EP1383929A2 - Genes exprimes dans l'epithelium intestinal et les cellules m des plaques de peyer - Google Patents

Genes exprimes dans l'epithelium intestinal et les cellules m des plaques de peyer

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Publication number
EP1383929A2
EP1383929A2 EP02763973A EP02763973A EP1383929A2 EP 1383929 A2 EP1383929 A2 EP 1383929A2 EP 02763973 A EP02763973 A EP 02763973A EP 02763973 A EP02763973 A EP 02763973A EP 1383929 A2 EP1383929 A2 EP 1383929A2
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EP
European Patent Office
Prior art keywords
polynucleotide
gene
polypeptide
cells
seq
Prior art date
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EP02763973A
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German (de)
English (en)
Other versions
EP1383929A4 (fr
Inventor
David J. Brayden
Daragh Byrne
Daniel J. O'mahony
Claire F. Evans
Steven P. Mah
David D. Lo
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NEUROME Inc
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NEUROME Inc
Digital Gene Technologies Inc
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Publication of EP1383929A2 publication Critical patent/EP1383929A2/fr
Publication of EP1383929A4 publication Critical patent/EP1383929A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/06—Anti-spasmodics, e.g. drugs for colics, esophagic dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Peyer's Patches are dome-shaped aggregates of lymphoid follicles that are covered by an epithelial layer (FAE, or follicle associated epithelium) that contains M cells.
  • FAE epithelial layer
  • M cells are a specialized epithelial phenotype that can transport particles and microorganisms from the lumen to the organized lymphoid follicles to initiate the mucosal immune response (See References 1-3. A list of the References cited in this application is attached at the end of this application, and such References are incorporated herein by this reference). However, many pathogenic microorganisms exploit the properties of the M cells as a mechanism for invasion (See References 4-6). Thus, M cells play an important role in microbial pathogenesis, mucosal immune response and are potential targets for drug and vaccine delivery.
  • a reproducible in vitro model of the M cell phenotype would facilitate the study of the Human M cell by providing a relatively unlimited supply of material with which to study the genes and mechanisms by which the transepithelial transport of microbes and particles occurs in a controlled laboratory setting. Additionally, an in vitro model of M cell function would abrogate the problems associated with the acquisition of and the processing of fresh human tissues. Recently, co-cultures of the human carcinoma cell line Caco-2 and mouse Peyer's Patch lymphocytes were found to induce the phenotypic expression of the M cell phenotype (See Reference 10). These phenotypically transformed cells show multiple characteristics of M cells in vivo, including morphological and transepithelial transport properties.
  • transcytosis properties of the in vitro co-culture model are remarkably similar to that of in vivo uptake of particles in mouse and rabbit models (See References 12, 13).
  • This new human derived co-culture model opens up novel possibilities to study the regulation of genes involved in the phenotypic transformation and/or maintenance of the human M-cell phenotype.
  • M cell function can be garnered from studying other species that are similar to man.
  • One of the more accessible animal models used to study M cell function is the mouse. As described above, the mouse and human Peyer's Patch show similarities in both M cell number and function (See References 3, 8). This similarity in function suggests that it may be possible to use the mouse Peyer's Patch model to identify genes that play a role in both mouse and human M cell function.
  • a second animal model for the study of M cell function is monkey. Ultrastructural studies of macaques described the presence of M cells that are morphologically similar to human M cells (See Reference 22). Functional studies of M cells have not yet been reported in monkeys. Given that mouse and human M cells share similar functions, it is likely that human and monkey M cells also have similar properties.
  • M cell mediated vectorial transport can be studied if the genes associated with the M cell phenotype are known.
  • transport mechanisms exist in epithelial cells (See Reference 15).
  • One mechanism is carrier mediated, whereby molecules directly bind and actively transport ligands through the epithelial cell.
  • transcytosis whereby a ligand is bound by a specific cell surface molecule and then internalized by endocytosis into vesicles.
  • a third method of transport is paracellular transport that is a passive process whereby molecules travel across the tight junctions separating neighboring epithelial cells.
  • M cells are unique among epithelial cells in that the major pathway for transport is transcytosis. Indeed, the M cell basolateral surface is highly invaginated and forms an intra-epithelial pocket that shortens the apical to basal distance through which the vesicles must travel (See References 1, 16). Identification of genes that encode molecules involved in the transport mechanism(s) in M cells will lead to the screening for and development of novel ligands with high affinity for the molecule(s). These ligands can then be used for the development of novel methods for vaccine and/or drug delivery (See Reference 15).
  • oral reagents offer both the ease of administration and abolish the need for specialized instruments and trained personnel.
  • vaccines that target the mucosal immune system in the gastro-intestinal tract will facilitate how vaccines are administered and may actually increase the use of vaccines in countries without extensive medical resources.
  • vaccine delivery by the oral route will prime an effective mucosal immunity; where the target pathogen infects via the mucosal route or mucosal surfaces then such a mucosal immunity is important.
  • orally administered reagents suffer from problems of bioavailability (See Reference 17) and also stability in the gastrointestinal tract ("GIT").
