Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform.

We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete "hot spots" with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope.

Epitope Mapping of a Monoclonal Antibody Directed against Neisserial Heparin Binding Antigen Using Next Generation Sequencing of Antigen-Specific Libraries.

We explore here the potential of a newly described technology, which is named PROFILER and is based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb) epitopes. For this purpose, we used a novel mAb (designated 31E10/E7) directed against Neisserial Heparin-Binding Antigen (NHBA), a component of the anti-group B meningococcus Bexsero® vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7, followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128) in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods.

Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using the PROFILER technology.

There is a strong need for rapid and reliable epitope mapping methods that can keep pace with the isolation of increasingly larger numbers of mAbs. We describe here the identification of a conformational epitope using Phage-based Representation OF ImmunoLigand Epitope Repertoire (PROFILER), a recently developed high-throughput method based on deep sequencing of antigen-specific lambda phage-displayed libraries. A novel bactericidal monoclonal antibody (mAb 9F11) raised against Neisseria meningitidis adhesin A (NadA), an important component of the Bexsero(®) anti-meningococcal vaccine, was used to evaluate the technique in comparison with other epitope mapping methods. The PROFILER technology readily identified NadA fragments that were capable of fully recapitulating the reactivity of the entire antigen against mAb 9F11. Further analysis of these fragments using mutagenesis and hydrogen-deuterium exchange mass-spectrometry allowed us to identify the binding site of mAb 9F11 (A250-D274) and an adjoining sequence (V275-H312) that was also required for the full functional reconstitution of the epitope. These data suggest that, by virtue of its ability to detect a great variety of immunoreactive antigen fragments in phage-displayed libraries, the PROFILER technology can rapidly and reliably identify epitope-containing regions and provide, in addition, useful clues for the functional characterization of conformational mAb epitopes.

Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.

There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.

Yeast killer toxin-like candidacidal Ab6 antibodies elicited through the manipulation of the idiotypic cascade.

A mouse anti-anti-anti-idiotypic (Id) IgM monoclonal antibody (mAb K20, Ab4), functionally mimicking a Wyckerhamomyces anomalus (Pichia anomala) killer toxin (KT) characterized by fungicidal activity against yeasts presenting specific cell wall receptors (KTR) mainly constituted by ß-1,3-glucan, was produced from animals presenting anti-KT Abs (Ab3) following immunization with a rat IgM anti-Id KT-like mAb (mAb K10, Ab2). MAb K10 was produced by immunization with a KT-neutralizing mAb (mAb KT4, Ab1) bearing the internal image of KTR. MAb K20, likewise mAb K10, proved to be fungicidal in vitro against KT-sensitive Candida albicans cells, an activity neutralized by mAb KT4, and was capable of binding to ß-1,3-glucan. MAb K20 and mAb K10 competed with each other and with KT for binding to C. albicans KTR. MAb K20 was used to identify peptide mimics of KTR by the selection of phage clones from random peptide phage display libraries. Using this strategy, four peptides (TK 1-4) were selected and used as immunogen in mice in the form of either keyhole limpet hemocyanin (KLH) conjugates or peptide-encoding minigenes. Peptide and DNA immunization could induce serum Abs characterized by candidacidal activity, which was inhibited by laminarin, a soluble ß-1,3-glucan, but not by pustulan, a ß-1,6-glucan. These findings show that the idiotypic cascade can not only overcome the barrier of animal species but also the nature of immunogens and the type of technology adopted.

A structural model of human ferroportin and of its iron binding site.

A structural model of human ferroportin has been built using two Escherichia coli proteins belonging to the major facilitator superfamily of transporters. A potential iron binding site was identified in the inward-open conformation of the model, and its relevance was tested through measurement of iron export of HEK293T cells expressing wild-type or mutated ferroportin. Aspartates 39 and 181 were found to be essential for the transport ability of the protein. Noteworthy, the D181V mutation is naturally found in type 4 hemochromatosis with reticuloendothelial system iron retention phenotype. The outward-open conformation of ferroportin was also predicted, and showed that significant conformational changes must occur in the inward- to outward-open transition of ferroportin. In particular, putative iron ligands move several ångströms away from each other, leading to the logical conclusion that the iron binding site is not occupied by the metal in the outward-open conformation of ferroportin.

