Breast Cancer-associated Antigen Research Articles

Abstract 4110: Identification of PD1 B cell mimotopes with functional PD1-PDL1 blocking capacity: New strategy for cancer immunotherapy

Abstract Immune checkpoint (ICP) blockade has become a major focus in cancer immunotherapy, and the application of monoclonal antibodies (mAbs) as ICP inhibitors shows impressive results in the therapy against cancers like melanoma, renal cell carcinoma or non-small cell lung cancer. Active immunization with B cell epitopes/mimotopes of ICP inhibitors rather than application of the corresponding mAbs for passive immunotherapy may, however, provide advantages such as induction of antibodies by the patient’s own immune system and overcoming the costly treatment of the mAbs. Applying a platform for bacterial surface-display of overlapping peptides spanning the extracellular domains of human PD1 (hPD1) and mouse PD1 (mPD1), as well as in vitro solid phase-based ELISA assay and cellular-based assays involving human Jurkat T cells expressing human/mouse PD1 and/or PDL1, B cell epitopes/mimotopes of Nivolumab and an anti-mPD1 mAb (with a blocking capacity) were identified. The mimotopes were not only shown to inhibit the binding of the corresponding mAbs but also to revert the PD1-PDL1 blocking inhibitory capacity of the respective mAbs. More importantly, to proof the antitumor activity of antibodies against PD1 mimotopes in vivo, rabbits were immunized with the mimotope of anti-mPD1 mAb to generate mimotope-specific IgGs. The specific IgGs not only inhibited the interaction of mPD1 and mPDL1 in vitro, but also significantly reduced tumor growth in a syngeneic model of mice grafted with cells expressing the breast cancer-associated tumor antigen Her-2/neu. Our results show systematic identification and characterization of mimotopes of anti-PD1 ICP inhibitor B cell mimotopes conferring in vivo anti-tumor activity, and also indicate the potential of such mimotopes for active immunization in cancer therapy. Based on the results, enhancement of our second generation of anti-Her-2/neu vaccine (HerVAXX; Tobias et al, 2017*) by combining with mimotopes of ICP inhibitors is also proposed, which may potentially pave the way to a paradigm change in active immunotherapy against Her-2/neu overexpressing cancers using an effective multi-level vaccine. The study is granted by Imugene Limited, Australia, and Medical University of Vienna. * Tobias J. et al. BMC Cancer, 2017, 17:118. Citation Format: Joshua Tobias, Claire Battin, Annika De Sousa Linhares, Baier Karin, Katharina AMbroye, Mirjana Drinic, Erika Garner-Spitzer, Christoph Zielinski, Michael Kundi, Peter Steinberger, Ursula Wiedermann. Identification of PD1 B cell mimotopes with functional PD1-PDL1 blocking capacity: New strategy for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4110.

Gold nanoprisms as a hybrid in vivo cancer theranostic platform for in situ photoacoustic imaging, angiography, and localized hyperthermia

The development of high-resolution nanosized photoacoustic contrast agents is an exciting yet challenging technological advance. Herein, antibody (breast cancer-associated antigen 1 (Brcaa1) monoclonal antibody)- and peptide (RGD)-functionalized gold nanoprisms (AuNprs) were used as a combinatorial methodology for in situ photoacoustic imaging, angiography, and localized hyperthermia using orthotopic and subcutaneous murine gastric carcinoma models. RGD-conjugated PEGylated AuNprs are available for tumor angiography, and Brcaa1 monoclonal antibody-conjugated PEGylated AuNprs are used for targeting and for in situ imaging of gastric carcinoma in orthotopic tumor models. In situ photoacoustic imaging allowed for anatomical and functional imaging at the tumor site. In vivo tumor angiography imaging showed enhancement of the photoacoustic signal in a time-dependent manner. Furthermore, photoacoustic imaging demonstrated that tumor vessels were clearly damaged after localized hyperthermia. This is the first proof-of-concept using two AuNprs probes as highly sensitive contrasts and therapeutic agents for in situ tumor detection and inhibition. These smart antibody/peptide AuNprs can be used as an efficient nanotheranostic platform for in vivo tumor detection with high sensitivity, as well as for tumor targeting therapy, which, with a single-dose injection, results in tumor size reduction and increases mice survival after localized hyperthermia treatment.

