Treatment with 5-Aza-2'-Deoxycytidine Induces Expression of NY-ESO-1 and Facilitates Cytotoxic T Lymphocyte-Mediated Tumor Cell Killing.

BACKGROUND: NY-ESO-1 belongs to the cancer/testis antigen (CTA) family and represents an attractive target for cancer immunotherapy. Its expression is induced in a variety of solid tumors via DNA demethylation of the promoter of CpG islands. However, NY-ESO-1 expression is usually very low or absent in some tumors such as breast cancer or multiple myeloma. Therefore, we established an optimized in vitro treatment protocol for up-regulation of NY-ESO-1 expression by tumor cells using the hypomethylating agent 5-aza-2'-deoxycytidine (DAC). METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated de novo induction of NY-ESO-1 in MCF7 breast cancer cells and significantly increased expression in U266 multiple myeloma cells. This effect was time- and dose-dependent with the highest expression of NY-ESO-1 mRNA achieved by the incubation of 10 µM DAC for 72 hours. NY-ESO-1 activation was also confirmed at the protein level as shown by Western blot, flow cytometry, and immunofluorescence staining. The detection and quantification of single NY-ESO-1 peptides presented at the tumor cell surface in the context of HLA-A*0201 molecules revealed an increase of 100% and 50% for MCF7 and U266 cells, respectively. Moreover, the enhanced expression of NY-ESO-1 derived peptides at the cell surface was accompanied by an increased specific lysis of MCF7 and U266 cells by HLA-A*0201/NY-ESO-1(157-165) peptide specific chimeric antigen receptor (CAR) CD8+ T cells. In addition, the killing activity of CAR T cells correlated with the secretion of higher IFN-gamma levels. CONCLUSIONS/SIGNIFICANCE: These results indicate that NY-ESO-1 directed immunotherapy with specific CAR T cells might benefit from concomitant DAC treatment.

RP1 is a phosphorylation target of CK2 and is involved in cell adhesion.

RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser(236) in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP(236) show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser(236) by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.

Radioimmunotherapy of fibroblast activation protein positive tumors by rapidly internalizing antibodies.

PURPOSE: Fibroblast activation protein (FAP) is a serine protease that has emerged as a promising target for cancer therapy, either by direct abrogation of its proinvasive activity or by specific targeting of FAP-expressing cells with cytotoxic immunoconjugates. We aimed to select novel human-mouse cross-reactive antibodies and to test suitability for tumor therapy as radioimmunoconjugates in a preclinical model. EXPERIMENTAL DESIGN: Human Fab fragments that bind to human and murine FAP were selected from an antibody phage library. Two candidates (ESC11 and ESC14) were engineered into fully human IgG1 antibodies and further characterized. We investigated the intracellular trafficking of ESC11 and ESC14 in live cells by confocal microscopy and analyzed the biodistribution and therapeutic effects of anti-FAP antibodies labeled with the ß-emitting radionuclide (177)Lu in a melanoma xenograft nude mouse model. Results were compared with vF19, a humanized variant of an anti-FAP antibody that has been previously used in clinical trials. RESULTS: The two antibodies bound selectively to both human and mouse FAP, with affinities in the low nanomolar range. Binding to FAP-expressing melanoma cells resulted in rapid internalization of FAP-antibody complexes. (177)Lu-labeled ESC11 specifically accumulated in melanoma xenografts in vivo, with a higher tumor uptake than ESC14 and vF19. Radioimmunotherapy with 8 MBq (177)Lu-labeled anti-FAP antibodies delayed growth of established tumors, whereas (177)Lu-ESC11 extended mouse survival more pronounced than (177)Lu-ESC14 and (177)Lu-vF19. CONCLUSION: Our results show the potential of ESC11 and ESC14 as potent radioimmunoconjugates or antibody-drug conjugates for diagnostic and therapeutic use in patients with FAP-expressing tumors.

Inhibiting HLA-B27 homodimer-driven immune cell inflammation in spondylarthritis.