Immunogenic properties of Streptococcus agalactiae FbsA fragments.

Several species of Gram-positive bacteria can avidly bind soluble and surface-associated fibrinogen (Fng), a property that is considered important in the pathogenesis of human infections. To gain insights into the mechanism by which group B Streptococcus (GBS), a frequent neonatal pathogen, interacts with Fng, we have screened two phage displayed genomic GBS libraries. All of the Fng-binding phage clones contained inserts encoding fragments of FbsA, a protein displaying multiple repeats. Since the functional role of this protein is only partially understood, representative fragments were recombinantly expressed and analyzed for Fng binding affinity and ability to induce immune protection against GBS infection. Maternal immunization with 6pGST, a fragment containing five repeats, significantly protected mouse pups against lethal GBS challenge and these protective effects could be recapitulated by administration of anti-6pGST serum from adult animals. Notably, a monoclonal antibody that was capable of neutralizing Fng binding by 6pGST, but not a non-neutralizing antibody, could significantly protect pups against lethal GBS challenge. These data suggest that FbsA-Fng interaction promotes GBS pathogenesis and that blocking such interaction is a viable strategy to prevent or treat GBS infections.

The Spr1875 protein confers resistance to the microglia-mediated killing of Streptococcus pneumoniae.

By screening a whole-genome λ-display library of Streptococcus pneumoniae, we have previously identified a novel surface protein, named Spr1875, that exhibited immunogenic properties and was closely related to pneumococcal virulence. In the present study, we investigated the role of the Spr1875 antigen in the interaction of S. pneumoniae with microglia, the resident brain macrophages. By using an in vitro infection model, the BV2 microglial cell line was challenged with the S. pneumoniae strain DP1004 and its isogenic spr1875-deleted mutant (Δspr1875). Both strains were phagocytosed by microglia efficiently and to a similar extent; however, the DP1004 strain was more resistant than the Δspr1875 mutant to the intracellular killing, as assessed by antibiotic protection and phagosome maturation assays. Moreover, significant differences between the two strains were also observed in terms of susceptibility to microglia-mediated killing. Taken together, these results indicate that S. pneumoniae-microglial cell interplay is influenced by the presence of Spr1875, suggesting that this protein may play a role in the pathogenesis of pneumococcal meningitis.

Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen.

Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen-antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of ∼1,000 Å(2) on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen-antibody interfaces are required to understand and design effective immunogens.

Protective activity of Streptococcus pneumoniae Spr1875 protein fragments identified using a phage displayed genomic library.

There is considerable interest in pneumococcal protein antigens capable of inducing serotype-independent immunoprotection and of improving, thereby, existing vaccines. We report here on the immunogenic properties of a novel surface antigen encoded by ORF spr1875 in the R6 strain genome. An antigenic fragment encoded by spr1875, designated R4, was identified using a Streptococcus pneumoniae phage displayed genomic library after selection with a human convalescent serum. Immunofluorescence analysis with anti-R4 antisera showed that Spr1875 was expressed on the surface of strains belonging to different serotypes. Moreover, the gene was present with little sequence variability in 27 different pneumococcal strains isolated worldwide. A mutant lacking Spr1875 was considerably less virulent than the wild type D39 strain in an intravenous mouse model of infection. Moreover, immunization with the R4 recombinant fragment, but not with the whole Spr1875 protein, induced significant protection against sepsis in mice. Lack of protection after immunization with the whole protein was related to the presence of immunodominant, non-protective epitopes located outside of the R4 fragment. In conclusion, our data indicate that Spr1875 has a role in pneumococcal virulence and is immunogenic. As the R4 fragment conferred immunoprotection from experimental sepsis, selected antigenic fragments of Spr1875 may be useful for the development of a pneumococcal protein-based vaccine.

Novel immunogenic peptides elicit systemic anaphylaxis in mice: implications for peptide vaccines.