  • the transport property of M cells has great biological significance for drug/vaccine delivery strategies in that the directed transcytosis of paniculate reagents to M cells may potentially increase bioavailability. Specifically, particulates actively targeted to the M cells will be more likely to be absorbed and will less likely be exposed to the hostile environment of the digestive tract (See Reference 12). Additionally, with respect to vaccine delivery, the transport and antigen presenting properties of the M cell is an ideal route through which to initiate response via the mucosal immune system.
  • TOGA ® Total Gene Expression Analysis
  • Patent No. 6,309,834 all of which are incorporated herein by reference and available from Digital Gene Technologies, Inc., La Jolla, California to different cellular experiments directed to intestinal epithelium differentiation, development or function.
  • the TOGA ® method is an improved method for the simultaneous sequence-specific identification of mRNAs in an mRNA population which allows the visualization of nearly every mRNA expressed by a tissue as a distinct band on a gel whose intensity corresponds roughly to the concentration of the mRNA.
  • the PCR-based Total Gene Expression Analysis (TOGA ® ) differential display system was used to identify M cell specific gene expression. Molecules were identified that correspond to genes that are upregulated in a human cell culture system that reproduces the morphology as well as the transepithelial transport properties of M cells. We also identified molecules from mouse Peyer's Patch tissue, from FAE enriched cells from mouse Peyer's Patch tissue, from a mouse Peyer's Patch model which were enriched for M cells by Salmonella infection, and from FAE enriched cells from monkey Peyer's Patch tissue. These molecules are useful in the identification of antibodies, peptides, peptidomimetics, small molecules or other binding partners that bind to these molecules.
  • These antibodies, peptides, peptidomimetics, small molecules, or other binding partners are then used to develop targeting agents for the delivery of drug formulations (including, without limitation, peptides including insulin, LHRH, buserelin, vasopressin, recombinant interleukins including IL-2, IL-12, etc., genes delivered by vectors such as adeno-associated virus, lipsomes, PLGA, canarypox virus, adenovirus, retrovimses including IL-1 and GM-CSF antagonists) and/or vaccines (including, without limitation, vaccines for H. pylori, C.
  • drug formulations including, without limitation, peptides including insulin, LHRH, buserelin, vasopressin, recombinant interleukins including IL-2, IL-12, etc.
  • genes delivered by vectors such as adeno-associated virus, lipsomes, PLGA, canarypox virus, adenovirus, retrovi
  • the present invention provides human, mouse, and monkey polynucleotide sequences, their corresponding genes and regions thereof and the encoded polypeptides that are associated with the M cell phenotype and allows for comparisons between different species.
  • inventions provide a method for assessing, modifying, modulating or regulating intestinal epithelium or M cell development, differentiation or function using at least one polynucleotide, polypeptide, antibody, binding partner or gene of the invention or regions thereof.
  • Other embodiments of the invention provide a method of assessing, modifying, modulating or regulating drug, oral vaccine or mucosal immunogen delivery through the intestinal epithelium or M cells by using at least one polynucleotide, polypeptide, antibody, binding partner or gene of the invention, or regions thereof.
  • polypeptides, antibodies and genes of the present invention or regions thereof can also be used to identify or use targets for mucosal immunity, drug delivery, oral vaccine delivery or disease diagnosis, or for the manufacture of a medicament, oral vaccine or drug delivery system.
  • Other embodiments of the present invention involve using the polynucleotides, polypeptides, antibodies, binding partners or genes of the present invention or regions thereof to assess, modify, modulate or regulate active transport through the intestinal epithelium or M cells.
  • the polypeptides and antibodies can be used to identify binding partners to the polypeptides of the invention.
  • the present invention associates identified polynucleotides, genes, gene regions and their encoded polypeptides with differentiated intestinal epithelium cells and M cells such that the polynucleotides and polypeptides are useful in the identification of intestinal epithelium cells and more specifically M cells, and for use as targets or to identify targets for drug delivery across Peyer's Patch epithelium.
  • One embodiment of the invention provides an isolated nucleic acid molecule comprising a polynucleotide chosen from the group consisting of SEQ ID NOs: 1 -345 or variants thereof associated with intestinal epithelium development, differentiation or function.
  • an isolated nucleic acid molecule comprising a polynucleotide at least 95% identical to any one of these isolated nucleic acid molecules and an isolated nucleic acid molecule at least ten bases in length that is hybridizable to any one of these isolated nucleic acid molecules under stringent conditions. Any one of these isolated nucleic acid molecules can comprise sequential nucleotide deletions from either the 5 '-terminus or the 3'- terminus. Also provided is the gene corresponding to the cDNA sequence of any one of these isolated nucleic acids.
  • Another embodiment of the invention provides an isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NO: 1-345 or their corresponding genes or variants thereof or post-translational variants thereof associated with intestinal epithelium or M cell development, differentiation or function. Also provided is an isolated nucleic acid molecule encoding any of these polypeptides, an isolated nucleic acid molecule encoding a fragment of any of these polypeptides, an isolated nucleic acid molecule encoding a polypeptide epitope of any of these polypeptides, and an isolated nucleic acid encoding a homologous polypeptide of any of these polypeptides. Preferably, any one of these polypeptides has biological activity. Also provided is any one of the isolated polypeptides comprising a polypeptide with sequential amino acid deletions from either the C-terminus or the N-terminus.