OBJECTIVE: Spondylarthritides (SpA), including ankylosing spondylitis (AS), are common inflammatory rheumatic diseases that are strongly associated with positivity for the HLA class I allotype B27. HLA-B27 normally forms complexes with ß(2) -microglobulin (ß(2) m) and peptide to form heterotrimers. However, an unusual characteristic of HLA-B27 is its ability to form ß(2) m-free heavy chain homodimers (HLA-B27(2) ), which, unlike classic HLA-B27, bind to killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). Binding of HLA-B27(2) to KIR-3DL2-positive CD4+ T and natural killer (NK) cells stimulates cell survival and modulates cytokine production. This study was undertaken to produce an antibody to HLA-B27(2) in order to confirm its expression in SpA and to inhibit its proinflammatory properties. METHODS: We generated monoclonal antibodies by screening a human phage display library positively against B27(2) and negatively against B27 heterotrimers. Specificity was tested by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) assay, and fluorescence-activated cell sorting (FACS) analysis of B27(2) -expressing cell lines and peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with SpA. Functional inhibition of KIR-3DL2-B27(2) interactions was tested using cell lines and PBMCs from patients with SpA. RESULTS: Monoclonal antibody HD6 specifically recognized recombinant HLA-B27(2) by ELISA and by SPR assay. HD6 bound to cell lines expressing B27(2) . FACS revealed binding of HD6 to PBMCs and SFMCs from patients with AS but not from controls. HD6 inhibited both the binding of HLA-B27(2) to KIR-3DL2 and the survival and proliferation of KIR-3DL2-positive NK cells. Finally, HD6 inhibited production of the proinflammatory disease-associated cytokine interleukin-17 by PBMCs from patients with AS. CONCLUSION: These results demonstrate that antibody HD6 has potential for use in both the investigation and the treatment of AS and other B27-associated spondylarthritides.

Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad antibody responses in humans, a RAYS-based analysis.

Antibody responses to tumor antigens play an important role in initiating a cellular antitumor response with respect to antigen cross-presentation and T cell cross-priming. Successful vaccination strategies rely on an optimally timed activation of the humoral and cellular immune system by using appropriate adjuvant stimulation. The LUD99-008 trial used the cancer testis antigen NY-ESO-1 formulated with ISCOMATRIX adjuvant injected into patients intramuscularly. It was shown that this vaccination strategy is a safe and highly potent activator of the humoral and cellular immune system. Using the RAYS technology, we analyzed in detail the humoral immune response in these patients before and after vaccination: during the course of repeated vaccinations with the adjuvant, antibody titers against NY-ESO-1 and cross-reactivity to LAGE 1A and B increased as an indicator of an enhanced immune response, whereas no antibody response could be detected after vaccination without the adjuvant. Analysis of single fragments of the NY-ESO-1 protein revealed that the humoral response was almost exclusively directed against the N-terminal fragments and the number of fragments and their length being recognized by the NY-ESO-1-specific antibodies increased during the course of vaccination. The humoral immune response mainly consisted of antibodies of the IgG1 and IgG3 subclass. We rarely found IgG2 and IgG4 subclass antibodies. Our results support the implication that target-specific antibodies raised after vaccination contribute to the stimulation of an effective T cell response against the target antigen.

Cryptic epitopes induce high-titer humoral immune response in patients with cancer.

In search of novel markers for diagnosis, prognosis, and therapy of cancer, screening of rcDNA expression libraries with patient's sera has been established as a valuable tool for identification of cancer-specific Ags. Interestingly, besides the expected humoral responses to annotated proteins, patients with cancer were frequently found to have serum Abs that bind to peptides without homology to known proteins. So far, the nature of these unconventional epitopes and their possible significance in tumor immunology have never been thoroughly investigated. In our study, we specifically analyzed humoral immune response toward such peptides in patients with pancreatic or breast cancer using yeast-displayed cDNA expression libraries derived from tumor tissue. A detailed analysis of the identified peptides revealed that they originated from translation of sequences outside annotated open reading frames and may derive from the use of alternative start codons or from DNA indel mutations. In several cases, the corresponding mRNA templates have a known association with cancer. In a final analysis, we were able to detect one of these tumor Ags in cancer tissue arrays by a selected Fab-Ab. We conclude that cryptic epitopes may elicit specific humoral immune responses in patients with cancer and thus play a role in immunologic surveillance. Due to the high prevalence of immune responses against some of the peptides, they may also be valuable markers for cancer diagnosis, prognosis, or therapy monitoring.