Peptide-based therapies are showing increasing potential for the development of vaccines and in the treatment of many important diseases. We previously reported two peptide conjugate vaccines that protected mice against pneumococcal disease. During this study, we observed an unexpected phenomenon; several vaccine candidates induced a rapid, fatal anaphylaxis after booster injection of the peptide conjugate. Further investigation indicated the reaction was mediated by the production of peptide-specific IgE and the release of histamine. Notably, among seven peptides tested, all of which bound the same mAb that selected them from a phage library, only four elicited this severe reaction. Sequence alignment analysis of all peptides revealed unique clusters of acidic amino acid residues in the allergenic peptides. Substitution of the acidic amino acid residues, ED, of peptide MP2 with their amine equivalents, QN, eliminated the anaphylactic effects but did not affect the production of peptide-specific IgG. These results have important implications for both the study of allergens and the development of future peptide-based therapies.

Plasminogen- and fibronectin-binding protein B is involved in the adherence of Streptococcus pneumoniae to human epithelial cells.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The ability of this bacterium to adhere to epithelial cells is considered as an essential early step in colonization and infection. By screening a whole genome phage display library with sera from infected patients, we previously identified three antigenic fragments matching open reading frame spr0075 of the strain R6 genome. This locus encodes for an approximately 120-kDa protein, herein referred to as plasminogen- and fibronectin-binding protein B (PfbB), which displays an LPXTG cell wall anchoring motif and six repetitive domains. In this study, by using isogenic pfbB-deleted mutants of the encapsulated D39 and of the unencapsulated DP1004 type 2 pneumococcal strains, we show that PfbB is involved in S. pneumoniae adherence to various epithelial respiratory tract cell lines. Our data suggest that PfbB directly mediates bacterial adhesion, because fluorescent beads coated with the recombinant PfbB sp17 fragment (encompassing one of the six repetitive domains and the C-terminal region) efficiently bound to epithelial cells. Mutants lacking PfbB bound to fibronectin and plasminogen considerably less efficiently than wild type bacteria, whereas sp17-coated beads specifically bound to both of these substrates. Taken together, our data suggest that, by directly interacting with fibronectin, PfbB significantly increases the ability of S. pneumoniae to adhere to human epithelial cells.

Immunogenic mimics of Brucella lipopolysaccharide epitopes.

Brucella melitensis and Brucella abortus are responsible for brucellosis in bovine and ovine species and for Malta fever in humans. The lipopolysaccharide (LPS) of Brucella is an important virulence factor and can elicit protective antibodies. Because of their potential importance in vaccine design and in serological diagnosis, we developed peptides mimicking the antigenic properties of distinctive antigenic determinants of Brucella LPS. These peptides were selected from several phage display random peptide libraries for their ability to bind monoclonal antibodies directed against the A- or C-type epitopes of Brucella LPS. Plasmids encoding for two of the isolated peptides induced, after DNA immunization, LPS-specific antibody responses. Although these responses were only moderate in extent, these data further suggest the feasibility of using peptide mimics of carbohydrate epitopes as immunogens, a property which may be useful in the design of novel anti-Brucella vaccines.

Peptide mimics of two pneumococcal capsular polysaccharide serotypes (6B and 9V) protect mice from a lethal challenge with Streptococcus pneumoniae.

Anti-polysaccharide immunity is a key facet of protection against several bacterial pathogens. Problems exist with current polysaccharide vaccines and alternative strategies that deliver a protective response are needed. We have identified immunological peptide mimics of type 6B and 9V pneumococcal capsular polysaccharides that could be used as vaccine antigens. Peptides mimicking antigenic properties of serotype 6B capsular polysaccharide were obtained from a phage-displayed peptide library expressing dodecameric peptides, using a human monoclonal antibody (Db3G9). A murine monoclonal antibody (206, F-5) against the serotype 9V capsular polysaccharide identified three peptide mimotopes from the dodecameric peptide library and one from a random pentadecameric peptide library. In ELISA, binding of 206, F-5 and Db3G9 to phage displaying the selected mimotopes was significantly inhibited by type-specific pneumococcal polysaccharide. Peptides were conjugated to keyhole limpet haemocyanin and were used to immunise mice. Two peptides, MP13 and MP7, induced specific anti-6B and 9V polysaccharide antibodies, respectively. Mice immunised with MP7-keyhole limpet hemocyanin or MP13-keyhole limpet hemocyanin conjugate were significantly and specifically protected against a lethal challenge with pneumococci of the appropriate serotype. This study provides strong in vivo evidence that peptide mimics are alternatives to polysaccharide vaccines.