  • Yet another embodiment of the invention comprises an isolated antibody or fragment thereof, synthetic peptide, synthetic peptidomimetic or small molecule ligand that binds specifically to an isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NO: 1-345 or their corresponding genesassociated with intestinal epithelium or M cell development, differentiation or function.
  • the isolated antibody can be a monoclonal antibody or a polyclonal antibody or variant or fragment thereof.
  • a further embodiment of the invention provides an isolated antibody or variants or fragments thereof that bind specifically to the isolated polypeptide of the invention.
  • a preferred embodiment of the invention provides a method for targeting the delivery of a compound, or encapsulated compound, across the intestinal epithelium barrier, e.g., by active transport through Peyer's Patch epithelial M cells.
  • Another embodiment of the invention provides a method for identifying a binding partner to a polypeptide of the invention as a means for developing targeting formulations for drug delivery across the intestinal epithelium or Peyer's Patch M cells.
  • a polypeptide of the invention is contacted with a binding partner and it is determined whether the binding partner effects an activity of the polypeptide.
  • Yet another embodiment of the invention is a method of identifying an activity of an expressed polypeptide in a biological assay such as an assay for active transport of molecules or particles into the intestinal epithelium or Peyer's Patch epithelium.
  • a polypeptide of the invention is expressed in a cell and isolated. The expressed polypeptide is tested for an activity in a biological assay and the activity of the expressed polypeptide is identified based on the test results.
  • Still another embodiment of the invention provides a substantially pure isolated DNA molecule suitable for use as a probe for genes useful in identifying intestinal epithelial or M cells, chosen from the group consisting of the DNA molecules shown in SEQ ID NO: 1-345 or their corresponding genes or regions thereof. Further embodiments of the present invention are described below and would be apparent to a person skilled in the art.
  • Figure 1 is a graphical representation of the results of duplicate TOGA ® runs using a 5' PCR primer with parsing bases AGGA (SEQ ID NO: 362) and the universal 3 ' PCR primer (SEQ ID NO:351) showing PCR products produced from mRNA extracted from the Caco-2 (top panel), co-cultured ("Caraj” is a co-culture of Caco-2 and RajiB) (middle panel) and RajiB (bottom panel) cell lines, where the vertical index line indicates a PCR product of about 114 b.p.
  • the horizontal axis represents the number of base pairs of the molecules in these samples and the vertical axis represents the fluorescence measurement in the TOGA ® analysis (which corresponds to the relative expression of the molecule of that address).
  • the length of the PCR product corresponding to SEQ ID NO:4 was directly sequenced and a 5' PCR primer was built from the directly sequenced DST (SEQ ID NO : 364) .
  • the product obtained from PCR with this primer (SEQ ID NO : 364) and the universal 3' PCR primer (SEQ ID NO: 351) was compared to the length of the original PCR product that was produced in the TOGA ® reaction with Caraj co-culture cells using a 5' PCR primer with parsing bases AGGA (SEQ ID NO: 362) and the universal 3' PCR primer (SEQ LO NO:351) (as shown in the middle panel).
  • Figure 3 compares the results from Real Time PCR validation (as described in the Materials and Methods section) to duplicate runs of TOGA ® .
  • the DST EDD1 6 (SEQ ID NO:4) is found in a much greater proportion in the Caraj co-culture than in the Caco-2 and RajiB cultures in both TOGA ® runs and Real Time PCR.
  • the data in Figure 3 illustrate an increased expression level DST EDD1_6 (SEQ ID NO: 4) in the Caraj cells relative to the Caco-2 cells. The relative abundance is measured as compared to Caco-2.
  • FIG. 4 shows sectioned tissue from normal mouse Peyer's Patch which was processed for in situ hybridization.
  • In situ hybridization was performed using a digoxigenin-labeled antisense probe.
  • EDD1 6 SEQ ID NO: 4
  • SEQ ID NO: 254 was a human polynucleotide
  • a probe was generated based on the homologous extended mouse sequence (SEQ ID NO: 254) and was used to insure a more specific hybridization signal in the mouse tissue.
  • the FAE is indicated by the arrowed-arc. As illustrated by the arrowed straight lines, in situ hybridization signal was detected in specific cells of the FAE.
  • Figure 5 is a graphical representation similar to Fig. 1 of the results of duplicate TOGA ® runs using a 5' PCR primer with parsing bases CCCT (SEQ ID NO:363) and the universal 3' PCR primer (SEQ ID NO:351) showing PCR products produced from mRNA extracted from the Caco-2 (top panel), Caraj co-culture (middle panel) and RajiB (bottom panel) cell lines where the vertical index line indicates a PCR product of about 257 b.p.
