Most patients with multiple myeloma (MM) will relapse after an initial response and eventually succumb to their disease. This is due to the persistence of chemotherapy-resistant tumor cells in the patients’ bone marrow (BM) and immunotherapeutic approaches could contribute to eradicating these remaining cells. We evaluated SLLP1 as a potential immunotherapeutic target for MM.

Cancer-testis antigens belonging to the MAGE family of genes, such as MAGEC2, are commonly and specifically expressed in Multiple Myeloma (MM) and are associated with a more aggressive clinical course and chemotherapy resistance. MAGEC2 is thought to be an excellent candidate for cancer immunotherapy; however, the biological role of MAGEC2 in MM has remained unclear. We investigated the biological role of MAGEC2 in myeloma cells determining the effect of MAGEC2 knockdown on proliferation and apoptosis. Loss of MAGEC2 resulted in reduced proliferation, viability, and anchorage-independent growth of myeloma cells irrespective of the functional status of TP53 (p53). The anti-proliferative effect of MAGEC2 silencing was due to a decrease of cells in the S phase, cell cycle delay at both G0/G1 and/or G2/M, and an increase in the sub-G0/G1 diploid population related to apoptotic cell death. Importantly, overexpression of short hairpin (sh)RNA-refractory MAGEC2 rescued the anti-proliferative effect of mRNA knockdown and protected cells from apoptotic cell death. Our findings support a TP53-independent role of MAGEC2 in promoting the survival of myeloma cells suggesting that MAGEC2-specific immunotherapies have the potential to eradicate the most malignant cells within the myeloma tumour bulk leading to durable clinical responses.

Multiple myeloma (MM) is the malignancy with the most frequent expression of the highly immunogenic cancer–testis antigens (CTA), and we have performed the first analysis of longitudinal expression, immunological properties, and fine specificity of CTA-specific antibody responses in MM.

1 Department of Internal Medicine II, Oncology/Hematology/Bone Marrow Transplantation with the Section Pneumology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 2 University Cancer Center Hamburg (Hubertus Wald Tumorzentrum), University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 3 Division of Clinical Pharmacology, Department of Internal Medicine, Ludwig-Maximilian University, 80336 Munich, Germany 4 Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 5 Institute for Pathology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany 6 Institute for Immunology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany

Acute psychological stress has primarily been investigated regarding its effects on conventional lymphocytes such as natural killer (NK) cells and CD4(+) and CD8(+) T cells. However, it might be important to focus on more "specialized" lymphocyte subsets, playing a role, for instance, in allergic conditions and autoimmunity, to identify links between stress, the immune system and somatic diseases. Using flow cytometry we determined frequencies of circulating T helper (Th)1-type (CD226(+)) and Th2-type (CRTH2(+)) T cells, γδ T cells, conventional CD56(+) natural killer T (NKT) cells and invariant NKT cells (iNKT) in healthy young males (N = 31; median age 26 years) undergoing a laboratory computer-based stressor lasting 12 min. We found that acute psychological stress induced a prolonged increase in CD4(+) and CD8(+) T cells expressing a Th2 phenotype. We also detected an acute increase in CD4(-) and CD8(-) double negative γδ T cells. Finally, we found that the well-known increase in NK cells under stressful conditions was paralleled by a significant increase in numbers of conventional CD56(+) NKT cells. In contrast, numbers of iNKT was not altered by stress. This study adds further evidence to a psychoneuroimmunological model proposing that under stressful conditions certain lymphocyte subsets, including iNKT and less mature T cells, are retained in lymphoid tissues while antigen-experienced effector-type T cells with a Th2 phenotype, γδ T cells and conventional CD56(+) NKT cells are mobilized into the peripheral blood. We suggest that in the case of frequent stress exposure, this might result in the promotion of, for example, allergic conditions.