A region of the N-terminal domain of meningococcal factor H-binding protein that elicits bactericidal antibody across antigenic variant groups.

Meningococcal factor H-binding protein (fHbp) is a promising vaccine antigen. Previous studies described three fHbp antigenic variant groups and identified amino acid residues between 100 and 255 as important targets of variant-specific bactericidal antibodies. We investigated residues affecting expression of an epitope recognized by a murine IgG2a anti-fHbp mAb, designated JAR 4, which cross-reacted with fHbps in variant group 1 or 2 (95% of strains), and elicited human complement-mediated, cooperative bactericidal activity with other non-bactericidal anti-fHbp mAbs with epitopes involving residues between 121 and 216. From filamentous bacteriophage libraries containing random peptides that were recognized by JAR 4, we identified a consensus tripeptide, DHK that matched residues 25-27 in the N-terminal domain of fHbp. Since DHK was present in both JAR 4-reactive and non-reactive fHbps, the tripeptide was necessary but not sufficient for reactivity. Based on site-directed mutagenesis studies, the JAR 4 epitope could either be knocked out of a reactive variant 1 fHbp, or introduced into a non-reactive variant 3 protein. Collectively, the data indicated that the JAR 4 epitope was discontinuous and involved DHK residues beginning at position 25; YGN residues beginning at position 57; and a KDN tripeptide that was present in variant 3 proteins beginning at position 67 that negatively affected expression of the epitope. Thus, the region of fHbp encompassing residues 25-59 in the N-terminal domain is important for eliciting antibodies that can cooperate with other anti-fHbp antibodies for cross-reactive bactericidal activity against strains expressing fHbp from different antigenic variant groups.

Structural mimicry of O-antigen by a peptide revealed in a complex with an antibody raised against Shigella flexneri serotype 2a.

The use of carbohydrate-mimicking peptides to induce immune responses against surface polysaccharides of pathogenic bacteria offers a novel approach to vaccine development. Factors governing antigenic and immunogenic mimicry, however, are complex and poorly understood. We have addressed this question using the anti-lipopolysaccharide monoclonal antibody F22-4, which was raised against Shigella flexneri serotype 2a and shown to protect against homologous infection in a mouse model. In a previous crystallographic study, we described F22-4 in complex with two synthetic fragments of the O-antigen, the serotype-specific saccharide moiety of lipopolysaccharide. Here, we present a crystallographic and NMR study of the interaction of F22-4 with a dodecapeptide selected by phage display using the monoclonal antibody. Like the synthetic decasaccharide, the peptide binds to F22-4 with micromolar affinity. Although the peptide and decasaccharide use very similar regions of the antigen-binding site, indicating good antigenic mimicry, immunogenic mimicry by the peptide was not observed. The F22-4-antigen interaction is significantly more hydrophobic with the peptide than with oligosaccharides; nonetheless, all hydrogen bonds formed between the peptide and F22-4 have equivalents in the oligosaccharide complex. Two bridging water molecules are also in common, adding to partial structural mimicry. Whereas the bound peptide is entirely helical, its structure in solution, as shown by NMR, is helical in the central region only. Moreover, docking the NMR structure into the antigen-binding site shows that steric hindrance would occur, revealing poor complementarity between the major solution conformation and the antibody that could contribute to the absence of immunogenic mimicry.

Specific and selective probes for Pseudomonas aeruginosa from phage-displayed random peptide libraries.

