NY-ESO-1 and LAGE-1 cancer-testis antigens are potential targets for immunotherapy in epithelial ovarian cancer.

Cancer-testis (CT) antigens are expressed in a variety of cancers, but not in normal adult tissues, except for germ cells of the testis, and hence appear to be ideal targets for immunotherapy. In an effort to examine the potential of NY-ESO-1 and LAGE-1 CT antigens for immunotherapy in epithelial ovarian cancer (EOC), we examined the expression of these antigens by reverse transcription-PCR (RT-PCR) and immunohistochemistry (IHC) in a large panel of EOC tissues and cell lines. Sera from a subgroup of the patients were tested for NY-ESO-1/LAGE-1 antibody by ELISA. The data indicated that four ovarian cancer cell lines were positive for one or both CT antigens. Expression of NY-ESO-1 in EOC was demonstrated by RT-PCR and/or IHC in 82 of 190 (43%) specimens. NY-ESO-1 expression by IHC ranged from homogeneous to heterogeneous pattern. LAGE-1 mRNA expression was present in 22 of 107 (21%) tumor tissues. Overall, the expression of either NY-ESO-1 or LAGE-1 mRNA was present in 42 of 107 (40%) EOC specimens and coexpression of both antigens was demonstrated in 11% of specimens. Antibody to NY-ESO-1/LAGE-1 was present in 11 of 37 (30%) patients whose tumors expressed either NY-ESO-1 or LAGE-1. Detectable antibodies were present for up to 3 years after initial diagnosis. Although there was no statistically significant relation between expression of NY-ESO-1/LAGE-1 antigen and survival, the data showed aberrant expression of NY-ESO-1 and LAGE-1 by IHC/RT-PCR in a significant proportion of EOC patients. These findings indicate that NY-ESO-1 and LAGE-1 are attractive targets for antigen-specific immunotherapy in EOC.

SCP-1 cancer/testis antigen is a prognostic indicator and a candidate target for immunotherapy in epithelial ovarian cancer.

SCP-1 is a novel tumor antigen that belongs to the growing family of cancer/testis (CT) antigens, and it is a potential target for immunotherapy. In an effort to determine the expression of SCP-1 in epithelial ovarian cancer (EOC), one-step RT-PCR was performed with RNA from epithelial ovarian tumor tissues and with two normal ovarian surface epithelial cell lines. We used immunohistochemistry (IHC) to investigate SCP-1 expression in paraffin-fixed EOC samples and ELISA to test sera from a subgroup of patients for SCP-1 antibody. SCP-1 was expressed in 15 out of 100 (15%) primary tumors, as determined by RT-PCR. The normal ovarian surface epithelial cell lines were negative for SCP-1 expression, as were a panel of other normal tissues. None of the patients whose tumors were determined to be SCP-1 positive by RT-PCR expressed the antigen by IHC or demonstrated a humoral immune response by ELISA. Tumors that expressed SCP-1 mRNA tended to have a higher grade than those that did not (P = 0.03). There was a significant decrease in survival time (P = 0.004) for patients with SCP-1 mRNA-positive tumors compared to those with SCP-1 mRNA-negative tumors [median 25 mo, 95% confidence interval (CI) 0-56 mo; and median 97 mo, CI 32-162 mo, respectively]. The present study shows that SCP-1 mRNA expression in patients with EOC is associated with a poorer chance of survival. These findings imply that further evaluation of SCP-1 as a potential target for vaccine therapy in EOC is warranted.

Frequency of NY-ESO-1 and LAGE-1 expression in bladder cancer and evidence of a new NY-ESO-1 T-cell epitope in a patient with bladder cancer.