  • the vertical line drawn through the three panels represents the DST molecule identified as EDD1 S_48 (SEQ ID NO:76). As Figure 5 indicates, this DST had a relatively low expression in the Caco-2 and RajiB cultures, and a relatively increased value in the Caraj co-culture.
  • Figure 6 illustrates another graphical example of verification by the Extended TOGA ® Method similar to Fig. 2 where a DST is verified using a primer generated from a sequence obtained through a database (as described below in the Materials and Methods section).
  • the length of the PCR product corresponding to SEQ ID NO:76 (EDD1 S_48) was generated with the 5' PCR primer built based on a DST match obtained from a database (SEQ ID NO:365).
  • the product obtained from PCR with this primer (SEQ ID NO: 365) and the universal 3' PCR primer (SEQ ID NO:351) (as shown in the top panel) was compared to the length of the original PCR product that was produced in the TOGA ® reaction with Caraj co-culture cells using a 5' PCR primer with parsing bases CCCT (SEQ LO NO: 363) and the universal 3' PCR primer (SEQ ID NO: 351) (as shown in the middle panel).
  • the bottom panel presents an overlay of the top and middle panels to illustrate that the peak found using an extended primer from the database-matched sequence is the same length as the original PCR product obtained through TOGA ® .
  • Figure 7 compares the results of Real Time PCR validation of EDD1S_48 (SEQ ID NO: 76) to duplicate runs of TOGA ® .
  • the DST EDD1S_48 (SEQ ID NO: 76) is found in a greater proportion in the Caraj co-culture than in the Caco-2 and RajiB cultures in both TOGA ® runs and Real Time PCR.
  • the data in Figure 7 illustrate an increased expression level of DST EDD1S_48 (SEQ ID NO: 76) in the Caraj cells relative to the Caco-2 cells. The relative abundance is measured as compared to Caco-2.
  • Figure 8 demonstrates the results of colocalization of in situ hybridization (ISH) signal for EDD1S 48 and M cell-specific lectin staining in sectioned tissue from normal mouse Peyer's Patch.
  • Immunofluorescence in situ hybridization was performed using a digoxigenin-labeled antisense probe. Because EDD1S 48 (SEQ ID NO: 76) was a human polynucleotide, a probe was generated based on the homologous extended mouse sequence (SEQ ID NO: 299) and was used to insure a more specific hybridization signal in the mouse tissue.
  • In situ hybridization signal was detected in the FAE as exemplified by the arrows in the top panel.
  • M cells were identified using a fluorescently-labeled lectin, Ulex Europaeus agglutinin I (UEA), a standard marker of murine M cells (arrows, bottom panel).
  • UAA Ulex Europaeus agglutinin I
  • UEA Ulex Europaeus agglutinin I
  • colocalization Coloc.
  • the photographic images are negatives of black and white images; in the original photographs, the lectin UEA was labeled with a red fluorophore and in situ signal was identified by a green fluorophore (see Materials and Methods).
  • Figure 9 demonstrates colocalization of the protein product of the coding sequence corresponding to the extended mouse sequence homologous to EDD1S 48 (SEQ ID NO: 299) with UEA. Colocalization was done on sectioned tissue from normal mouse Peyer's Patch. Using database searches (see Materials and Methods), EDD1 S 48 (SEQ ID NO: 76) was identified as the human Clostridium perfringens enterotoxin receptor, claudin-4. As exemplified by the arrows in the top panel, claudin- 4 was detected by immunofluorescent labeling using an antibody to murine claudin-4. M cells were identified with fluorescently-labeled UEA (bottom panel, arrows point to two examples of M cells).
  • the anti-claudin- 4 staining colocalized with M cells.
  • the photographic images are negatives of black and white images; in the original photographs, the lectin UEA-1 was labeled with a red fluorophore and EDD1S 48 antibodies were labeled with a green fluorophore.
  • Figure 10 shows sectioned tissue from a human small intestine biopsy containing a Peyer's Patch which was processed for non-radioactive digoxigenin in situ hybridization using an antisense probe based on the extended sequence corresponding to EDD 1 S_48 (SEQ ID NO 298). As indicated by the arrows, in situ hybridization signal was detected in the FAE of the human tissue.
  • Figure 11 demonstrates the results of colocalization of in situ hybridization (ISH) signal for EDD1_6 and M cell-specific lectin staining in sectioned tissue from normal mouse Peyer's Patch. Immunofluorescence in situ hybridization was performed using a digoxigenin-labeled antisense probe.
  • ISH in situ hybridization
  • EDD1 6 (SEQ ID NO: 4) was a human polynucleotide
  • a probe was generated based on the homologous extended mouse sequence (SEQ ID NO: 254) and was used to insure a more specific hybridization signal in the mouse tissue.
  • In situ hybridization signal was detected in the FAE as exemplified by the arrow in the top panel. M cells were identified using fluorescently-labeled UEA (arrow, bottom panel). As seen in the middle panel, colocalization (Coloc.) of some of the in situ signal with the UEA signal resulted in the identification of M cells containing mRNA for EDD1_6.