Abstract 1987: Molecular characterization of the breast cancer-associated antigen NY-BR-1

Abstract Breast cancer is one of the most common malignancies with increasing incidence every year and a leading cause of death among women. Although early stage breast cancer can be effectively treated, there are limited numbers of treatment options available for patients with advanced and metastatic disease. The breast cancer associated antigen NY-BR-1 was identified by a serological screening strategy (SEREX). NY-BR-1 is expressed in the majority (>70%) of breast tumors as well as metastases, in normal breast tissue, in testis, and occasionally in prostate tissue. NY-BR-1 was confirmed as an immunogenic antigen as we could detect NY-BR-1 specific spontaneous humoral and cellular immune responses in several breast cancer patients. However, biological function, regulatory mechanisms, and interaction partners of NY-BR-1 are still unknown. Therefore, we combined integrative functional analysis (genomics, transcriptomics and epigenetics) and experiments to unravel the transcriptional regulation of NY-BR-1 and to detect its function within the mammary gland. As numerous binding sites for nuclear hormone receptors (e.g. Estrogen Receptor alpha, Progesterone receptor and Glucocorticoid receptor) and CpG islands were predicted within the promoter sequence of NY-BR-1, primary-tissues from breast reductions or tumor cells obtained from pleural effusions were treated with different hormones (Estradiol, Dexamethasone, Progesterone, Retinoic acid, Vitamin D) or with the HDAC inhibitors Valproic acid, Trichostatin A and the demethylating agent 5´Aza-Deoxycytidine in order to analyze the transcriptional regulation of NY-BR-1. Additionally, we analyzed the methylation status at various sites within the promoter region of NY-BR-1 using EpiTyper MassARRAY technology and publicly available resources such as the TCGA database. Here, we present preliminary results of our analysis and evaluate their implication for further investigation of NY-BR-1. Citation Format: Julia Bitzer, Zeynep Kosaloglu, Niels Halama, Claudia Ziegelmeier, Tina Lerchl, Anna Spille, Maria Pudenz, Eva Koellensperger, Stefan Eichmueller, Wolfram Osen, Andreas Schneeweiss, Frederik Marmé, Inka Zoernig, Dirk Jaeger. Molecular characterization of the breast cancer-associated antigen NY-BR-1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1987. doi:10.1158/1538-7445.AM2015-1987

Recombinant Mammaglobin A Adenovirus-Infected Dendritic Cells Induce Mammaglobin A-Specific CD8+ Cytotoxic T Lymphocytes against Breast Cancer Cells In Vitro

Mammaglobin A (MGBA) is a novel breast cancer-associated antigen almost exclusively over-expressed in primary and metastatic human breast cancers, making it a potential therapeutic target for breast cancer. The development of dendritic cell (DC)-induced tumor antigen specific CD8+ cytotoxic T lymphocytes (CTLs) may hold promise in cancer immunotherapy. In this study we constructed recombinant replication-defective adenoviral (Ad) vectors encoding MGBA and evaluated their ability to trigger anti-tumor immunity in vitro. DCs were isolated from the human peripheral blood monocyte cells (PBMCs) of two HLA-A33+ healthy female volunteers, and infected with adenovirus carrying MGBA cDNA (Ad-MGBA). After that, the Ad-MGBA-infected DCs were used to stimulate CD8+ CTLs in vitro and the latter was used for co-culture with breast cancer cell lines. The data revealed that infection with Ad-MGBA improved DC maturation and up-regulated the expression of co-stimulatory molecules and the secretion of interleukin-12 (IL-12), but down-regulated interleukin-10 (IL-10) secretion from DCs. Ad-MGBA-infected DC-stimulated CD8+CTLs displayed the highest cytotoxicity towards HLA-A33+/MGBA+ breast cancer MDA-MB-415 cells compared with other CD8+CTL populations, and compared with the cytotoxicity towards HLA-A33−/MGBA+ breast cancer HBL-100 cells and HLA-A33−/MGBA− breast cancer MDA-MB 231 cells. In addition, Ad-MGBA-infected DC-stimulated CD8+ CTLs showed a high level of IFNγ secretion when stimulated with HLA-A33+/MGBA+ breast cancer MDA-MB-415 cells, but not when stimulated with HLA-A33−/MGBA+ HBL-100 and HLA-A33−/MGBA−MDA-MB-231 cells. In addition, killing of CD8+CTLs against breast cancer was in a major histocompability complex (MHC)-limited pattern. Finally, the data also determined the importance of TNF-α in activating DCs and T cells. These data together suggest that MGBA recombinant adenovirus-infected DCs could induce specific anti-tumor immunity against MGBA+ breast cancers, which could provide a novel strategy in the immunotherapy of breast cancer.