Cancer-testis (CT) antigens are ideal vaccine targets since their expression is restricted in adult tissues to testicular germ cells and a subset of cancers. The frequency of expression in transitional cell carcinomas (TCCs) of NY-ESO-1, the most immunogenic CT antigen to date, and its closely related gene LAGE-1 was studied. NY-ESO-1 and LAGE-1 antigen expression were found to occur frequently in high-grade TCC tumors. On an MSKCC IRB-approved protocol, 68 patient specimens were collected prospectively at the time of transurethral resection or cystectomy, of which 43 were read pathologically as high-grade tumors (pCIS, pTaG3, pT1, pT2, pT3, and pT4), 8 as low-grade tumors (pTaG1, pTaG2), and 17 as disease-free samples. These 68 samples were analyzed by immunohistochemistry (IHC) and/or RT-PCR. There were also an additional 53 paraffin-embedded specimens studied retrospectively by IHC, of which 39 were high-grade tumors and 14 were low-grade tumors. Cumulatively, our data indicate that NY-ESO-1 and/or LAGE-1 are expressed in 39/82 (48%) high-grade TCC and 3/22 (14%) low-grade TCC samples when analyzed by RT-PCR and/or IHC. Immunological assessment of these patients' sera identified one patient, whose tumor homogeneously expressed NY-ESO-1, which had detectable antibodies against NY-ESO-1 and LAGE-1. Further analysis of this patient, who remains clinically without evidence of disease 24 months after cystectomy for high-grade pT4 disease, revealed T-cell immunity against NY-ESO-1. This patient's T-cell response was determined to be specific for a new NY-ESO-1 epitope, p94-102, in the context of HLA-B35.

Th1/Th2 CD4+ T cell responses against NY-ESO-1 in HLA-DPB1*0401/0402 patients with epithelial ovarian cancer.

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.

Cancer/testis antigen expression and autologous humoral immunity to NY-ESO-1 in gastric cancer.

Gastric cancer has the highest mortality rate and the second-highest morbidity rate of all malignant tumors in China. Since cancer/testis (CT) antigens are expressed in various types of human tumors but generally not in normal tissue except for testis, they are promising antigens for cancer immunotherapy. NY-ESO-1, in particular, is the most immunogenic of the CT antigens. To study the feasibility of developing a CT antigen vaccine for gastric cancer, 101 gastric cancer samples were analyzed for the presence of NY-ESO-1 mRNA and that of 10 other CT antigen genes. Twelve out of 101 samples (11.9%) were found to be NY-ESO-1 mRNA-positive, 11 of them from advanced stage patients. In 7 of the 12 NY-ESO-1 mRNA-positive samples, the NY-ESO-1 protein was also detected by immunohistochemistry. An autologous humoral immune response to NY-ESO-1 was detected in 6 of 12 advanced stage NY-ESO-1 mRNA-positive patients, indicating that NY-ESO-1 is immunogenic in advanced stage gastric cancer. The serum from a patient with an NY-ESO-1 negative but LAGE-1 positive tumor was also found to be NY-ESO-1 antibody positive, possibly due to cross-reactivity between NY-ESO-1 and LAGE-1. All NY-ESO-1 mRNA-positive gastric cancer samples also expressed one to seven additional CT genes, revealing a tendency toward a clustered expression pattern, regardless of disease stage. About 74% of the samples expressed at least one CT antigen, most frequently MAGE-3 (41.6%). NY-ESO-1 and MAGE-3 are thus potential targets for a multivalent CT antigen vaccine.

Recombinant antigen expression on yeast surface (RAYS) for the detection of serological immune responses in cancer patients.

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.

Vaccine-induced CD4+ T cell responses to MAGE-3 protein in lung cancer patients.

MAGE-3 is the most commonly expressed cancer testis Ag and thus represents a prime target for cancer vaccines, despite infrequent natural occurrence of MAGE-3-specific immune responses in vivo. We report in this study the successful induction of Ab, CD8(+), and CD4(+) T cells in nonsmall cell lung cancer patients vaccinated with MAGE-3 recombinant protein. Two cohorts were analyzed: one receiving MAGE-3 protein alone, and one receiving MAGE-3 protein with adjuvant AS02B. Of nine patients in the first cohort, three developed marginal Ab titers and another one had a CD8(+) T cell response to HLA-A2-restricted peptide MAGE-3 271-279. In contrast, of eight patients from the second cohort vaccinated with MAGE-3 protein and adjuvant, seven developed high-titered Abs to MAGE-3, and four had a strong concomitant CD4(+) T cell response to HLA-DP4-restricted peptide 243-258. One patient simultaneously developed CD8(+) T cells to HLA-A1-restricted peptide 168-176. The novel monitoring methodology used in this MAGE-3 study establishes that protein vaccination induces clear CD4(+) T cell responses that correlate with Ab production. This development provides the framework for further evaluating integrated immune responses in vaccine settings and for optimizing these responses for clinical benefit.