a high-titer antibody response against the "cancer-testis" antigen NY-ESO-1. Concurrently, a strong MHC class I-restricted CTL reactivity against the autologous NY-ESO-1-positive tumor cell line was found. A stable CTL line (NW38-IVS-1) was established from this patient that reacted with autologous melanoma cells and with allogeneic human histocompatibility leukocyte antigen (HLA)-A2(-), NY-ESO-1-positive, but not NY-ESO-1-negative, melanoma cells. Screening of NY-ESO-1 transfectants with NW38-IVS-1 revealed NY-ESO-1 as the relevant CTL target presented by HLA-A2. Computer calculation identified 26 peptides with HLA-A2-binding motifs encoded by NY-ESO-1. Of these, three peptides were efficiently recognized by NW38-IVS-1. Thus, we show that antigen-specific humoral and cellular immune responses against human tumor antigens may occur simultaneously. In addition, our analysis provides a general strategy for identifying the CTL-recognizing peptides of tumor antigens initially defined by autologous antibody.