Molecular cloning of hMena (ENAH) and its splice variant hMena+11a: epidermal growth factor increases their expression and stimulates hMena+11a phosphorylation in breast cancer cell lines

hMena (ENAH), an actin regulatory protein involved in the control of cell motility and adhesion, is modulated during human breast carcinogenesis. In fact, whereas undetectable in normal mammary epithelium, hMena becomes overexpressed in high-risk benign lesions and primary and metastatic tumors. In vivo, hMena overexpression correlates with the HER-2(+)/ER(-)/Ki67(+) unfavorable prognostic phenotype. In vitro, neuregulin-1 up-regulates whereas Herceptin treatment down-modulates hMena expression, suggesting that it may couple tyrosine kinase receptor signaling to the actin cytoskeleton. Herein, we report the cloning of hMena and of a splice variant, hMena(+11a), which contains an additional exon corresponding to 21 amino acids located in the EVH2 domain, from a breast carcinoma cell line of epithelial phenotype. Whereas hMena overexpression consistently characterizes the transformed phenotype of tumor cells of different lineages, hMena(+11a) isoform is concomitantly present only in epithelial tumor cell lines. In breast cancer cell lines, epidermal growth factor (EGF) treatment promotes concomitant up-regulation of hMena and hMena(+11a), resulting in an increase of the fraction of phosphorylated hMena(+11a) isoform only. hMena(+11a) overexpression and phosphorylation leads to increased p42/44 mitogen-activated protein kinase (MAPK) activation and cell proliferation as evidenced in hMena(+11a)-transfected breast cancer cell lines. On the contrary, hMena knockdown induces reduction of p42/44 MAPK phosphorylation and of the proliferative response to EGF. The present data provide new insight into the relevance of actin cytoskeleton regulatory proteins and, in particular, of hMena isoforms in coupling multiple signaling pathways involved in breast cancer.     

Inducible Hsp70 as target of anticancer immunotherapy: Identification of HLA-A*0201-restricted epitopes

The design of a broad application tumor vaccine requires the identification of tumor antigens expressed in a majority of tumors of various origins. We questioned whether the major stress-inducible heat shock protein Hsp70 (also known as Hsp72), a protein frequently overexpressed in human tumors of various histological origins, but not in most physiological normal tissues, constitutes a tumor antigen. We selected the p391 and p393 peptides from the sequence of the human inducible Hsp70 that had a high affinity for HLA-A*0201. These peptides were able to trigger a CTL response in vivo in HLA-A*0201-transgenic HHD mice and in vitro in HLA-A*0201+ healthy donors. p391- and p393-specific human and murine CTL recognized human tumor cells overexpressing Hsp70 in a HLA-A*0201-restricted manner. Tetramer analysis of TILs showed that these Hsp70 epitopes are targets of an immune response in many HLA-A*0201+ breast cancer patients. Hsp70 is a tumor antigen and the Hsp70-derived peptides p391 and p393 could be used to raise a cytotoxic response against tumors of various origins.     

The cytoskeleton regulatory protein hMena (ENAH) is overexpressed in human benign breast lesions with high risk of transformation and human epidermal growth factor receptor-2-positive/hormonal receptor-negative tumors.

PURPOSE: hMena (ENAH), a cytoskeleton regulatory protein involved in the regulation of cell motility and adhesion, is overexpressed in breast cancer. The aim of this study was to define at what stage of breast carcinogenesis hMena is overexpressed and to correlate hMena overexpression with established prognostic factors in breast cancer, focusing on human epidermal growth factor receptor-2 (HER-2). EXPERIMENTAL DESIGN: hMena expression was assessed immunohistochemically in a prospective cohort of cases (n = 360) encompassing a highly representative spectrum of benign breast diseases associated with different risk of transformation, in situ, invasive, and metastatic tumors. Correlations with conventional pathologic and prognostic variables, such as proliferation index, hormonal receptor status, and HER-2 overexpression, were also evaluated. In vitro experiments were done to study the effect of neuregulin-1 and Herceptin treatments on hMena expression. RESULTS: hMena protein is undetectable in normal breast and is weakly expressed in a small percentage of low-risk benign diseases (9%), but displays a progressive and significant increase of positivity in benign lesions at higher risk of transformation (slightly increased risk 43%; moderate increased risk 67%), in in situ (72%), invasive (93%), and metastatic breast cancer (91%). A significant direct correlation with tumor size (P = 0.04), proliferation index (P < 0.0001), and HER-2 overexpression (P < 0.0001) and an inverse relationship with estrogen (P = 0.036) and progesterone receptors (P = 0.001) are found in invasive carcinomas. In vitro experiments show that neuregulin-1 up-regulates, whereas Herceptin down-regulates, hMena expression. CONCLUSIONS: Our data provide new insights into the relevance of actin-binding proteins in human breast carcinogenesis and indicate hMena overexpression as a surrogate indicator in breast disease management.     