Abstract 1259: Murine HLA-restricted CD4+ T cell lines as source of high affinity TCRs specific for the human breast cancer-associated tumor antigen NY-BR-1.

Abstract Out of all malignancies, breast cancer is the second most common cancer worldwide and the leading cause of cancer death in females. Passive immunization approaches such as adoptive transfer of TCR-transduced autologous T cells specific for tumor associated antigens, provide an innovative strategy for cancer immunotherapy. The differentiation antigen NY-BR-1 has been described to be expressed in 60% of all invasive mammary carcinomas. Since NY-BR-1 protein levels are highly elevated in malignant breast tissues compared to healthy breast tissue, NY-BR-1 might represent a suitable target antigen for T cell based immunotherapy approaches against breast cancer. Successful immunological eradication of tumors depends on the presence of activated tumor antigen-specific CD4+ effector T cells as documented by numerous reports. Therefore, NY-BR-1 was screened for the presence of CD4+ T cell epitopes using HLA-transgenic mice immunized with a NY-BR-1-encoding expression plasmid and subsequent ex vivo analysis of the NY-BR-1-specific T cell responses against a synthetic peptide library covering the entire NY-BR-1 protein. Stable CD4+ T cell lines specific for six newly identified epitopes could be established from peptide-immunized DRB1*0301 and HLA-DRB1*0401-transgenic mice and HLA-DR-restriction of the cell lines was confirmed on peptide loaded T2/DR3 and T2/DR4 target cells. Next, natural processing of the epitopes identified in HLA transgenic mice will be tested in the human system using human HLA-matched cells lines infected with NY-BR-1 encoding adenovirus as targets for recognition by the murine NY-BR-1-specific CD4+ T cell lines. Clinical relevance of the newly identified NY-BR-1-specific epitopes will be investigated by screening of blood samples from NY-BR-1+, HLA-matched breast cancer patients for the presence of cognate CD4+ T cells. High affinity TCRs from NY-BR-1 specific CD4+ T cells will be sequenced and subsequently used to generate autologous TCR-transduced T cell lines ready for adoptive transfer of breast cancer patients Citation Format: Adriane Gardyan, Wolfram Osen, Stefan B. Eichmüller, Inka Zoernig, Dirk Jaeger. Murine HLA-restricted CD4+ T cell lines as source of high affinity TCRs specific for the human breast cancer-associated tumor antigen NY-BR-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1259. doi:10.1158/1538-7445.AM2013-1259

Abstract 1581: Identification of novel HLA-restricted T-cell epitopes specific for the breast cancer-associated tumor antigen NY-BR-1

Abstract Out of all malignancies, breast cancer resembles the most common malignant caner type (26%) of women in the western world and the second most frequent cancer related cause of death (15%) in women. Efficient treatment options are available for early stage breast cancers yielding five year survival rates of up to 85%, however for metastasized breast cancer types, five year survival rates are resided at much lower levels (20%) and therapy options are very limited. It is therefore of great importance to establish innovative therapy strategies which will affect the late stage breast cancer tumors. Immunotherapy approaches, particularly adoptive T cell transfer, constitute an attractive strategy to develop new therapy options for metastasized mammary carcinomas. The differentiation antigen NY-BR-1 was found to be expressed on mRNA level in breast tissue, testis, prostate, and most notably in 80% of tested breast cancers. Among normal tissues, expression of the NY-BR-1 protein was present solely in ductal epithelium of the breast. Compared to healthy breast tissue NY-BR-1 protein levels are highly elevated in invasive breast tumors and their corresponding metastasis. Thus, NY-BR-1 can be considered as a breast cancer associated tumor antigen that might represent a suitable target antigen for T cell based immunotherapy approaches. The main focus of this project is to identify novel HLA-restricted T cell epitopes that could be used in clinical trials. Therefore, various HLA-transgenic (tg) mouse strains (DR4tg, DR3tg and HHDtg mice), were immunized with the NY-BR-1- encoding expression plasmid pcDNA3.1-NY-BR-1 followed by ex vivo analyses of the NY-BR-1-specific T cell responses with a synthetic peptide library covering the entire NY-BR-1 protein. Applying this strategy, four potential HLA-DRB1*0301 restricted epitopes and three HLA-DRB*0401 restricted candidate epitopes could be determined. The relevance of these new epitopes for the human system will be analyzed by screening of PBMCs from tumor patients and healthy donors for the presence of cognate T cells. Such T cells will be expanded and used as donors for high affinity NY-BR-1-specific TCRs in order to establish autologous TCR-transduced T cells ready for adoptive transfer of breast cancer patients. Currently, blood-samples of NY-BR-1+ patients are being collected for subsequent in vitro analyses of HLA-restricted, NY-BR-1-specific T cell responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1581. doi:1538-7445.AM2012-1581