  • FIG. 12 demonstrates the results of colocalization of in situ hybridization (ISH) signal for EDD1S 42 and M cell-specific lectin staining in sectioned tissue from normal mouse Peyer's Patch. Immunofluorescence in situ hybridization was performed using a digoxigenin-labeled antisense probe.
  • ISH in situ hybridization
  • EDD1S 42 (SEQ ID NO: 71) was a human polynucleotide
  • a probe was generated based on the homologous extended mouse sequence (SEQ ID NO: 295) and was used to insure a more specific hybridization signal in the mouse tissue.
  • In situ hybridization signal was detected in the FAE as exemplified by the arrow in the top panel. M cells were identified using fluorescently-labeled UEA (arrow, bottom panel). As seen in the middle panel, colocalization (Coloc.) of some of the in situ signal with the UEA signal resulted in the identification of M cells containing mRNA for EDD1 S_42.
  • the photographic images are negatives of black and white images; in the original photographs, the lectin UEA was labeled with a red fluorophore and in situ signal was identified by a green fluorophore.
  • Figure 13 demonstrates the results of colocalization of in situ hybridization (ISH) signal for EDD2_13 and M cell-specific lectin staining in sectioned tissue from normal mouse Peyer's Patch.
  • Immunofluorescence in situ hybridization was performed using a digoxigenin-labeled antisense probe that was generated based on the mouse extended sequence of EDD2 13 (SEQ ID NO: 318).
  • In situ hybridization signal was detected in the FAE as exemplified by the arrow in the top panel.
  • M cells were identified using fluorescently-labeled UEA (arrow, bottom panel).
  • Figure 14 demonstrates the results of colocalization of in situ hybridization (ISH) signal for EDD34S_25 and M cell-specific lectin staining in sectioned tissue from normal mouse Peyer's Patch.
  • Immunofluorescence in situ hybridization was performed using a digoxigenin-labeled antisense probe containing the mouse extended sequence of EDD34S 25 (SEQ ID NO: 339).
  • In situ hybridization signal was detected in the FAE as exemplified by the arrow in the top panel.
  • M cells were identified using fluorescently-labeled UEA (arrow, bottom panel).
  • Figure 15 demonstrates the results of colocalization of in situ hybridization (ISH) signal for EDD4_17 and M cell-specific lectin staining in sectioned tissue from normal mouse Peyer's Patch.
  • Immunofluorescence in situ hybridization was performed using a digoxigenin-labeled antisense probe that was generated based on the mouse extended sequence of EDD4_17 (SEQ ID NO: 329).
  • In situ hybridization signal was detected in the FAE as exemplified by the arrow in the top panel.
  • M cells were identified using fluorescently-labeled UEA (arrow, bottom panel).
  • Figure 16 demonstrates the results of colocalization of in situ hybridization (ISH) signal for the murine peptidoglycan recognition protein, PGRP-L (SEQ ID NO: 332; also called TAGL-alpha) and M cell-specific lectin staining in sectioned tissue from normal mouse Peyer's Patch.
  • Immunofluorescence in situ hybridization was performed using a digoxigenin-labeled antisense probe that was generated based on the nucleotide sequence corresponding to PGRP-L (SEQ ID NO: 332).
  • PGRP-L is a gene whose identity is related to EDD4 17 (SEQ ID NO: 192; see Results).
  • In situ hybridization signal was detected in the FAE as exemplified by the tagged arrow in the top panel.
  • M cells were identified using fluorescently-labeled UEA (arrow, bottom panel).
  • labeling of M cells was distinct from the in situ signal, indicating that PGRP-L mRNA is expressed by FAE but not by M cells.
  • the photographic images are negatives of black and white images; in the original photographs, the lectin UEA-1 was labeled with a red fluorophore and in situ signal was identified by a green fluorophore.
  • FIG 17 shows sectioned tissue from normal mouse Peyer's Patches that were harvested at various intervals along the small intestine.
  • Proximal refers to the Peyer's Patch that was closest to the stomach (duodenum)
  • Mesiddle refers to the Peyer's Patch that was mid-way between the stomach and cecum (jejunum)
  • distal refers to the Peyer's Patch that was in the ileum, closest to the cecum.
  • the tissue was processed for non-radioactive in situ hybridization to determine whether there was any regional distribution of mRNA expression. Digoxigenin in situ hybridization was performed using antisense and sense probes generated based on the mouse extended sequence of EDD34S_41 (SEQ ID NO:342).
  • EDD34S_41 corresponds to an mRNA that is regionally expressed in the small intestine.
  • Figure 18 shows sectioned tissue from a human small intestine biopsy containing a Peyer's Patch which was processed for non-radioactive digoxigenin in situ hybridization using an antisense probe based on the extended sequence corresponding to EDD 1 S_l 05 (SEQ ID NO 308). As indicated by the arrows, in situ hybridization signal was detected in the FAE of the human tissue.
  • isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC). Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments.
  • Typical blocking reagents include Denhardt's reagent, BLOTTO (5% w/v non-fat dried milk in phosphate buffered saline (“PBS”) , heparin, denatured salmon sperm DNA, and other commercially available proprietary formulations.