Identification of HLA-A3-restricted CD8+ T cell epitopes derived from mammaglobin-A,a tumor-associated antigen of human breast cancer

Mammaglobin-A is highly overexpressed in breast cancer cell lines and primary breast tumors. This pattern of expression is restricted to mammary epithelium and metastatic breast tumors. Thus, mammaglobin-A-specific T cell immune responses may provide an important approach for the design of breast cancer-specific immunotherapy. The purpose of our study was to define the T cell-mediated immune response to mammaglobin-A. We determined that the frequency of mammaglobin-A-reactive CD8+ and CD4+ T cells in breast cancer patients is significantly higher than that observed in healthy female controls using limiting dilution analyses (p = 0.026 and p = 0.02, respectively). We identified 8 mammaglobin-A-derived 9-mer peptides with the highest binding affinity for the HLA-A3 molecule (Mam-A3.1-8) using a computer-assisted analysis of the mammaglobin-A protein sequence. Subsequently, we determined that CD8+ T cells from breast cancer patients reacted to peptides Mam-A3.1 (23-31, PLLENVISK), Mam-A3.3 (2-10, KLLMVLMLA), Mam-A3.4 (55-63, TTNAIDELK) and Mam-A3.8 (58-66, AIDELKECF) using an IFN-gamma enzyme-linked immunospot assay. A CD8+ T cell line generated in vitro against HLA-A*0301-transfected TAP-deficient T2 cells loaded with these peptides showed significant cytotoxic activity against the Mam-A3.1 peptide. This CD8+ T cell line showed a significant HLA-A3-restricted cytotoxic activity against mammaglobin-A-positive but not mammaglobin-A-negative breast cancer cells. In summary, our study identified four HLA-A3-restricted mammaglobin-A-derived epitopes naturally expressed by breast cancer cells, indicating the immunotherapeutic potential of this novel antigen for the treatment and prevention of breast cancer.     

Antibody and CD8+ T cell responses against HER2/neu required for tumor eradication after DNA immunization with a Flt-3 ligand fusion vaccine.

PURPOSE: HER2/neu is frequently overexpressed in breast cancer. In a mouse model, vaccination with HER2/neu DNA elicits antibodies that confer partial protection against tumor challenge. EXPERIMENTAL DESIGN: To enhance antitumor immunity, we fused cDNA encoding Flt-3 ligand (FL) to the rat HER2/neu extracellular domain (neu), generating a chimeric FLneu molecule. FLneu and neu DNA vaccines were compared for immunogenicity and their ability to protect mice from tumor challenge. RESULTS: The neu vaccine generated a HER2/neu-specific antibody response. In contrast, vaccination with FLneu induced CD8+ T cells specific for HER2/neu but a negligible anti-HER2/neu antibody response. The switch from an antibody-mediated to T cell-mediated response was due to different intracellular localization of neu and FLneu. Although the neu protein was secreted, the FLneu protein was retained inside the cell, co-localizing with the endoplasmic reticulum, facilitating processing and presentation to T cells. The neu and FLneu vaccines individually conferred only weak tumor immunity. However, efficient tumor rejection was seen when neu and FLneu were combined, inducing both strong anti-HER2/neu-specific antibody and T cell responses. Adoptive transfer of both immune CD8+ T cells and immune sera from immunized mice was required to confer tumor immunity in na?ve hosts. CONCLUSIONS: These results show that active induction of both humoral and cellular immunity to HER2/neu is required for efficient tumor protection, and that neither response alone is sufficient.     