Identification of HLA-A24-restricted CD8+ cytotoxic T-cell epitopes derived from mammaglobin-A, a human breast cancer–associated antigen

Human breast cancer-associated antigen, mammaglobin-A (Mam-A), potentially offers a novel therapeutic target as a breast cancer vaccine. In this study, we define the CD8(+) cytotoxic T lymphocyte (CTL) response to Mam-A-derived candidate epitopes presented in the context of HLA-A24 (A*2402). HLA-A24 has a frequency of 72% in Japanese, 27% in Asian Indian, and 18% in Caucasian populations. Using a human leukocyte antigen (HLA)-binding prediction algorithm we identified 7 HLA-A24-restricted Mam-A-derived candidate epitopes (MAA24.1-7). Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-A2402 (T2.A24) indicated that MAA24.2 (CYAGSGCPL) and MAA24.4 (ETLSNVEVF) have the highest HLA-A24 binding affinity. Further, 2 CD8(+) CTL cell lines generated in vitro against T2.A24 cells individually loaded with Mam-A-derived candidate epitopes demonstrated significant cytotoxic activity against MAA24.2 and MAA24.4. In addition, the same CD8(+) CTL lines lysed the HLA-A24(+)/Mam-A(+) stable transfected human breast cancer cell lines AU565 and MDA-MB-361. However, these CTLs had no cytotoxicity against HLA-A24(-)/Mam-A(+) and HLA-A24(+)/Mam-A(-) breast cancer cell lines. In summary, our results define HLA-A24-restricted, Mam-A-derived, CD8(+) CTL epitopes that can potentially be employed for Mam-A-based breast cancer vaccine therapy to breast cancer patients with HLA-A24 phenotype.

Abstract LB-154: Anti-tumor effect of dendritic cell-based immunotherapy in the patients with advanced breast cancer.

Abstract Background and objective: Tumor-specific cytotoxic T lymphocytes (CTLs) can be activated in vivo by dendritic cell (DC)-based vaccination. However, clinical responses to the immunotherapy with DC vaccination have only been observed in a minority of patients with solid cancer. In the current study, the clinical efficacy of the DC vaccine pulsed with the peptide derived from breast cancer-associated antigens has been evaluated in the patients with advanced, inoperable breast cancer. Patients and Methods: 21patients with advanced, inoperable breast cancer refractory to standard treatment were entered the study. DCs which were generated from CD14+ monocytes from leukapheresis by 6-day cultivation with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4, were matured by OK-432, a streptococcal agent, and were pulsed with the pancreatic cancer-associated antigens, such as WT1, MUC1. These DCs (1 × 107) were intradermally administered more than 5 times at 14-day intervals. Results: Of the 21 patients, complete response (CR) was observed in 0 (0%), partial response (PR) in 2 (9.6%), stable disease (SD) in 7 (33.3%), progressive disease (PD) in 12 (57.1%). Response rate was 9.6%. Tumor control rate was 42.9%. Mean survival time after DC therapy was 514days. Severe side effects of more than grade 3 which were assessed in accordance with NCI-Common Toxicity Criteria v.2.0, were not observed. Conclusions: It was strongly suggested that the DC vaccination pulsed with cancer associated-peptides was safety and effective in the patients with the inoperable breast cancer refractory to standard treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-154. doi:10.1158/1538-7445.AM2011-LB-154

Generation and Characterization of a High-affinity Monoclonal Antibody for MUC1 Measurement in Breast Cancer