  • BLOTTO 5% w/v non-fat dried milk in phosphate buffered saline
  • heparin 5% w/v non-fat dried milk in phosphate buffered saline
  • denatured salmon sperm DNA and other commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double- stranded cDNA clone).
  • Constant Amino Acid Substitution refers to a substitution between similar amino acids that preserves an essential chemical characteristic of the original polypeptide. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g., Lesk, Ed., Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, Ed., Biocomputing: Informatics And Genome Projects, Academic Press, New York, (1993); Griffin and Griffin, Eds., Computer Analysis Of Sequence Data, Parti, Humana Press, New Jersey, (1994); von Heinje, Sequence Analysis In Molecular Biology, Academic Press, (1987); and Gribskov and Devereux, Eds., Sequence Analysis Primer, M Stockton Press, New York, (1991)).
  • identity is well known to skilled artisans (Carillo et al., SIAM J Applied Math. , 48: 1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in "Guide to Huge Computers,” Martin J. Bishop, Ed., Academic Press, San Diego, (1994) and Carillo et al., (1988), Supra.
  • EST refers to an Expressed Sequence Tag, i.e. a short sequence of a gene made from cDNA, typically in the range of 200 to 500 base pairs. Since an EST corresponds to a specific region of a gene, it can be used as a tool to help identify unknown genes and map their position in the genome.
  • DST refers to a Digital Sequence Tag, i.e., a polynucleotide that is an expressed sequence tag of the 3' end of an mRNA.
  • PPW means whole Peyer's Patch tissue.
  • PPD means Peyer's Patch domes.
  • NPPW non-Peyer's Patch associated tissue.
  • MNW whole mesenteric lymph nodes.
  • FAE means follicle associated epithelium.
  • NFAE means non-follicle associated epithelium.
  • HBSS Hank's Buffered Saline Solution (Gibco Life Sciences).
  • LB refers to Luria Broth (5 g yeast extract, lOg bacto-typtone and 5g NaCl per liter).
  • DHB refers to Dihydroxybenzoic acid.
  • PST refers to FAE isolated from Salmonella-infected mice.
  • NPPST refers to NFAE from Salmonella-infected mice.
  • This model displays a similar degree of M cell phenotypic transformation as in the original model of Kernais et al., 1997 (Reference 10).
  • This new human derived co-culture model opens up novel possibilities to study the regulation of genes involved in the phenotypic transformation and/or maintenance of intestinal epithelium and, more specifically, human M-cell phenotype.
  • EDD1 and EDD1S we have isolated total RNA from phenotypically transformed co-cultured Caco-2 and RajiB cells (which we refer to as the "Caraj" co-culture), pure Caco-2 cells, and pure RajiB cells.
  • Duplicate sets of isolated RNA were analyzed using a method of simultaneous sequence-specific identification of mRNAs known as TOGA (as referenced and described below).
  • TOGA a method of simultaneous sequence-specific identification of mRNAs known as referenced and described below.
  • EDD1 polynucleotides were identified by Mspl cleavage of sample cDNA during the TOGA ® process
  • EDD1S polynucleotides were identified by Sau3AI cleavage of sample cDNA during the TOGA ® process (see Materials and Methods).
  • DSTs Digital Sequence Tags
  • EDD1_ Digital Sequence Tags
  • EDD1S_ The intensities of the laser induced fluorescence of the labeled PCR products were compared across the 3 cell culture samples.
  • the relative gene expression values of several DSTs identified in EDD1 and EDD1S are set forth in Tables 1 and 2, respectively.
  • An example of the DST data is set forth in Figures 1 and Figure 5, which are both described in more detail below. DSTs were chosen if they were upregulated 2- fold or greater in the Caraj co-culture sample compared to the Caco-2 sample and are claimed herein. These selected DSTs were analyzed further to generate a prioritized list for further study (as described below in the Materials and Methods section).
  • DSTs that have been through the validation process were then selected for further study by satisfying either of 2 criteria.
  • the first criterion is that the DST must be upregulated in the Caraj co-culture sample compared to the Caco-2 sample.
  • the second criterion is that the DST is epithelial specific, that is, the DST must be expressed in both Caco-2 and co- culture samples, but not RajiB cells. These DSTs were chosen as being molecules expressed on M cells in addition to other intestinal epithelium cells. Validated DSTs that meet either of the 2 above criteria are claimed herein.
  • An alternative approach to identify regulated molecules associated with the M cell phenotype is to apply TOGA ® to Peyer's Patch tissues and non-Peyer's Patch associated tissues in a related species.
  • TOGA ® analysis was performed on total RNA isolated from whole Peyer's Patch (PPW), microdissected Peyer's Patch domes (PPD), whole non-Peyer's Patch associated tissue (NPPW) and whole mesenteric lymph nodes (MLNW). The sample cDNA was cleaved with Mspl during the TOGA ® process.