Overexpression of human ortholog of mammalian enabled (hMena) is associated with the expression of mutant p53 protein in human breast cancers

The human ortholog of mammalian enabled (hMena), a member of the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family, is an actin regulatory protein involved in the regulation of cell motility. Increasing evidence suggests that hMena overexpression is involved in human cancers, but the upstream events that influence the expression of hMena remain to be elucidated. In this study, we performed immunohistochemical analysis of the expression of hMena protein in paraffin-embedded archival tissues of infiltrating ductal carcinomas (IDCs) obtained from 52 cases. We found that elevated hMena expression is associated with larger tumor size (>2.5 cm, p<0.01), HER2 expression (p<0.05), p53 index (p<0.03) and Ki67 index (p<0.01), suggesting that hMena is a predictor of poor prognosis in IDCs. The histological characteristics of each specimen showed that hMena was overexpressed in the tumor cells at the invasive front of IDCs, indicating that hMena expression is at least partly mediated by tumor cell-matrix interactions. To explore the role of the absence of p53 function in hMena overexpression of IDCs, wild-type p53 cDNA was introduced into SW620 cells, which originally express mutant p53. In wild-type p53-transfected cells, hMena mRNA expression was decreased to 70% of the levels in mock transfected cells (p<0.01). In conclusion, our study indicates that hMena overexpression is involved in the progression of IDCs, and raises the possibility that wild-type p53 may suppress hMena expression.     

Identification of a gene coding for a protein possessing shared tumor epitopes capable of inducing HLA-A24-restricted cytotoxic T lymphocytes in cancer patients.

Genes encoding tumor epitopes that are capable of inducing CTLs against adenocarcinomas and squamous cell carcinomas, two major human cancers histologically observed in various organs, have rarely been identified. Here, we report a new gene from cDNA of esophageal cancer cells that encodes a shared tumor antigen recognized by HLA-A2402-restricted and tumor-specific CTLs. The sequence of this gene is almost identical to that of the KIAA0156 gene, which has been registered in GenBank with an unknown function. This gene encodes a Mr 140,000 protein that is expressed in the nucleus of all of the malignant tumor cell lines tested and the majority of cancer tissues with various histologies, including squamous cell carcinomas, adenocarcinomas, melanomas, and leukemia cells. However, this protein was undetectable in the nucleus of any cell lines of nonmalignant cells or normal tissues, except for the testis. Furthermore, this protein was expressed in the cytosol of all of the proliferating cells, including normal cells and malignant cells, but not in normal tissues, except for the testis and fetal liver. Two peptides of this protein were recognized by HLA-A2402-restricted CTLs and were able to induce HLA-A24-restricted and tumor-specific CTLs from peripheral blood mononuclear cells of most of HLA-A24+ cancer patients tested, but not from peripheral blood mononuclear cells of any healthy donors. These peptides may be useful in specific immunotherapy for HLA-A24+ cancer patients with various histological types.     

CD4+ T helper responses in squamous cell carcinoma of the head and neck

Anti-tumor immunity plays an important role in the development of and protection from malignancy. However, there is a lack of information regarding induction of CD4+ T helper responses in patients with squamous cell carcinoma (SCCHN). To explore anti-tumor immune responses against SCCHN, a permanent cell line, Gun-1 was established from a squamous cell carcinoma of the hypopharynx. In addition to its characterization, we performed mixed lymphocyte-tumor cell cultures (MLTC) using peripheral blood lymphocytes and autologous tumor cells. Furthermore, T cell responses to wild type (wt) p53-derived peptides were assessed. Gun-1 cells overexpressed p53 and were negative for HLA-A2 expression. No tumor-specific or wt p53-specific CD8+ CTL lines could be established from peripheral blood mononuclear cells (PBMCs) of this patient. Autologous tumor-specific HLA-DR-restricted CD4+ T helper clone was obtained by limiting dilutions using bulk populations from MLTC. This clone produced IFN-gamma but not IL-5 in response to autologous tumor cells. In addition, CD4+ T cells were generated from the patient's PBMCs which responded to two HLA-DP5-restricted wt p53-derived peptides. Our results suggest that the immune cells specific for autologous tumor as well as wt p53-derived epitopes are present in the peripheral circulation of this cancer patient. However, helper-type CD4+ T lymphocytes represent the predominant anti-tumor response.     