Breast mucin is secreted by breast tumor cells and serves as a marker for breast cancer. Thus, antibodies against breast mucin will be valuable in the development of immunotherapy and laboratory diagnostic tests. Monoclonal antibodies (MAbs) against breast cancer-associated antigen were generated and characterized. Balb/c mice were immunized with breast cancer-associated antigen CA15-3, and subsequently splenocytes from immunized mice were fused with myeloma cells. After fusion, culture supernatants from hybridomas surviving HAT medium were screened by enzyme-linked immunosorbent assay (ELISA). A total of eight hybridomas producing MAbs against breast cancer showed significant levels of antibody activity against CA15-3. Two selected stable hybridomas were adapted into CELLine CL 350 bioreactors, and the MAbs produced were characterized for their subclass, specificity, and affinity. The MAbs were of high specificity and affinity as shown by ELISA. The MAbs produced may represent a powerful tool and are considered promising reagents for use in diagnosis and detection of early stage of the disease.

Abstract P1-11-16: Her2/neu Overexpressing Breast Cancer Patients Retain an Immunological Proficiency Compromised in Her2/neu-Negative Cases

Abstract Although breast cancers overexpressing HER2/neu are associated with a worse prognosis, a high rate of complete responses is usually induced in these patients by current therapeutic strategies, such as Trastuzumab, that also directly mediates antibody-dependent cellular cytotoxicity and possesses immunomodulating properties. Moreover, other chemotherapeutics used in this setting, i.e. doxorubicin and Taxol, were shown to enhance antigen-specific immune responses. On these grounds, a thorough evaluation of the immunologic profile of breast cancer patients may be helpful to predict clinical responses to such therapies. We therefore analyzed at diagnosis different immunological parameters of patients with locally advanced HER2+ (n=18) and HER2- (n=30) breast cancer eligible for neoadjuvant chemotherapy, respectively treated with weekly Paclitaxel (80 mg/mq/ev) and Trastuzumab (4mg/kg/ev day 1 followed by 2 mg/kg/ev weekly), or Epirubicin (90 mg/mq/ev) and Docetaxel (75mg/mq/ev) every 3 weeks. Immunophenotyping of circulating immune cells disclosed several differences in HER2- cases as compared with HER2+ patients and healthy controls (n=20). In particular, HER2- patients showed a higher percentage of NK and B cells (10.2 and 10.0%, respectively) and a lower CD4/CD8 T cell ratio (1.8; with a prevalence of naïve and central memory cells in the CD8+ population) if compared to HER2+ patients (NK 5.8%, B cells 8.2%, and CD4/CD8=2.3) and healthy controls (NK 5.5%, B cells 7.7%, and CD4/CD8=2.2). In addition, HER2- cases had higher numbers of circulating CD4+ regulatory T cells (1.6% FoxP3+CD25+CD127- of CD4+ T cells) with respect to donors (0.9%). Moreover, IFN-gamma ELISPOT assay demonstrated spontaneous T cell responses to a broad spectrum of HLA-A*0201-restricted peptides derived from breast cancer-associated antigens (HER2/neu, MUC-1, mammaglobin-A, Taxol resistance-associated gene-3, survivin, and Bcl-xL) in both groups of tumour patients, although the extent of these responses was usually higher in HER2+ cases. Finally, a multiparametric evaluation of the serum levels of 9 different cytokines revealed a comparable profile in HER2+ cases and in controls, whereas HER2- patients showed significantly lower cytokine amounts compared to healthy donors (IL-2, IL-6, and IL-8) and to HER2+ breast cancers (IL-6, IL-8, and IL-10). Preliminary data revealed a pathological complete response (pCR) in 44.4% of HER2+ patients (8/18), compared to 16.7% of the HER2- arm (5/30). These results are consistent with a retained immunologic proficiency of HER2+ patients, showing an innate tumor-specific immune response. Such features may at least partly account for the clinical benefit of drugs acting through, but also promoting, immunemediated effects. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-11-16.