The design of novel biosensors for the detection of biological threats, such as Pseudomonas aeruginosa, requires probes that specifically bind biological agents and insure their immediate and efficient recognition. Advanced bio-selective sensors may meet the requests for isolation, concentration of the agents and their real-time detection. There is a need for robust and inexpensive affinity probes alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we identified from two phage-displayed random peptide libraries phage clones displaying peptides capable of specific and strong binding to P. aeruginosa cell surface. The ability of the phage clones to interact specifically with P. aeruginosa was demonstrated by using enzyme-linked immunosorbent assay (ELISA). We assessed selectivity of phage-bacteria-binding by comparing the binding ability of the selected clones to the selector bacterium and a panel of other bacterial species; we also demonstrated by dot spot and immunoblotting that the most reactive and selective phage peptide bound with high avidity the bacterial cell surface. In addition, as proof-of-concept, we tested the possibility to immobilize the affinity-selected phage to a putative biosensor surface. The quality of phage deposition was monitored by ELISA, and phage-bacterial-binding was confirmed by high-power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including clinical-based diagnostics and possibly biological warfare applications.

Identification of a human immunodominant B-cell epitope within the immunoglobulin A1 protease of Streptococcus pneumoniae.

BACKGROUND: The IgA1 protease of Streptococcus pneumoniae is a proteolytic enzyme that specifically cleaves the hinge regions of human IgA1, which dominates most mucosal surfaces and is the major IgA isotype in serum. This protease is expressed in all of the known pneumococcal strains and plays a major role in pathogen's resistance to the host immune response. The present work was focused at identifying the immunodominant regions of pneumococcal IgA1 protease recognized by the human antibody response. RESULTS: An antigenic sequence corresponding to amino acids 420-457 (epiA) of the iga gene product was identified by screening a pneumococcal phage display library with patients' sera. The epiA peptide is conserved in all pneumococci and in two out of three S. mitis strains, while it is not present in other oral streptococci so far sequenced. This epitope was specifically recognized by antibodies present in sera from 90% of healthy adults, thus representing an important target of the humoral response to S. pneumoniae and S. mitis infection. Moreover, sera from 68% of children less than 4 years old reacted with the epiA peptide, indicating that the human immune response against streptococcal antigens occurs during childhood. CONCLUSION: The broad and specific recognition of the epiA polypeptide by human sera demonstrate that the pneumococcal IgA1 protease contains an immunodominant B-cell epitope. The use of phage display libraries to identify microbe or disease-specific antigens recognized by human sera is a valuable approach to epitope discovery.

Peptide mimics of the group B meningococcal capsule induce bactericidal and protective antibodies after immunization.

Neisseria meningitidis serogroup B (MenB) is a leading cause of sepsis and meningitis in children. No vaccine is available for the prevention of these infections because the group B capsular polysaccharide (CP) (MenB CP) is unable to stimulate an immune response, due to its similarity with human polysialic acid. Because the MenB CP bears both human cross-reactive and non-cross-reactive determinants, we developed immunogenic peptide mimics of the latter epitopes. Peptides were selected from phage display libraries for their ability to bind to a protective anti-MenB CP mAb. One of these peptides (designated 9M) induced marked elevations in serum bactericidal activity, but not polysialic acid cross-reacting Abs, after gene priming followed by carrier-conjugate boosting. Moreover, the occurrence of bacteremia was prevented in infant rats by administration of immune sera before MenB challenge. 9M is a promising lead candidate for the development of an effective and affordable anti-MenB vaccine.

A novel approach for identification of tumor-associated antigens expressed on the surface of tumor cells.

To improve tumor targeting in a subset of patients, where tumor cells do not express the well-known tumor antigens widely used in immunotherapy, we have developed a novel biotechnological tool. It is useful for tumors of various origins for the identification of tumor-associated proteins, which are differentially expressed in tumor cells with respect to normal tissue, and exposed on the cell surface. For this purpose, a combination of techniques, such as "suppression subtractive hybridization" and "transmembrane trapping," was employed. In applying this novel approach to breast cancer, we identified a large panel of cDNA fragments encoding for the well-known tumor-associated surface antigens, such as erb-B2, erbB3 and the urokinase receptor and, more importantly, for several clones overexpressed in breast cancer, whose cDNA fragments match the sequences of hypothetical transmembrane proteins with unknown function. The latter may represent novel tumor-specific targets.