Human Mena+11a isoform serves as a marker of epithelial phenotype and sensitivity to epidermal growth factor receptor inhibition in human pancreatic cancer cell lines

PURPOSE: hMena, member of the enabled/vasodilator-stimulated phosphoprotein family, is a cytoskeletal protein that is involved in the regulation of cell motility and adhesion. The aim of this study was to determine whether or not the expression of hMena isoforms correlated with sensitivity to EGFR tyrosine kinase inhibitors and could serve as markers with potential clinical use. EXPERIMENTAL DESIGN: Human pancreatic ductal adenocarcinoma cell lines were characterized for in vitro sensitivity to erlotinib, expression of HER family receptors, markers of epithelial to mesenchymal transition, and expression of hMena and its isoform hMena(+11a). The effects of epidermal growth factor (EGF) and erlotinib on hMena expression as well as the effect of hMena knockdown on cell proliferation were also evaluated. RESULTS: hMena was detected in all of the pancreatic tumor cell lines tested as well as in the majority of the human tumor samples [primary (92%) and metastatic (86%)]. Intriguingly, in vitro hMena(+11a) isoform was specifically associated with an epithelial phenotype, EGFR dependency, and sensitivity to erlotinib. In epithelial BxPC3 cells, epidermal growth factor up-regulated hMena/hMena(+11a) and erlotinib down-regulated expression. hMena knockdown reduced cell proliferation and mitogen-activated protein kinase and AKT activation in BxPC3 cells, and promoted the growth inhibitory effects of erlotinib. CONCLUSIONS: Collectively, our data indicate that the hMena(+11a) isoform is associated with an epithelial phenotype and identifies EGFR-dependent cell lines that are sensitive to the EGFR inhibitor erlotinib. The availability of anti-hMena(+11a)-specific probes may offer a new tool in pancreatic cancer management if these results can be verified prospectively in cancer patients.     

Cancer immunotherapy in head and neck region

There is no one common immunotherapy for the treatment of head and neck cancer (H&N cancer). A streptococcal agent, OK-432, which is classified as a biological response modifier (BRM), is occasionally used by means of local administration for recurrent H&N cancer, and the response rate is approximately 18%. In regard to specific immunotherapy, a murine monoclonal antibody (named mAb 225) against the epidermal growth factor receptor (FGFR) that is frequently overexpressed in H&N cancer has been produced in the U.S.A. Furthermore, to obviate human anti-mouse antibody responses, a chimeric human-to-murine version of mAb 225 (C225) was developed by exchanging the constant regions of mAb 225 to counterparts in human immune globulin. Phase I clinical trials of C225 in the U.S.A. demonstrated that treatment with C225 was well tolerated and that C225 given in combination with cisplatin has biologic activity. On the other hand, many tumor antigens recognized by cytotoxic T lymphocytes (CTL) have been identified from a variety of malignant tumors and some of them, including the MAGE-3 antigen, are frequently expressed in H&N cancer. We identified an MAGE-3-derived epitope recognized by HLA-A24-restricted CTL from peripheral blood mononuclear cells (PBMC). In contrast we failed to generate CTL specific for MAGE-3+/HLA-A24+ tumors from PBMC in any of 5 HLA-A24+ cancer patients whose tumors expressed the MAGE-3 gene. Therefore, we did not apply MAGE-3-derived CTL epitope in clinical uses such as peptide vaccine and peptide pulsed dendritic cell infusion for H&N cancer.     

Recognition of an epitope of a breast cancer antigen by human antibody

A novel tumor-associated peptide epitope KASIFLK expressed preferentially by breast cancer cells was identified using an IgG antibody from a breast cancer patient. A cDNA library from a MCF-7 breast cancer cell line was screened to isolate three cDNA clones that were immunoreactive with this antibody. KASIFLK was located in clones 27 and 40, both of which were identical to the cDNA and protein sequence of retinoblastoma binding protein 1 (RBP1, 250–256). An affinity-purified IgG antibody against the peptide epitope was completely absorbed by cytoplasmic extracts of MCF-7 cells. Immunohistochemical staining using this antibody revealed the antigen in MCF-7 cells and in 12 of 15 primary breast cancer tissues and 3 of 34 other cancer tissues, but in none of 6 normal breast tissues. Anti-KASIFLK antibody titers were significantly higher in sera of 55 breast cancer patients than in sera from 30 normal healthy donors (P > 0.001). These results suggest that KASIFLK or its cross-reactive epitope is a breast cancer antigen and is immunogenic in humans.     

More efficient induction of HLA-A*0201-restricted and carcinoembryonic antigen (CEA)-specific CTL response by immunization with exosomes prepared from heat-stressed CEA-positive tumor cells.