Background: Patients with gastric cancer benefit from perioperative chemotherapy, however, treatment is toxic and many patients will relapse. The trifunctional antibody catumaxomab targets EpCAM on tumor cells, CD3 on T cells, and the Fcγ-receptor of antigen-presenting cells. While in Europe catumaxomab is approved for treating malignant ascites, it has not been investigated in the perioperative setting and its exact immunological mode of action is unclear. Methods: In our study, gastric cancer patients received neoadjuvant platinum-based chemotherapy, one intraoperative application of catumaxomab, and 4 postoperative doses of intraperitoneal catumaxomab. Immunomonitoring was performed in 6 patients before surgery, after completion of catumaxomab treatment, and one month later. Results: Intraperitoneal application of catumaxomab caused an increased expression of activation markers on the patients’ T cells. This was accompanied by a transient decrease in numbers of CXCR3+ effector T cells with a T-helper (Th)-1 phenotype in the peripheral blood. All patients evidenced pre-existing EpCAM-specific CD4+ and/or CD8+ T cells. While these cells transiently disappeared from the blood stream after intraperitoneal application of catumaxomab, we detected increased numbers of peripheral EpCAM-specific cells and a modified EpCAM-specific T-cell repertoire 4 weeks after completion of treatment. Finally, catumaxomab also amplified humoral immunity to tumor antigens other than EpCAM. Conclusions: Our findings suggest that catumaxomab exerts its clinical effects by (1) activating peripheral T cells, (2) redistributing effector T cells from the blood into peripheral tissues, (3) expanding and shaping of the pre-existing EpCAM-specific T-cell repertoire, and (4) spreading of anti-tumor immunity to different tumor antigens.

Abstract Abstract 1808 Background: Cancer-testis antigens belonging to the MAGE class I family of genes are commonly expressed in Multiple Myeloma (MM). Expression of MAGE class I genes is associated with an aggressive clinical course of MM and resistance to chemotherapy, suggesting that MAGE genes may confer a survival advantage on myeloma cells. MAGE-C2/CT10 is member of the MAGE class I family of genes thought to be a good candidate for cancer immunotherapy given its very frequent expression in primary myeloma. In normal cells, MAGE-C2/CT10 seems to suppress p53 expression by promoting its polyubiquitination and degradation. However, the function of MAGE-C2/CT10 in malignancies is completely unknown. We, therefore, investigated for the first time the role of MAGE-C2/CT10 in tumor cells derived from patients with MM. Material and Methods: MAGE-C2/CT10 expression was analysed by real-time PCR and western blot in myeloma cell lines (N=8) and in PBMC from healthy donors (N=8). For the functional evaluation of MAGE-C2/CT10 we decided to use myeloma cell line U-266 which constitutively expresses MAGE-C2/CT10 and a missense mutant p53 (A161T) that has partially lost its transcriptional activity. The biological role of MAGE-C2/CT10 was investigated by stably silencing its expression using lentiviral short hairpin RNA (shRNA). The effects of silencing MAGE-C2/CT10 expression on myeloma cell biology were examined by determining the number of viable or apoptotic cells using a colorimetric MTT assay and annexin-V/7AAD staining followed by flow cytometry, In addition, we measured myeloma cell proliferation and the anchorage-independent growth using a BrdU incorporation assay and a colony formation assay, respectively. Finally, we investigated cell cycle phase distribution by flow cytometry and we analyzed the expression of key molecules involved in cell cycle progression and apoptosis using a real-time PCR array as well as western blot. Results: We found MAGE-C2/CT10 to be constitutively expressed in all myeloma cells lines but not in PBMC from healthy donors. Lentivirus-mediated silencing of MAGE-C2/CT10 inhibited significantly the proliferation and the anchorage-independent growth of myeloma cells. Cell cycle analysis demonstrated that the anti-proliferative effect of MAGE-C2/CT10 silencing in U-266 was due to a 70% decrease of cells in the S phase, a cell cycle arrest at both G0/G1 and G2/M transitions and an increase in the subG0/G1 population due to an activation of apoptotic cell death. The serine-threonine checkpoint effector kinase 2 (CHK2) and its substrate, the tumor suppressor protein p53, are essential for cell cycle control, DNA repair and apoptosis. We found that the loss of MAGE-C2/CT10 expression was associated with the activation of CHK2 through phosphorylation at Thr68 as well as the activation of p53 by phosposphorylation at Ser20. Furthermore, we observed a three-fold increase in the endogenous level of p53 protein which correlated with an up-regulation of two transcriptional targets of p53, the cyclin-dependant kinase inhibitor p21WAF1 and the growth arrest and DNA-damage-inducible alpha protein (GADD45A), known to be essential for p53-induced G1 and G2 arrest, respectively. Finally, using the Human Apoptosis Profiler PCR array that contains a number of p53 target genes, we found that apoptosis induced by MAGE-C2/CT10 knockdown was due to a more than two-fold increase in the transcription of pro-apoptotic genes like BAX (Bcl2-associated × protein), BAD (BCL2-associated agonist of cell death), Cytochrome c, APAF1 (Apoptosis activating factors) as well as several caspases, which are the down-stream mediators of p53-dependant apoptosis in response to DNA damage. Conclusions: Our collected findings support an anti-apoptotic function of MAGE-C2/CT10 in MM, likely through the regulation of key molecules involved in cell cycle progression and p53-mediated apoptosis. The central role of MAGE-C2/CT10 in the biology of myeloma strongly suggest that this cancer-testis antigen represents a promising target for myeloma-specific immunotherapies or other targeted modes of therapy for MM. Disclosures: Kröger: Fresenius Biotech: Honoraria, Research Funding.