  • FAE Follicle associated epithelium
  • NFAE Non-Peyer's Patch associated epithelium
  • DSTs that are claimed herein from EDD2/3 were selected based on 2 criteria. The first criterion is that the DST must have a two-fold or greater upregulation in the FAE or the PPD samples compared to the NFAE or NPPW. The second criterion is that the DST must not be represented in the MLNW sample except for four peaks that were upregulated in MLNW (with digital addresses ACGC441, CAGC364, CGGC436, GCCA192). These selected DSTs were analyzed further to generate a prioritized list for further study (as described below in the Materials and Methods section).
  • M cells are a relatively rare cell population
  • any enrichment in the proportion of M cells to intestinal epithelium cells in the Peyer's Patch will facilitate identification of rare M cell specific genes.
  • oral infection of mice with bacteria will result in a conversion of a subset of Peyer's Patch intestinal epithelium cells into M cells (See References 19-21).
  • Peyer's Patch from infected mice will be enriched for M cells up to 60-70% compared to normal Peyer's Patch (unpublished results).
  • the transformed intestinal epithelium cells show multiple characteristics of normal M cells in vivo, including morphological and transport properties (See Reference 21 and unpublished results).
  • EDD4 we have isolated RNA from FAE (PPST) and NFAE (NPPST) from the Peyer's Patch of Salmonella infected mice and have analyzed duplicate samples using the TOGA ® process.
  • the DSTs identified in EDD4 are designated using the format EDD4_#.
  • the sample cDNA was cleaved with Mspl during the TOGA ® process.
  • the relative gene expression values of several DSTs identified in EDD4 are set forth in Table 4 and described in greater detail below.
  • DSTs have a two-fold or greater upregulation in the PPST sample and are not represented in the FAE or PPD samples described above. This suggests that these DSTs are M cell specific molecules that were not identified without the M cell enrichment from Salmonella infection or the M cell priming events that ensue following activation by or interaction with the Salmonella bacteria. Furthermore, these polynucleotides, polypeptides, or variants or derivatives thereof, if co-delivered with the antigen, DNA vaccine or drug delivery vehicle or vector, are important in fulfilling, achieving, sustaining, or priming the desired immune outcome. Validated DSTs that meet the above criteria are claimed herein.
  • EDD3/4S polynucleotides were identified by Sau3AI cleavage of sample cDNA during the TOGA ® process (see Materials and Methods).
  • the DSTs identified in EDD3/4S are designated using the format EDD34S_#.
  • EDD2/3 mouse normal follicle associated epithelium (FAE) was enriched for M cell specific RNA compared to whole Peyer's Patch. This enrichment was accomplished by isolating Peyer's Patch associated epithelium from the lymphoid follicle by an EDTA dissociation technique. RNA from non-Peyer's Patch associated epithelium (NFAE) was used as a control.
  • FAE non-Peyer's Patch associated epithelium
  • a final approach used to identify molecules expressed specifically by M cells was to apply TOGA to Peyer's Patch tissues and non-Peyer's Patch associated tissues from the primate, Macaca cynomolgus (also called Macacafascicularis).
  • TOGA ® analysis was performed on total RNA isolated from cynomolgus monkey Peyer's Patch follicle associated epithelium (FAE) and non-follicle associated epithelium (NFAE). The sample cDNA was cleaved with Mspl during the TOGA ® process.
  • the results of duplicate TOGA ® runs for several DSTs identified in EDD5 are set forth in Table 6.
  • the DSTs identified in EDD5 are designated using the format EDD5_#.
  • a Real-Time Quantitative PCR procedure was performed on the FAE and NFAE samples (as described in the Materials and Methods section).
  • Validated DSTs that meet the above criterion are claimed herein. Materials and Methods
  • Caco-2 cells of passage 30-40 were maintained and grown on polycarbonate filters (3.0um) as described previously (See Reference 11).
  • the Caraj co-culture conditions were as follows. 5 xlO 5 RajiB cells were resuspended in RPMLDMEM 1 :2 and added to the basolateral chamber of 14-day-old Caco-2 cell monolayers and the co- cultures were maintained for 4-6 days. The corresponding mono-cultures of Caco-2 cells on matched filter supports were used as controls. The integrity of the cell monolayers was measured by transepithelial resistance (TER) before and after co- culture with RajiB cells (See Reference 11).
  • TER transepithelial resistance
  • follicle associated epithelium FAE
  • NFAE non-follicle associated epithelium
  • HBSS Hank's Buffered Saline Solution
  • the Peyer's Patches were examined under a light dissection microscope and villi microdissected out as previously described. Normal intestine not containing Peyer's Patches were used for isolation of NFAE.
  • the FAE and NFAE tissues were prepared in the following manner. Each sample was pooled and placed in HBSS with 0.011M glucose and 25mM Hepes. The samples were then each placed in 15-20ml of HBSS (with 0.011M glucose and 25 mM Hepes) along with 40mM EDTA and stirred vigorously with a magnetic stir bar for 15 minutes in a sterilized Erlenmeyer Flask. Each cell suspension was then pipetted up and down with a wide bore 3 ml plastic Pasteur Pipet for several minutes.
  • Each cell suspension was then poured through a lOOum nylon cell strainer (Falcon) taking care to leave behind the bulk of the large paniculate matter that is mainly lymphoid follicles in the FAE sample.