Targeting the epidermal growth factor receptor (HER) family by T cell receptor gene-modified T lymphocytes

Human epidermal growth factor receptor 2 (HER2) has been successfully targeted as a breast cancer-associated antigen by various strategies. HER2 is also overexpressed in other solid tumors such as stomach cancer, as well as in hematological malignancies such as acute lymphoblastic leukemia. HER2-targeted therapies are currently under clinical investigation for a panel of malignancies. In this study, we isolated the T cell receptor (TCR) genes of a HER2-reactive allo-human leukocyte antigen-A2-restricted CTL clone and introduced the TCRα- and β-chain genes into the retrovirus vector MP71. Murinization and codon optimization of the HER2-reactive TCR was required for efficient TCR expression in primary human T cells. The tumor recognition efficiency of HER2-TCR gene-modified T cells was similar to the parental CTL clone from which the TCR genes were isolated. The known cross-reactivity of the HER2-reactive TCR with HER3 and HER4 was retained when the TCR was transduced into primary T cells. Our results could contribute to the development of a TCR-based approach for the treatment of HER2-positive breast cancer, as well as of other malignancies expressing HER2, HER3, and/or HER4.

Mam-A2.4 DNA vaccination leads to generation of effector phenotype CD8+ T cells and results in tumor regression. (131.1)

Abstract Mammaglobin-A (Mam-A) is a breast cancer-associated antigen expressed by approximately 80% of human breast tumors. Recently, seven epitopes derived from Mam-A were identified that bind HLA-A2 and are naturally presented by breast tumors, making these potential targets for immunotherapy. We show that epitope-specific, rather then full-length Mam-A DNA vaccination induces more effective tumor regression in vivo. Spleen cells from C57Bl/6 HLA-A2 mice, vaccinated with either full-length Mam-A DNA or epitope DNA (Mam-A2.1, Mam-A2.2, Mam-A2.4 and Mam-A2.6), were transferred into SCID-beige mice bearing tumors derived from the AU-565 (HLA-A2+/mammaglobin-A+) breast cancer cell line. Our results show that antigen-specific CD8+ T-cells derived from both full-length and epitope-specific DNA vaccination were initially capable of causing regression. However, only vaccination with Mam-A2.4 leads to long-term tumor regression, which can be further enhanced by low-dose total body irradiation of the tumor-bearing host. Furthermore, vaccination with Mam-A2.4 leads to the generation of CD8+ T-cells with an effector phenotype. These data demonstrate the potential of Mam-A single epitope DNA vaccination combined with low-dose total body irradiation as a therapy for the treatment and/or prevention of breast cancer.

A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen

BackgroundWe have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients.MethodsThe current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification.ResultsBy application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.ConclusionWe have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.

Redirection of primary T lymphocytes towards HER2 by T cell receptor gene transfer

3061 Background: The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated antigen by T cell-based immunotherapeutical strategies. Until now several vaccination studies against HER2 which is overexpressed in about 25% of breast cancer were performed but the objective response rate achieved was only low. The goal of our studies is the adoptive transfer of primary T cells transduced with a HER2-specific T cell receptor (TCR) for patients with HER2-overexpressing breast cancer. Methods: CD8+ T cells isolated from an HLA-A2- donor were stimulated with allogeneic HLA-A2+ dendritic cells (DC) pulsed with the peptide HER2369–377 known to be naturally presented with HLA-A2. HER2369–377 specific T cells were screened via ELISpot and FACS sorting, respectively, cloned and further tested in 51Chromium release assays (51CRA) and ELISA using the corresponding HER3 and HER4 peptides pulsed on as well as HER2-, HER3- and HER4 overexpressing cell lines. Retroviral vectors containing the TCRα or β chain of a HER2-, HER3- and HER4 reactive cytotoxic T cell (CTL) clone were constructed. Following virus production in HEK293 T cells CD8+ T cells were transduced with the retrovirus and tested in IFN-γ ELISA and 51CRA. Results: The CD8+ T cells transduced with the HER2-specific TCR recognized the same panel of HLA-A2-matched, HER2-overexpressing cell lines as the parental CTL clone. As the parental CTL clone, the TCR- transduced T cells were not only able to recognize HER2369–377 but also the corresponding peptides from HER3 and HER4 as determined by lytic activity and cytokine secretion. Conclusions: The fine specificity of a HER2-reactive TCR which also cross-reacts with the corresponding peptides from HER3 and HER4 is conserved following its transduction into CD8+ T cells. This is a promising result for the HER2-directed design of immunotherapies based on TCR-transduced T cells. Cross-recognition especially of HER3 may be beneficial as HER2 and HER3 overexpressing tumors are particularly aggressive. No significant financial relationships to disclose.