Background. Multiple myeloma (MM) and its therapies may induce a severely compromised humoral immunity. We have performed a longitudinal analysis of IgG-antibody responses against influenza virus (FLU) and tetanus toxoid (TT) as surrogate markers for the B cell-mediated immunity in MM patients. Methods. 1094 serum samples of 190 MM patients and samples from 100 healthy donors were analyzed by ELISA for FLU-and TT-specific antibodies. Results. MM patients evidenced lower levels of FLU-and TT-specific antibodies than healthy controls (P < 0.001). Immunoreactivity decreased with progressing disease and worsening clinical status. Levels of FLU-and TT-specific antibodies increased shortly (0-6 months) after alloSCT (P < 0.001), a time-period during which intravenous immunoglobulin (IVIG) is routinely applied. Thereafter, antibody concentrations declined and remained suppressed for 3 years in the case of FLU-specific and for more than 5 years in the case of TT-specific antibodies. Conclusions. We found that MM is associated with a profound disease-and therapy-related immunosuppression, which is compensated for a few months after alloSCT, most likely by application of IVIG. This and the differences regarding the recovery of anti-FLU and anti-TT antibody titers during the following years need to be taken into account for optimizing IVIG application and immunization after alloSCT.

BACKGROUND Multiple myeloma is a malignancy characterized by the expansion of a plasma cell clone that localizes to the human bone marrow. Myeloma cells and bone marrow stromal cells produce soluble factors that promote the survival and progression of multiple myeloma. Interleukin 16 (IL-16) is involved in regulating the migration and proliferation of normal leukocytes. However, the role of IL-16 in human cancers, including multiple myeloma, is unclear. METHODS We investigated IL-16 expression in cell lines (n = 10) and in the bone marrow of myeloma patients (n = 62) and healthy bone marrow donors (n = 12) by quantitative reverse transcription-polymerase chain reaction, immunoblot analysis, enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemistry. Transfection of two human multiple myeloma cell lines with small interfering RNAs was used to examine the effect of IL-16 gene silencing on apoptosis by flow cytometry, on proliferation by bromodeoxyuridine incorporation, and on colony formation. Protein neutralization assays were performed by treating multiple myeloma cells with a monoclonal antibody against the carboxyl-terminal fragment of IL-16. All statistical tests were two-sided. RESULTS IL-16 was strongly overexpressed in the bone marrow of myeloma patients compared with healthy donors. Myeloma cell lines as well as primary tumor cells from myeloma patients constitutively expressed IL-16 and its receptors CD4 and/or CD9 and spontaneously secreted soluble IL-16. Silencing of IL-16 reduced the proliferative activity of myeloma cells by approximately 80% compared with untreated cells (mean relative proliferative activity IL-16 siRNA vs untransfected cells, EJM cells: 20.1%, 95% confidence interval [CI] = 14.3% to 26.0%, P = .03; KMS-12-BM cells: 22.8%, 95% CI = 5.5% to 40.0%, P = .04), and addition of a recombinant carboxyl-terminal IL-16 peptide reversed that effect. A monoclonal antibody directed against IL-16 or its receptors had a comparably strong growth-inhibiting effect on the tumor cells. CONCLUSIONS IL-16 is an important growth-promoting factor in multiple myeloma and a candidate for novel diagnostic, prognostic, and therapeutic applications for this incurable human malignancy.