PURPOSE: Tumor-derived exosomes are proposed as a new type of cancer vaccine. Heat shock proteins are potent Th1 adjuvant, and heat stress can induce heat shock protein and MHC-I expression in tumor cells, leading to the increased immunogenicity of tumor cells. To improve the immunogenicity of exosomes as cancer vaccine, we prepared exosomes from heat-stressed carcinoembryonic antigen (CEA)-positive tumor cells (CEA+/HS-Exo) and tested the efficacy of these exosomes in the induction of CEA-specific antitumor immunity. EXPERIMENTAL DESIGN: First, we identified the composition of CEA+/HS-Exo and observed their effects on human dendritic cell maturation. Then, we evaluated their ability to induce a CEA-specific immune response in vivo in HLA-A2.1/Kb transgenic mice and CEA-specific CTL response in vitro in HLA-A*0201+ healthy donors and HLA-A*0201+CEA+ cancer patients. RESULTS: CEA+/HS-Exo contained CEA and more heat shock protein 70 and MHC-I and significantly induced dendritic cell maturation. Immunization of HLA-A2.1/Kb transgenic mice with CEA+/HS-Exo was more efficient in priming a CEA-specific CTL, and the CTL showed antitumor effect when adoptively transferred to SW480-bearing nude mice. Moreover, in vitro incubation of lymphocytes from HLA-A*0201+ healthy donors and HLA-A*0201+CEA+ cancer patients with CEA+/HS-Exo-pulsed autologous dendritic cells induces HLA-A*0201-restricted and CEA-specific CTL response. CONCLUSIONS: Our results show that CEA+/HS-Exo has superior immunogenicity than CEA+/Exo in inducing CEA-specific CTL response and suggest that exosomes derived from heat-stressed tumor cells may be used as efficient vaccine for cancer immunotherapy.     

Immune infiltrates in the breast cancer microenvironment: detection,characterization and clinical implication

Although unlike melanoma, breast cancer is not generally viewed as a highly immunogenic cancer, recent studies have described a rich tumor immune microenvironment in a subset of breast cancers. These immune infiltrates, comprised cells from the innate and adaptive immune response, can be detected and characterized in biopsy specimens and have prognostic value. Tumor-infiltrating lymphocytes (TILs) represent the majority of mononuclear immune infiltrates in the breast tumor microenvironment and can be easily identified in formalin-fixed paraffin-embedded tissues after standard hematoxylin and eosin staining. High levels of TILs are most common in HER2+ and basal-like subtypes where they are associated with good prognosis and with response to certain therapies such as the anti-HER2 antibody trastuzumab. International collaborative efforts are underway to standardize the assessment of TILs so as to facilitate their implementation as a breast cancer biomarker. Using immunohistochemistry to further characterize TILs, recent reports describe the presence of important lymphocyte populations including CD8+ cytotoxic, FOXP3+ regulatory, and CD4+ helper and follicular T cells which have overlapping associations with prognosis and response to therapies. Moreover, recently identified immune checkpoint markers (PD-1, PD-L1) are present in some breast cancers, implying some cases might be especially amenable to immune checkpoint inhibitor treatment strategies which are being evaluated in a number of active clinical trials.     

Identification of a novel tumor-associated antigen, cadherin 3/P-cadherin, as a possible target for immunotherapy of pancreatic, gastric, and colorectal cancers

PURPOSE: To establish cancer immunotherapy, it is important to identify the tumor-associated antigens (TAA) that are strongly expressed in the tumor cells but not in the normal cells. In this study, to establish an effective anticancer immunotherapy, we tried to identify the useful TAA of pancreatic cancer. EXPERIMENTAL DESIGN: Based on a previous genome-wide cDNA microarray analysis of pancreatic cancer, we focused on cadherin 3 (CDH3)/P-cadherin as a novel candidate TAA for anticancer immunotherapy. To identify the HLA-A2 (A*0201)-restricted CTL epitopes of CDH3, we used HLA-A2.1 (HHD) transgenic mice (Tgm). Furthermore, we examined the cytotoxicity against the tumor cells in vitro and in vivo of CTLs specific to CDH3 induced from HLA-A2-positive healthy donors and cancer patients. RESULTS: CDH3 was overexpressed in the majority of pancreatic cancer and various other malignancies, including gastric and colorectal cancers, but not in their noncancerous counterparts or in many normal adult tissues. In the experiment using HLA-A2.1 Tgm, we found that the CDH3-4(655-663) (FILPVLGAV) and CDH3-7(757-765) (FIIENLKAA) peptides could induce HLA-A2-restricted CTLs in Tgm. In addition, peptides-reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA-A2-positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both CDH3 and HLA-A2. Furthermore, the adoptive transfer of the CDH3-specific CTLs could inhibit the tumor growth of human cancer cells engrafted into nonobese diabetic/severe combined immunodeficiency mice. CONCLUSIONS: These results suggest that CDH3 is a novel TAA useful for immunotherapy against a broad spectrum of cancers, including pancreatic cancer.     