P83 Systematic validation of novel breast cancer progression-associated biomarkers via highthroughput antibody generation and application of tissue microarray technology: an initial report

A plethora of promising breast cancer-associated autoantigens have been cloned by immunoscreening cDNA expression libraries with breast cancer sera or identified using proteomics, yet no biomarkers, whether individual autoantigens or panels of antigens developed using antibody-based methods have been validated and incorporated to routine oncologic practice for the early diagnosis of breast cancer. Recently, the addition of genomics, proteomics and high throughput technology to traditional immunological techniques has revived the interest in this field, and some of the most promising breast cancer autoantigens are in the process of being validated prospectively in large cohorts of patients with breast cancer. In addition, some of the identified breast cancer-associated autoantigens are recognized by T-cells and may prove to have a role in the treatment of breast cancer in the future. Autoantibodies found in breast cancer patient sera provide important clues about their significance. The discovery of breast cancer-associated antigens has provocative implications beyond the quest for novel diagnostic biomarkers, because autoantibodies target molecules involved in signal transduction, cell cycle regulation, cell proliferation and apoptosis, all of them key processes in carcinogenesis. Molecular components of the DNA double-strand break repair machinery as well as several members of the rapamycin-sensitive pathway elicit an autoantibody response in breast cancer. Data obtained by screening cDNA expression libraries of breast cancer antigens with autoantibodies present in breast cancer sera suggest that autoantibodies in cancer sera may be linked to the process of apoptosis. The studies reviewed here, clearly demonstrate the participation of autoimmunity in breast cancer to an extent previously unsuspected, which may have broad implications for the discovery of molecular targets for drug therapy and cancer biomarkers in general.

Generation of mammaglobin-A-specific CD4 T cells and identification of candidate CD4 epitopes for breast cancer vaccine strategies

Mammaglobin-A (MGB) is a breast cancer-associated antigen that is an attractive target for immune intervention. MGB has been shown to induce a specific CD8 T cell response in breast cancer patients, but little is known about a possible MGB-specific CD4 T cell response. Peripheral blood-derived CD4(+)CD25(-) T cells were stimulated in vitro with MGB-pulsed antigen-presenting cells (APC). The MGB and human leukocyte antigen (HLA) class II specificity of the CD4 T cell lines was confirmed by cytokine release following restimulation with autologous and allogenic APC pulsed with MGB from different sources. Candidate HLA class II-restricted epitopes were identified by computer algorithm and validated in cytokine release assays. MGB-specific CD4 T cells were successfully generated in cultures from six of seven donors. Restimulation of MGB-specific CD4 T cells with MGB-pulsed APC induced significantly higher levels of interferon (IFN)-gamma release than APC pulsed with an irrelevant protein (P = 0.0004). Cultures from five of seven donors showed a pure Th1 type response as evidenced by the absence of interleukin (IL)-4. MGB-specific CD4 T cells recognized both recombinant and naturally processed MGB presented by APC. This recognition was HLA class II-restricted, as HLA-DR mismatched APC were not recognized. MGB-specific CD4 T cells from three of four donors recognized MGB-derived, HLA class II-restricted peptides pulsed onto APC. We have successfully generated MGB-specific CD4 T cell cultures and identified candidate MGB HLA class II epitopes. These studies should facilitate study of the CD4 T cell response to MGB, and the development and monitoring of vaccine strategies targeting this unique antigen.

Focused antibody response in plasma cell-infiltrated non-medullary (NOS) breast cancers

Breast tumors with prominent plasma cell (PC) infiltrates often have a more favorable natural course that may plausibly be mediated by anti-tumor activity of the elicited antibodies. These breast tumor-associated PCs are typically IgG dominant in contrast to normal breast PCs, which are mainly IgA. It is our hypothesis that this PC infiltration represents a host immune response that is driven by one or more tumor antigens. Previously, we and others showed that medullary carcinoma (MC) had a focused repertoire and features suggestive of a protein antigen driven response. Infrequently, non-MC, not otherwise specified (NOS) breast tumors may exhibit heavy PC infiltrations, also of IgG isotype. In this first characterization of this favorable prognosis NOS subgroup, IgG heavy chain (Hc) and light chain (Lc) variable (V) regions from three PC-infiltrated NOS tumors were randomly cloned and sequenced. We found biased (V) gene usage by the infiltrating PCs and somatic hypermutation in the rearranged Ig Hc and Lc V regions that were compatible with antigenic selection of the progenitor B cells. The antibody response of NOS infiltrated breast cancer is repertoire-focused, with 13-68% of isolates being clonally reiterated in the samples. Each NOS patient used distinct Hc V-D-J and Lc V-J rearrangements, with her own immune response "footprint," but the overall pattern of gene usage followed that typical of exogenous antigen-induced immune responses. The data are consistent with the hypothesis that PC infiltrates infrequently arising in NOS tumors, as previously inferred for MC, are in response to one or more breast cancer-associated protein tumor antigens.