Cancer‐testis antigens (CTA) represent attractive targets for tumor immunotherapy. However, a broad picture of CTA expression in acute myeloid leukemia (AML) is missing. CTA expression was analyzed in normal bone marrow (BM) as well as in AML cell lines before and after treatment with demethylating agents and/or histone acetylase inhibitors. Presence of selected CTA with a strictly tumor‐restricted expression was then determined in samples of patients with AML before and after demethylating therapy. Screening AML cell lines for the expression of 20 CTA, we identified six genes (MAGE‐A3, PRAME, ROPN1, SCP‐1, SLLP1, and SPO11) with an AML‐restricted expression. Analyzing the expression of these CTA in blast‐containing samples from AML patients ( N = 64), we found all samples to be negative for MAGE‐A3 and SPO11 while a minority of patients expressed ROPN1 (1.6%), SCP‐1 (3.1%), or SLLP1 (9.4%). The only CTA expressed in substantial proportion of patients (53.1%) was PRAME. Following demethylating treatment with 5′‐aza‐2′‐deoxycytidine, we observed an increased or de novo expression of CTA, in particular of SSX‐2, in AML cell lines. In AML patients, we detected increased expression of PRAME and induction of SSX‐2 after demethylating therapy with 5‐azacytidine. With the exception of PRAME, CTA are mostly absent from AML blasts. However, demethylating treatment induces strong expression of CTA, particularly of SSX‐2, in vitro and in vivo. Therefore, we propose that CTA‐specific immunotherapy for AML should preferentially target PRAME and/or should be combined with the application of demethylating agents opening the perspective for alternative targets like CTA SSX‐2. Am. J. Hematol., 2011. © 2011 Wiley‐Liss, Inc.

Background To date, multiple myeloma remains an incurable malignancy due to the persistence of minimal residual disease in the bone marrow. In this setting, monoclonal antibodies against myeloma-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate target structures expressed across all pathogenic myeloma cell populations. We, therefore, investigated functionally relevant immunoreceptors specifically associated with myeloma cells as well as their clonogenic precursors.Design and Methods Potential target proteins were identified using antibody arrays against phosphorylated immunoreceptors with lysates from myeloma cell lines. CD229 expression was confirmed in primary myeloma cells by reverse transcriptase polymerase chain reaction, western blot, fluorescence-activated cell sorting, and immunohistochemistry. Apoptosis, clonogenic growth, and sensitivity to chemotherapy were determined following short-interfering RNA-mediated downregulation of CD229. Antibody-dependent cellular and complement-dependent cytotoxicity were analyzed using a monoclonal antibody against CD229 to demonstrate the antigen’s immunotherapeutic potential.Results Our screening assay identified CD229 as the most strongly over-expressed/phosphorylated immunoreceptor in myeloma cell lines. Over-expression was further demonstrated in the CD138-negative population, which has been suggested to represent myeloma precursors, as well as on primary tumor cells from myeloma patients. Accordingly, CD229 staining of patients’ bone marrow samples enabled the identification of myeloma cells by flow cytometry and immunohistochemistry. Down-regulation of CD229 led to a decreased number of viable myeloma cells and clonal myeloma colonies, and enhanced the anti-tumor activity of conventional chemotherapeutics. Targeting CD229 with a monoclonal antibody resulted in complement- and cell-mediated lysis of myeloma cells.Conclusions Our results demonstrate that the immunoreceptor CD229 is specifically over-expressed on myeloma cells including their clonogenic precursors and contributes to their malignant phenotype. Monoclonal antibodies against this protein may represent a promising diagnostic and immunotherapeutic instrument in this disease.

The occurrence of SOX2-specific autoantibodies seems to be associated with an improved prognosis in patients with monoclonal gammopathy of undetermined significance (MGUS). However, it is unclear if SOX2-specific antibodies also develop in established multiple myeloma (MM). Screening 1094 peripheral blood (PB) sera from 196 MM patients and 100 PB sera from healthy donors, we detected SOX2-specific autoantibodies in 7.7% and 2.0% of patients and donors, respectively. We identified SOX2211–230 as an immunodominant antibody-epitope within the full protein sequence. SOX2 antigen was expressed in most healthy tissues and its expression did not correlate with the number of BM-resident plasma cells. Accordingly, anti-SOX2 immunity was not related to SOX2 expression levels or tumor burden in the patients’ BM. The only clinical factor predicting the development of anti-SOX2 immunity was application of allogeneic stem cell transplantation (alloSCT). Anti-SOX2 antibodies occurred more frequently in patients who had received alloSCT (n=74). Moreover, most SOX2-seropositive patients had only developed antibodies after alloSCT. This finding indicates that alloSCT is able to break tolerance towards this commonly expressed antigen. The questions whether SOX2-specific autoantibodies merely represent an epiphenomenon, are related to graft-versus-host effects or participate in the immune control of myeloma needs to be answered in prospective studies.