Recognition of breast cancer cells by CD8+ cytotoxic T-cell clones specific for NY-BR-1

Immunotherapy for breast cancer using cytotoxic T cells (CTL) is hindered by the lack of well-characterized breast cancer antigens that are expressed in most breast tumor cells and recognized by CD8+ CTL. A recently described breast tissue differentiation antigen, NY-BR-1, is expressed in >80% breast tumors and elicits a humoral response in a subset of breast cancer patients. To identify potential NY-BR-1 epitopes that are recognized by CTL, CD8+ T cells were stimulated in vitro with autologous dendritic cells pulsed with NY-BR-1 peptides that were predicted to bind to HLA-A2. In multiple normal female donors and breast cancer patients, specific CD8+ CTL responses were detected by enzyme-linked immunospot assay against several NY-BR-1 peptides after two cycles of stimulation. CD8+ CTL clones against three NY-BR-1 epitopes were isolated and recognized peptide-pulsed target cells with high avidity. T-cell clones specific for one of the NY-BR-1 epitopes (p904) also recognized breast tumor cells expressing NY-BR-1, NY-BR-1(-) cells transfected with a cDNA encoding the NY-BR-1 protein, and autologous dendritic cells pulsed with opsonized NY-BR-1+ breast tumor cells. Taken together, these results show that the p904 epitope derived from NY-BR-1 is efficiently processed and presented endogenously and identify NY-BR-1 as a promising target for T-cell-based immunotherapy for breast cancer.     

Combined clinical trial results of a HER2/neu (E75) vaccine for the prevention of recurrence in high-risk breast cancer patients: U.S. Military Cancer Institute Clinical Trials Group Study I-01 and I-02

PURPOSE: E75 is an immunogenic peptide from the HER2/neu protein, which is overexpressed in many breast cancer patients. We have conducted two overlapping E75 vaccine trials to prevent recurrence in node-positive (NP) and node-negative (NN) breast cancer patients. EXPERIMENTAL DESIGN: E75 (HER2/neu 369-377) + granulocyte macrophage colony-stimulating factor was given intradermally to previously treated, disease-free NP breast cancer patients in a dose escalation safety trial and to NN breast cancer patients in a dose optimization study. Local and systemic toxicity was monitored. Immunologic responses were assessed using in vitro assays and in vivo delayed-type hypersensitivity responses. Clinical recurrences were documented. RESULTS: One hundred and eighty-six patients were enrolled in the two studies (NP, 95; NN, 91). Human leucocyte antigen A2 (HLA-A2) and HLA-A3 patients were vaccinated (n = 101), whereas all others (n = 85) were followed prospectively as controls. Toxicities were minimal, and a dose-dependent immunologic response to the vaccine was shown. Planned primary analysis revealed a recurrence rate of 5.6% in vaccinated patients compared with 14.2% in the controls (P = 0.04) at a median of 20 months follow-up. As vaccine-specific immunity waned over time, the difference in recurrence lost significance at 26 months median follow-up (8.3% versus 14.8%); however, a significant difference in the pattern of recurrence persisted. CONCLUSIONS: E75 is safe and effective in raising a dose-dependent HER2/neu immunity in HLA-A2 and HLA-A3 NP and NN breast cancer patients. More importantly, E75 may reduce recurrences in disease-free, conventionally treated, high-risk breast cancer patients. These findings warrant a prospective, randomized phase III trial of the E75 vaccine with periodic booster to prevent breast cancer recurrences.     