Chemiluminescent optical fiber immunosensor for detection of autoantibodies to ovarian and breast cancer-associated antigens

We report herein the development of an optical fiber based chemiluminescent immunosensor for detection of the native autoimmune response to GIPC-1, a PDZ containing protein involved in regulation of G-protein signaling. The recombinant protein GIPC-1 was expressed in bacteria, purified, refolded and conjugated to the tip of an optical fiber. A human monoclonal 27.B1 IgM isolated from a breast cancer patient, which targets the GIPC-1 protein, was used for calibration of the immunosensor and was detected down to a concentration of 30 pg/ml. We determined that the fiber-optic immunosensor had a detection limit 50 times lower than chemiluminescent ELISA, and approximately 500 times lower than colorimetric ELISA. In addition, sera from 11 ovarian cancer patients, 22 breast cancer patients and asymptomatic controls were tested for the presence of IgM anti-GIPC-1 autoantibodies in their serum using the two methods. The immunosensor assay detected 54% and 77% GIPC-1 positive sera within ovarian and breast cancer patients, respectively, as compared to chemiluminescent ELISA, which only detected 18% and 27%, respectively. We envision that this immunosensor may serve as a diagnostic tool for screening women for ovarian and breast cancer at an early stage, thus increasing their chance of survival.

Analysis of the Resveratrol-binding Protein using Phage-displayed Random Peptide Library

Resveratrol, a plant polyphenol, is found in significant amounts in the skin of grapes and in some traditional herbs. It is reported to exert different biological activities, such as inhibiting lipid peroxidation, scavenging free radicals, inhibiting platelet aggregation, and anticancer activity. In order to screen the resveratrol-binding proteins, we synthesized biotinylated resveratrol, purified by liquid chromatography and immobilized it into streptavidin-coated microplate wells. 3-(4,5-Demethylthiazol-)-2,5-diphenyl tetrazolium bromide assay showed little change in the anticancer activity of biotinylated resveratrol in vitro. A random library of phage-displayed peptides was screened for binding to immobilized resveratrol to isolate resveratrol-binding proteins. Several peptides were found to bind to resveratrol specifically, which was proven by enzyme-linked immunosorbent assay. Through amino acid sequence analysis of the selected peptides and human proteins using the BLAST program, the results showed that resveratrol has an affinity for various proteins such as breast cancer-associated antigen, breast cancer resistance protein, death-associated transcription factor, and human cyclin-dependent kinase. These results demonstrate that our study provides a feasible method for the study of binding proteins of natural compounds using a phage-displayed random peptide library.

Effect of short-term, low-dose treatment with tamoxifen in patients with primary dysmenorrhea

Current treatment of painful periods and other symptoms related to primary dysmenorrhea (PD) is usually commenced with non-steroidal anti-inflammatory drugs or oral contraceptives, which fails in about 10% of affected patients. Tamoxifen, a selective estrogen-receptor modulator (SERM), has been demonstrated to directly inhibit uterine contractions, causing improvement in uterine blood flow. It could be considered for application in selected groups of dysmenorrheic patients, for instance carriers of breast cancer-associated antigen (BRCA) genes, breast cancer survivors or women with advanced endometriosis. Thus the aim of the present study was to investigate the effect of short-term treatment with tamoxifen on PD and PD-related symptoms, as well as its direct effect on parameters of intrauterine pressure during the painful menstruation, in a group of dysmenorrheic patients. After two cycles of administration of tamoxifen we noted a significant decrease in bleeding together with reductions in the severity of menstrual cramps, diarrhea, headache, fatigue and anxiety. In intrauterine pressure assessments, tamoxifen significantly decreased propagation of uterine contractions. In conclusion, SERMs such as tamoxifen may constitute a therapeutic option in selected groups of patients, improving dysmenorrheic symptoms. Additionally to its receptor-mediated effects, tamoxifen was shown to exert a direct influence on uterine contractile activity that may explain the decrease of menstrual pain and cramps noted in the studied group.