NY-ESO-1 protein glycosylated by yeast induces enhanced immune responses.

Vaccine strategies that target dendritic cells to elicit potent cellular immunity are the subject of intense research. Here we report that the genetically engineered yeast Saccharomyces cerevisiae, expressing the full-length tumour-associated antigen NY-ESO-1, is a versatile host for protein production. Exposing dendritic cells (DCs) to soluble NY-ESO-1 protein linked to the yeast a-agglutinin 2 protein (Aga2p) protein resulted in protein uptake, processing and MHC class I cross-presentation of NY-ESO-1-derived peptides. The process of antigen uptake and cross-presentation was dependent on the glycosylation pattern of NY-ESO-1-Aga2p protein and the presence of accessible mannose receptors. In addition, NY-ESO-1-Aga2p protein uptake by dendritic cells resulted in recognition by HLA-DP4 NY-ESO-1-specific CD4(+) T cells, indicating MHC class II presentation. Finally, vaccination of mice with yeast-derived NY-ESO-1-Aga2p protein led to an enhanced humoral and cellular immune response, when compared to the bacterially expressed NY-ESO-1 protein. Together, these data demonstrate that yeast-derived full-length NY-ESO-1-Aga2p protein is processed and presented efficiently by MHC class I and II complexes and warrants clinical trials to determine the potential value of S. cerevisiae as a host for cancer vaccine development.

Yeast-based identification of prostate tumor antigens provides an effective vaccine platform.

BACKGROUND/AIM: To evaluate cancer/testis (CT) antigens as targets for immunotherapy or vaccine approaches in prostate cancer. PATIENTS AND METHODS: We investigated the antibody response in 181 patients with prostate cancer, 83 benign prostate hyperplasia (BPH) patients, and 39 healthy donors against 13 different CT antigens recombinantly expressed on yeast surface (RAYS) and compared the results to antigen expression in tumor tissue. We then used the yeast clone expressing the most promising antigen directly as a vaccine to elicit potent cellular immunity. RESULTS: The antibody response to NY-ESO-1 was more frequent (20%) and strong compared to other investigated antigens, and was associated with progressive disease. Interestingly, it was also detected in several BPH patients (9%). Feeding dendritic cells with NY-ESO-1-expressing yeast cells resulted in efficient HLA presentation and activation of specific CD3(+) T-cells. CONCLUSION: The RAYS approach offers a fast means of analyzing serological autoreacitvity in cancer patients and serves as an effective anticancer vaccine platform.

Rational development of high-affinity T-cell receptor-like antibodies.

T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1(157-165) peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW "peg" dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2-4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1(157-165) target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes.

MHC-peptide-specific antibodies reveal inefficient presentation of an HLA-A*0201-restricted, Melan-A-derived peptide after active intracellular processing.

MHC-peptide-specific Fab antibodies binding to HLA-A*0201 complexes presenting the wild-type EAAGIGILTV (EAA) or analogue Melan-A 10-mer ELAGIGILTV (ELA) peptide were generated to study efficacy of peptide processing and presentation. None of the selected Fab antibodies detected the naturally processed EAA/HLA-A*0201 complex on melanoma tumor cells, confirming the known low peptide number on the cell surface. To study the effect of peptide presentation and processing in more detail, genes coding for the A27L-mutated Melan-A protein or the processed ELA peptide were introduced into HLA-A*0201(+) B cells by infection with the respective recombinant vaccinia virus construct producing equimolar amounts of GFP-ubiquitin directly linked to the fragment of interest. Correlating GFP expression to actual numbers of peptide presented, 1100-2600 [corrected] ELA peptides had to be synthesized to be presented by a single MHC class I antigen-peptide complex. This number increased 10- to 20-fold when ELA peptide presentation from the A27L-mutated full length Melan-A protein was studied, since 16000-52000 [corrected] GFP molecules needed to be synthesized for the detection of one ELA peptide. Our results indicate that peptide processing rather than presentation is the rate-limiting step in our experimental setting and is much more ineffective for Melan-A than has been previously shown for other MHC class I-restricted epitopes.

Fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes.

Fibroblast activation protein (FAP), as described so far, is a type II cell surface serine protease expressed by fibroblastic cells in areas of active tissue remodelling such as tumour stroma or healing wounds. We investigated the expression of FAP by fibroblast-like synoviocytes (FLSs) and compared the synovial expression pattern in rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Synovial tissue from diseased joints of 20 patients, 10 patients with refractory RA and 10 patients with end-stage OA, was collected during routine surgery. As a result, FLSs from intensively inflamed synovial tissues of refractory RA expressed FAP at high density. Moreover, FAP expression was co-localised with matrix metalloproteinases (MMP-1 and MMP-13) and CD44 splice variants v3 and v7/8 known to play a major role in the concert of extracellular matrix degradation. The pattern of signals appeared to constitute a characteristic feature of FLSs involved in rheumatoid arthritic joint-destructive processes. These FAP-expressing FLSs with a phenotype of smooth muscle actin-positive myofibroblasts were located in the lining layer of the synovium and differ distinctly from Thy-1-expressing and non-proliferating fibroblasts of the articular matrix. The intensity of FAP-specific staining in synovial tissue from patients with RA was found to be different when compared with end-stage OA. Because expression of FAP by RA FLSs has not been described before, the findings of this study highlight a novel element in cartilage and bone destruction of arthritic joints. Moreover, the specific expression pattern qualifies FAP as a therapeutic target for inhibiting the destructive potential of fibroblast-like synovial cells.

Structure-activity profiles of Ab-derived TNF fusion proteins.

TNF application in humans is limited by severe side effects, including life-threatening symptoms of shock. Therefore, TNF can be successfully applied as a tumor therapeutic reagent only under conditions that prevent its systemic action. To overcome this limitation, genetic fusion of TNF to tumor-selective Abs is a favored strategy to increase site-specific cytokine targeting. Because wild-type TNF displays its bioactivity as noncovalently linked homotrimer, the challenge is to define structural requirements for a TNF-based immunokine format with optimized structure-activity profile. We compared toxicity and efficacy of a dimerized CH2/CH3 truncated IgG1-TNF fusion protein and a single-chain variable fragment-coupled TNF monomer recognizing fibroblast-activating protein. The former construct preserves its dimeric structure stabilized by the natural disulfide bond IgG1 hinge region, while the latter trimerizes under native conditions. Analysis of complex formation of wild-type TNF and of both fusion proteins with TNFR type 1 (TNF-R1) using surface plasmon resonance correlated well with in vitro and in vivo toxicity data. There is strong evidence that TNF subunits in a trimeric state display similar toxicity profiles despite genetic fusion to single-chain variable fragment domains. However, LD(50) of either immunodeficient BALB/c nu/nu or immunocompetent BALB/c mice was significantly decreased following administration of TNF in the formation of IgG1-derived dimeric fusion protein. Reduction of unspecific peripheral complexation of TNF-R1 resulted in higher anticancer potency by immunotargeting of fibroblast-activating protein-expressing xenografts. The broader therapeutic window of the IgG1-derived TNF fusion protein favors the dimeric TNF-immunokine format for systemic TNF-based tumor immunotherapy.

Cross-presentation of HLA class I epitopes from influenza matrix protein produced in Saccharomyces cerevisiae.