Parathyroid hormone-related protein as a common target molecule in specific immunotherapy for a wide variety of tumor types

Parathyroid hormone-related protein (PTH-rP) has been considered to be responsible for malignancy-associated hypercalcemia and is thought to participate in pathological changes in bone metastases of cancer. In this study, we determined whether or not PTH-rP could be a common target molecule in specific immunotherapy for patients with a wide variety of tumor types. Various types of tumor cell lines were examined for PTH-rP expression at the mRNA and protein levels by RT-PCR, flow cytometry, and immunocytochemistry. We also determined whether or not cancer-reactive cytotoxic T lymphocytes (CTLs) could be induced from the peripheral blood mononuclear cells (PBMCs) of HLA-A24+ patients with gastric, colon, renal, or cervical cancer by in vitro stimulation with two PTH-rP peptides. As a result, PTH-rP mRNA was expressed in the majority of gastric, breast, lung, colon, cervical, and renal cancer cell lines. Expression of the protein was confirmed by both flow cytometry and immunocytochemistry. Furthermore, PTH-rP peptide-specific and cancer-reactive CTLs were successfully generated from the PBMCs of HLA-A24+ patients with different tumor types using in vitro stimulation with either the PTH-rP(102-111) or PTH-rP(110-119) peptide. These findings indicate that PTH-rP could be a common target molecule in specific immunotherapy for patients with a wide variety of tumor types, particularly bone metastases.     

Dendritic cells transduced with full-length wild-type p53 generate antitumor cytotoxic T lymphocytes from peripheral blood of cancer patients.

Accumulation of wild-type or mutant p53 protein occurs in approximately 50% of human malignancies. This overexpression may generate antigenic epitopes recognized by CTLs. Because normal cells have undetectable levels of p53, these CTLs are likely to be tumor specific. Here, for the first time, we test the hypothesis that full-length wild-type p53 protein can be used for generation of an immune response against tumor cells with p53 overexpression. T cells obtained from nine HLA-A2-positive cancer patients and three HLA-A2-positive healthy individuals were stimulated twice with dendritic cells (DCs) transduced with an adenovirus wild-type p53 (Ad-p53) construct. Significant cytotoxicity was detected against HLA-A2-positive tumor cells with accumulation of mutant or wild-type p53 but not against HLA-A2-positive tumor cells with normal (undetectable) levels of p53 or against HLA-A2-negative tumor cells. This response was specific and mediated by CD8+ CTLs. These CTLs recognized HLA-A2-positive tumor cells expressing normal levels of p53 protein after their transduction with Ad-p53 but not with control adenovirus. Stimulation of T cells with Ad-p53-transduced DCs resulted in generation of CTLs specific for p53-derived peptide. These data demonstrate that DCs transduced with the wild-type p53 gene were able to induce a specific antitumor immune response. This offers a new promising approach to immunotherapy of cancer.     

Blocking of the response by human T-lymphocytes to extracts of autologous cancer by monoclonal antibody to Class-I major histocompatibility complex gene products in the leukocyte adherence inhibition assay

In the leukocyte adherence inhibition (LAI) assay, about 34% of adherent T-cells from patients with breast cancer exhibit nonadherence to glass when incubated with extracts of autologous cancer but not with HLA-A, -B, and -C mismatched extracts of breast cancer. To determine whether the recognition by T-cells of tumor antigen was major histocompatibility complex restricted, major histocompatibility complex antigens in the cancer extracts or on the T-cells were coated with monoclonal antibody (MAb) to nonpolymorphic determinants. Nonadherence of T-cells was antagonized by coating the target cancer extracts with MAb to a common framework determinant of Class I HLA-A, -B, and -C antigens or to the nonpolymorphic beta 2-microglobulin, which is noncovalently associated with Class I antigens. By contrast, a MAb to a monomorphic determinant on HLA-DR antigens did not change the positive T-cell response. Moreover, coating the T-cells with MAb to HLA-A, -B, and -C did not inhibit the positive T-cell response. The positive LAI response of buffy coat peripheral blood leukocytes from patients with breast cancer to extracts of allogeneic breast cancer was not affected by coating the cancer extracts with the same MAb, indicating that MAb inhibited T-cell LAI specifically and that the antibody-dependent LAI response of buffy coat peripheral blood leukocytes was not major histocompatibility complex restricted. The results indicate that HLA-A, -B, and -C antigens in extracts of autologous breast cancer restrict the LAI response to tumor antigens of T-cells from patients with breast cancer.