NY-BR-1 is a differentiation antigen of the mammary gland.

NY-BR-1 was recently identified by autologous serological typing of the recombinant expression library in a breast cancer patient. Extensive reverse transcriptase-polymerase chain reaction analysis revealed the presence of NY-BR-1 in normal breast tissue and tumors derived thereof. Except normal testis, no other normal tissue or tumors showed NY-BR-1 expression. However, nothing is known about the expression of its actual antigen. In the present study, we describe the generation of 2 new monoclonal antibodies, NY-BR-1#2 and NY-BR-1#3, to NY-BR-1 for the analysis of its expression on a protein level employing recombinant NY-BR-1 protein for the immunization of BALB/c mice. In normal tissues, immunohistochemical testing demonstrates NY-BR-1 in a mostly focal fashion in the epithelia of ducts and acini of the mammary gland. No other tissue was immunopositive including testis. In tumors, homogenous staining can be seen in almost all ductal carcinomas in situ and/or the intraductal component of invasive carcinomas. Invasive carcinomas show a lower number of NY-BR-1-positive tumors. Initial higher numbers of NY-BR-1 mRNA-positive invasive carcinomas are most likely based on sample error owing to the contamination of tumor tissue with remnants of normal breast epithelium. Sweat gland carcinomas, which are related to breast cancer, are also positive in about one-third of the cases. These data indicate that NY-BR-1 is a differentiation antigen of the mammary gland that could be useful for diagnosis and/or immunotherapy of breast carcinomas.

The differentiation antigen NY-BR-1 is a potential target for antibody-based therapies in breast cancer.

Antibody-based cancer immunotherapy relies on the identification and characterization of target antigens and the development of potent antibodies recognizing the target. Here we report the expression analysis and molecular characterization of the differentiation antigen NY-BR-1, which we previously identified by using the SEREX (serological analysis of recombinant cDNA expression libraries) method. Corroborating methodologies, including mRNA quantitation and immunoblotting show that NY-BR-1 is strongly expressed in >70% of 129 breast tumors. Application of a NY-BR-1 specific antibody demonstrated NY-BR-1 expression in primary and metastastic breast cancers. In contrast, most of the breast cancer cell lines tested, expressed only low NY-BR-1 levels. Importantly, confocal microscopy revealed that ectopically expressed NY-BR-1 localizes to the cytoplasm and the cell membrane. NY-BR-1 localization in breast cancer specimens was also confirmed by immunohistochemistry. Bioinformatic analysis and deletion mutagenesis further show that NY-BR-1 membrane localization is mediated by 2 cis-active membrane targeting domains. Biochemical surface labeling and FACS analysis of live cells further characterize NY-BR-1 as a transmembrane protein, which can be specifically recognized by the anti-NY-BR-1 antibody on the surface of vital cells. The strong expression of NY-BR-1 in breast tumors, its cytoplasmic and membrane localization and accessibility to an ectopically applied antibody now suggest to pursue NY-BR-1 as a potential target for antibody-based therapies in breast cancer patients.

Humoral and cellular immune responses against the breast cancer antigen NY-BR-1: definition of two HLA-A2 restricted peptide epitopes.

Cancer immunotherapy depends on the identification of tumor-specific target antigens that are predominantly expressed in cancer cells and not in normal tissues. Here, we report the cloning and the expression analysis of the differentiation antigen NY-BR-1 that we have identified in a previous SEREX (serological analysis of recombinant cDNA expression libraries) screening. The cloning of the full length NY-BR-1 sequence led to the prediction of an open reading frame of 4.2 kb, encoding a protein of 158.9 kDa. NY-BR-1 mRNA expression analysis revealed tissue-specific expression in normal testis and breast tissues, as well as in 70% of breast tumors. We now show that NY-BR-1 is also sporadically expressed in normal prostate and in 32% of prostate tumors. Furthermore, we were able to identify two HLA-A2 restricted NY-BR-1 epitopes (p158-167 and p960-968) that are recognized by CD8+ T cell clones (NW1100-CTL-7 and NW1100-CTL-43, respectively), as determined by ELISPOT analysis and tetramer staining. Cotransfection assays of COS-7 cells also demonstrated that these two peptides are naturally processed and presented on HLA-A2 molecules. The identification of these two naturally processed NY-BR-1-specific CD8+ T cell epitopes opens the perspective for active immunotherapy of HLA-A2 positive patients with NY-BR-1 expressing tumors.

Phylogenetic relationships of non-mitochondrial nucleotide transport proteins in bacteria and eukaryotes.

Current knowledge about the nucleotide metabolism of intracellular bacteria is very limited. Here we report on the identification of nucleotide transport proteins (NTT) of two obligate endoparasites, Caedibacter caryophila and Holospora obtusa, both alpha-proteobacteria, which reside in the vegetative macronucleus of Paramecium caudatum. For comparative studies, we also identified the first nucleotide transporter in chloroplasts of a red alga, i.e. Galdieria sulphuraria, and further homologs in plant chloroplasts. Heterologous expression of the NTT proteins from C. caryophila, H. obtusa, and G. sulphuraria in Escherichia coli demonstrate that the nucleotide influx mediated by these transporters is specific for ATP and ADP. The NTT proteins of C. caryophila and H. obtusa exhibit substantial sequence identity with their counterparts in chloroplasts and intracellular bacterial pathogens of humans, but not with the nucleotide transport system of mitochondria. Comprehensive phylogenetic analyses of bacterial and chloroplast NTT proteins showed that homologs in chloroplasts from plants, and green, red, stramenopile and glaucocystophyte algae are monophyletic. In contrast, the evolutionary relationships of the bacterial counterparts appear highly complex. In the presented phylogeny, NTT proteins of C. caryophila and H. obtusa are only distantly related to one another, although these two taxa are close relatives in 16S rRNA trees. The tree topology indicates that some bacterial NTT paralogs have arisen by gene duplications and others by horizontal transfer.