Here we report that genetically engineered yeast of the strain Saccharomyces cerevisiae expressing full-length influenza matrix protein (IMP) attached to the yeast cell wall are a very versatile host for antigen delivery. Feeding of dendritic cells with either intact yeast expressing IMP protein or soluble IMP protein cleaved off the cell wall resulted in protein uptake, processing and cross-presentation of IMP-derived peptides. This process was analysed using previously established T-cell lines recognizing the immuno-dominant 58-66 peptide when presented by HLA-A2*0201 complexes. In addition, IMP(58-66)/HLA-A2*0201-specific antibodies were selected from a naive phage library which confirmed that peptide presentation was an active process of endocellular uptake and not just a result of external peptide loading. Moreover, MHC peptide antibodies could block the recognition of peptide-presenting dendritic cells by IMP(58-66)-specific T-cells in a dose dependent manner. There was no difference in T-cell recognition when either intact yeast or yeast cell extracts were used for DC feeding. Together, these data demonstrate that yeast derived proteins either in their soluble form or as part of a whole yeast vaccine are taken up, processed and presented by dendritic cells in HLA class I context.

Serological immune response to cancer testis antigens in patients with pancreatic cancer.

Serological screening approaches have allowed for the identification of a large number of potentially relevant tumor antigens in cancer patients. Within this group, cancer testis antigens represent promising targets for cancer immunotherapy, since they are widely expressed in a variety of human cancer entities. In pancreatic cancer, however, there are only few data available about the expression pattern and serological response to cancer testis antigens and other serological-defined tumor antigens. Therefore, we investigated the IgG antibody response against 11 cancer testis antigens (SCP-1, GAGE, LAGE-1a,-1b, CT-7, NY-ESO-1, SSX-1-5) recombinantly expressed on yeast surface (RAYS) in patients with pancreatic cancer (n = 96), chronic pancreatitis (n = 18) and healthy donors (n = 48). We found in 14% of all patients antibody responses to SCP-1, but not to other cancer testis antigens (GAGE, LAGE-1a,-1b, CT-7, NY-ESO-1, SSX-1-5). Antibody response correlated with the expression of SCP-1 in the primary tumor of the respective patient as shown by RT-PCR, immunohistochemistry and Western blot. In contrast, no serological response to cancer testis antigens was observed in healthy donors. The humoral immune response against SCP-1 was associated with the size of tumor, but not with other clinico-pathological parameters such as histology, stage, presence of lymph node metastases, grading, age, gender or gemcitabine treatment. In conclusion, antibody response to cancer testis antigen SCP-1 is found in a proportion of pancreatic carcinoma patients. These results indicate that identification of additional tumor antigens by serological screening of tumor cDNA expression libraries by RAYS is a promising goal in pancreatic cancer.

Prospective study on the expression of cancer testis genes and antibody responses in 100 consecutive patients with primary breast cancer.

To determine the expression of cancer testis (CT) genes and antibody responses in a nonselected population of patients with primary breast cancer, we investigated the composite expression of 11 CT genes by RT-PCR in fresh biopsies of 100 consecutive cases of primary breast carcinoma and by immunohistology in selected RT-PCR-positive cases. Antibody responses against 7 CT antigens were analyzed using recombinant antigen expression on yeast surface. In 98 evaluable cases, SCP-1 and SSX-4 were expressed most frequently (both 65%), followed by HOM-TES-85/CT-8 (47%), GAGE (26%), SSX-1 (20%), NY-ESO-1 (13%), MAGE-3 (11%), SSX-2 (8%), CT-10 (7%), MAGE-4 (4%) and CT-7 (1%). One CT gene was expressed by 90% of the cases; 79% expressed > or =2, 48% > or =3, 29% > or =4, 12% > or =5, 6% > or =6, 3% > or =7, 2% > or =8 and one case coexpressed 9 antigens. Of 100 serum samples screened for CT antigen-specific antibodies, antibodies against NY-ESO-1 were detected in 4 patients, against SCP-1 in 6 patients and against SSX-2 in 1 patient, while no antibodies were detected against MAGE-3, CT-7 and CT-10. Expression of CT genes or antibody responses was not correlated with clinical parameters (menopausal status, tumor size, nodal involvement, grading, histology and estrogen receptor status) or the demonstration of CT gene expression at the protein level, by immunohistology. Our results show that breast carcinomas are among the tumors with the most frequent expression of CT antigens, rendering many patients potential candidates for vaccine trials.