Abstract 5087 Background: Cancer-testis antigens (CTA) are attractive targets for cancer immunotherapy based on their tumor-restricted expression and immunogenicity. A number of CTA, including Melanoma-associated antigen 3 (MAGE-A3), are already under clinical investigation and CTA have been shown to induce strong T cell and humoral immunity in cancer patients receiving active immunotherapy. However, little is known about the fine specificity and the function of vaccine-induced humoral immune responses and it is unclear how they relate to spontaneous CTA-specific immune responses occurring in a minority of patients. Methods: We have performed a longitudinal analysis of spontaneously occurring antibody responses against the CT antigen MAGE-A3 in sera (N=1537), which were collected from patients with multiple myeloma (N=355) over a period of 6 years. Antibody titers were determined by ELISA technique and a B cell ELISPOT assay was applied to estimate the number of MAGEA3-specific memory B cells in peripheral blood of the patients. Fine specificity of the antibody responses was examined using overlapping 20mer peptides spanning the whole sequence of MAGE-A3. The given IgG subtype was determined, and the quality of MAGE-A3-specific antibodies was analyzed using western blot as well as affinity assays. Results were compared to those obtained with MAGE-A3-specific antibody responses induced by vaccination with full-length MAGE-A3 protein and adjuvants AS02B or AS15 in patients with non-small cell lung cancer (NSCLC; N=15). Results: Out of 355 myeloma patients 4 (1.1%) evidenced spontaneous antibody responses against MAGE-A3 at least at one point during the course of their disease. Spontaneously occurring anti-MAGE-A3 humoral responses were usually of low titer. In contrast, all of the vaccinated patients showed high-titered and persisting antibody responses which usually appeared around week 6 after the first application of the vaccine. Accordingly, we found high frequencies of vaccine-induced MAGE-A3-specific memory B cells in the peripheral blood of NSCLC patients while they remained undetectable in most myeloma patients. Vaccine-induced antibody responses underwent affinity maturation reaching affinity levels of spontaneous immune responses after repeated cycles of treatment. MAGE-A3-specific antibodies consisted of IgG1 and IgG3>IgG2>IgG4 subtypes in vaccinated patients whereas spontaneously occurring antibodies were mainly of the IgG2 subtype. Spontaneous as well as vaccine-induced IgG antibodies both recognized the natural full-length protein. Analysis of the fine specificity of the antibody responses revealed that vaccine-induced antibodies recognized a much larger number of MAGE-A3 epitopes than spontaneously occurring antibodies. However, both, spontaneous as well as vaccine-induced responses, most frequently and strongly recognized a specific region within the MAGE-A3 protein corresponding to amino acids 51–70. Conclusions This study demonstrates for the first time important qualitative differences between spontaneously occurring and vaccine-induced antibody responses against the MAGE-A3 antigen in cancer patients. While the potential of both types of antibody responses to promote antigen uptake and induction of T cell responses by antigen-presenting cells might differ, they both recognized the same restricted region within the MAGE-A3 protein. The latter finding might be of importance for the design of future immunotherapies targeting MAGE-A3. Disclosures: No relevant conflicts of interest to declare.

2601 Background: The abundant expression of the epithelial cell-adhesion molecule EpCAM on the surface of epithelial cancer cells renders it an attractive target for immunotherapy of gastric cancer (GC). The trifunctional antibody (Ab) catumaxomab targets EpCAM on malignant cells and CD3 on T cells while triggering antigen (Ag)-presenting cells via their Fcγ-receptor. Recently, catumaxomab has received approval for the treatment of malignant ascites; however, the exact immune mechanisms behind its clinical effects are unknown. Methods: In a phase II study, patients with operable GC received neoadjuvant chemotherapy followed by 1 dose of catumaxomab during surgery and 4 consecutive doses applied intraperitoneally in the adjuvant setting. We measured the immunomodulatory effects of catumaxomab in 6 patients at 3 time points: before surgery, after application of the first dose, and 1 month post treatment. Results: None of the patients had evidence of autologous EpCAM-specific Ab responses. However, the majority of patients developed Abs against different classes of Ags, namely cancer-testis Ags, after catumaxomab treatment. Monitoring T cell subsets, we observed a drastic decrease in peripheral CD4+ T cells carrying an effector and T-helper (Th)-1 phenotype (CXCR3+CCR4–) directly after Ab application, whereas the CD4+ Th2 population remained unchanged. At 4 weeks after catumaxomab, the number of CD4+ effector-type T cells in the peripheral blood (PB) had returned to baseline levels. Interestingly, we could demonstrate pre-existing EpCAM-specific CD4+ and/or CD8+ T cells in all patients. While these T cells seemed to disappear from the PB immediately after Ab exposure, we detected significantly increased numbers of peripheral EpCAM-specific cells 4 weeks after immunomodulatory treatment. Conclusions: Our data suggest that catumaxomab might exert its clinical effects through at least 3 different immunological routes: (1) the spreading of humoral immune responses to other Ags apart from EpCAM, (2) the redistribution of effector and Th1-type cells from the PB into peripheral tissues, and (3) the expansion of pre-existing EpCAM-specific T cells.