Characterization of Hap/BAG-1 variants as RP1 binding proteins with antiapoptotic activity.

The MAPRE protein family (EB1, RP1, EB2) represents a highly conserved group of proteins that localize preferentially to the plus end of microtubules, both in the nucleus and cytoplasm. In addition, MAPRE family members are characterized by their capability to bind to the C-terminus of the adenomatous polyposis coli (APC) protein and tubulin in order to stabilize microtubules. Apart from the interaction with APC and tubulin, no other direct binding partners are known today. Because the RP1 gene product was identified in activated T cells, we set out to search for new interacting molecules in a yeast 2-hybrid system. We isolated a cDNA variant encoding for the antiapoptotic Hap/BAG-1 protein truncated by 34 amino acids at the C-terminus. In the original Hap/BAG-1 protein, the C-terminal domain is responsible for binding to Bcl-2 and Hsp/Hsc70, which is believed to be the reason for its antiapoptotic activity. Although this putative Hap/BAG-1 variant protein showed no interaction with Bcl-2 or Hsp/Hsc70, it was perfectly able to confer resistance to apoptosis. Subcellular distribution analysis revealed that the Hap/Bag-1 variant protein localized homogenously to the cytoplasm and shuttles into the nucleus in response to stress, a process that could be blocked by RP1 protein overexpression.

Characterization of the human GARP (Golgi associated retrograde protein) complex.

The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the "orphan" SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells.

Serological identification of breast cancer-related antigens from a Saccharomyces cerevisiae surface display library.

A yeast cell surface display technology was used for the isolation and characterization of tumor antigens recognised by autologous or allogeneic breast cancer serum. More than 100 clones recognized by patient serum were isolated using high-through-put fluorescence activated cell sorting. Combined serological and sequence analysis confirmed that a number of proteins known to be overexpressed in breast cancer tissue could be detected. A recently identified small breast epithelial mucin almost exclusively expressed in mammary gland tissue was isolated as a mutated protein variant. Subsequent serological analysis using the yeast expression system for the wild-type and mutant form showed a strong recognition by patient sera, whereas no significant recognition was observed for the respective prokaryotically expressed proteins. The small breast epithelial mucin is present to a large extent in a membrane bound format and might be used for tumor targeting strategies.

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

The serological analysis of antigens by recombinant expression cloning (SEREX) has identified a multitude of new tumor antigens in many different tumor entities. These antigens can be grouped into different classes according to their specificities, with cancer/testis antigens appearing to be the most attractive candidates for vaccine development. The observation that CD8 and CD4 T-cell responses against cancer/testis antigens such as NY-ESO-1 correlate with the presence of specific antibodies demonstrates the importance of serological monitoring patients participating in vaccine trials. However, all serological assays available (Western blot, phage display and ELISA) are hampered by the fact that the protein cannot be analyzed in its natural conformation. We have thus developed a yeast display system where the antigen is expressed on the yeast surface (RAYS), allowing for a more natural folding of the protein. To validate this approach we displayed the A33 colorectal cancer antigen on the yeast cell surface and demonstrated specific binding by an A33 monoclonal antibody recognizing a conformation-dependent epitope on the A33 antigen. We then compared RAYS with the more commonly used ELISA and Western blot serological monitoring methods by analyzing 50 sera from cancer patients with known NY-ESO-1 antibody status and 10 sera from patients with unknown SSX2 antibody status in a blind fashion. RAYS appears at least equivalent to both ELISA and Western blotting for the monitoring of antibodies against NY-ESO-1 as regards specificity and sensitivity, while antibodies against SSX2 were detected more frequently by RAYS than by ELISA or phage display.