e13045 Background: Cancer-testis (CT) antigens represent ideal candidates for tumor-specific therapy due to their inherent immunogenicity and exceptional tissue restriction. Despite these advantageous properties the development of targeted therapeutics has been hampered by their predominantly intracellular localization. CT antigen FMR1NB might represent a promising exception due to its predicted transmembrane topology. Methods: We screened acute (AML) and chronic (CML) myeloid leukemia samples as well as healthy tissues for the expression of FMR1NB. Mammalian cell lines were transfected with FLAG- or GFP-tagged FMR1NB fusion constructs. We evaluated the subcellular localization of FMR1NB by fractionation and confocal microscopy. Epitopes of commercial anti-FMR1NB antibodies were characterized by ELISA using overlapping peptides. Results: FMR1NB mRNA was expressed in AML cell lines and patient samples and confirmed by western blot on the protein level. Specificity was confirmed by the lack of FMR1NB expression in healthy tissues. Confocal fluorescence microscopy of GFP chimeras in mammalian cell lines, immunofluorescence staining of AML cell lines, and subcellular fractionation strongly support a membraneous localization of FMR1NB including the plasmamembrane. In contrast to results of structural prediction software our characterization of epitopes recognized by polyclonal anti-FMR1NB antibodies and the subsequent immunostaining of intact transfected and native AML cells suggest an extracellular amino-terminus of FMR1NB. Flow cytometric analysis of AML and CML cell lines as well as AML patient samples and healthy donors further confirmed a strong and specific expression of FMR1NB on leukemia samples. Conclusions: FMR1NB mRNA is specifically expressed in AML cells and the resulting protein localizes to the membrane compartment including the plasma membrane. A putative extracellular domain of over 100aa length of the protein can be detected using FMR1NB-specific antibodies. The membrane localization of FMR1NB is a rare exception within the CT antigen family and our data suggest that it represents an ideal target for the diagnosis and therapy of myeloid leukemias. No significant financial relationships to disclose.

NY-CO-58/KIF2C has been identified as a tumor antigen by screening antibody responses in patients with colorectal cancer. However, expression had not consequently been examined, and nothing was known about its ability to induce spontaneous T cell responses, which have been suggested to play a role in the development of colorectal cancer. We analyzed 5 colorectal cancer cell lines, and tumor samples and adjacent healthy tissues from 176 patients with epithelial cancers for the expression of NY-CO-58/KIF2C by RTPCR and Western Blot. T cell responses of 43 colorectal cancer patients and 35 healthy donors were evaluated by ELISpot following stimulation with 30mer peptides or full-length protein. All cell lines and tumor samples from colorectal cancer patients expressed NY-CO-58/KIF2C on the protein and RNA level, and expression levels correlated strongly with Ki-67 expression (r = 0.69; p = 0.0003). Investigating NY-CO-58/KIF2C-specific T cell responses, CD8(+) T cells directed against 1 or more peptides were found in less than 10% of patients, whereas specific CD4(+) T cells were detected in close to 50% of patients. These T cells were of high avidity, recognized the naturally processed antigen and secreted IFN-gamma and TNF-alpha. Depletion of CD4(+) CD25(+) T cells before stimulation significantly increased the intensity of the preexisting response. NY-CO-58/KIF2C is significantly overexpressed in colorectal and other epithelial cancers and expression levels correlate with the proliferative activity of the tumor. Importantly, NY-CO-58/KIF2C was able to induce spontaneous CD4(+) T cell responses of the Th1-type, which were tightly controlled by peripheral T regulatory cells.