  • the filter was moved to another 50ml tube and the residue material was washed out on the filter with HBSS (with 0.011M glucose and 25mM Hepes, no EDTA).
  • This residue in the FAE sample contains the majority of the Peyer's Patch dome epithelial sheaths.
  • the FAE residue retained was then washed back into a centrifuge tube, spun down at 3000 rpm for 5 minutes and the supernatant removed.
  • the NFAE tissue supernatant was also washed back into a centrifuge tube, spun down at 3000 rpm for 5 minutes and the supernatant removed.
  • Each pellet was used to isolate total RNA using standard methods well known in the art.
  • Liquid cultures of stationary phase Salmonella typhimurium were grown overnight at 37°C with shaking in Luria Broth ("LB") (5g yeast extract, lOg bacto- tryptone and 5g NaCl per liter) supplemented with 0.1% Dihydroxybenzoic acid (“DHB", 1 drop per 20ml).
  • LB Luria Broth
  • DHB Dihydroxybenzoic acid
  • the bacterial titer was determined by growing serial 10- fold dilutions of the overnight culture on LB agar plates supplemented with 1 drop 0.1% DHB. lOO ⁇ l of serial dilutions were spread on plates in duplicate and incubated overnight at 37°C. The average number of colonies per plate was determined, and the titer was calculated to be 1 x 10 10 /ml.
  • Total RNA was prepared from Caco-2, RajiB and co-culture preparations.
  • Caco-2 and co-culture preparations the cells were washed once in medium before being scraped off the filters and then spun down at 1,000 rpm for 5 minutes, snap frozen in liquid nitrogen and stored at -70°C until ready for total RNA isolation by standard methods well known in the Art.
  • For isolation of total RNA from RajiB cells the cells were pelleted, washed once in media and then snap frozen in liquid nitrogen and stored until ready for total RNA isolation by standard methods well known in the Art.
  • Total RNA from whole tissues and epithelial cell pellets were isolated by homogenization in Trizol (GibcoBRL) and subsequent purification using standard techniques well known in the art.
  • RNA from the cells and tissue samples was analyzed using a method of simultaneous sequence-specific identification of mRNAs using TOGA ® described in Sutcliffe, et al. Proc. Natl. Acad. Sci. USA, 97(5): 1976-1981 (2000); International published application WO 00/026406; U.S. application Serial No. 09/775,217, Patent No. 5,459,037; U.S. Patent No. 5,807,680; U.S. Patent No. 6,030,784; U.S. Patent No. 6,096,503, U.S. Patent 6,110,680, and U.S. Patent 6,309,834, hereby incorporated herein by reference.
  • a final PCR step that used 256 5' PCR primers in separate reactions with a universal 3' PCR primer (SEQ ID NO:351) produced PCR products that corresponded to the 3' ends of RNAs in the starting mRNA population.
  • the produced PCR products were then identified by: a) the initial 5' sequence comprising the sequence remainder of the recognition site of the restriction endonuclease used to cut and define the 5' end plus the sequence of the four parsing bases immediately 3' to the remainder of the recognition site, preferably the sequence of the entire fragment, and b) the length of the fragment. These two parameters, sequence and fragment length, were used to compare the obtained PCR products to a database of known polynucleotide sequences.
  • DSTs Digital Sequence Tags
  • double-stranded cDNA is generated from poly(A)-enriched cytoplasmic RNA extracted from the tissue samples of interest using an equimolar mixture or set of all 48 5 '-biotinylated anchor primers to initiate reverse transcription.
  • One such suitable set is G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-G-C-A-G-G-T-A-C-T-C-A-C-T-G-C-T-A-G-T-A-C-T-C-A-C-T-G-C-A-G-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 346), where V is A, C or G and N is A, C, G or T.
  • One member of this mixture of 48 anchor primers initiates synthesis at a fixed position at the 3' end of all copies of each mRNA species in the sample, thereby defining a 3 ' endpoint for each species, resulting in biotinylated double-stranded cDNA.
  • Each biotinylated double-stranded cDNA sample was cleaved with the restriction endonuclease Mspl, which recognizes the sequence CCGG, or with the restriction endonuclease Sau3 Al, which recognizes the sequence GATC.
  • the resulting fragments of cDNA corresponding to the 3' region of the starting mRNA were then isolated by capture of the biotinylated cDNA fragments on a streptavidin-coated substrate.
  • Suitable streptavidin-coated substrates include microtitre plates, PCR tubes, polystyrene beads, paramagnetic polymer beads, and paramagnetic porous glass particles.
  • a preferred streptavidin-coated substrate is a suspension of paramagnetic polymer beads (Dynal, Inc., Great Neck, NY).
  • suitable polynucleotides are G-A-T-C-C-T-C-A-C-C-A-C-C-A- C-A-G-A-G-C-T-T-C-G-A-G-G-T-C-C-C-T-T-A-G-T-G-A-G-G-G-T-T-A-A-T-T- G-G-T-A-C-C-G-A-A-T-T-T and A-A-T-T-C-G-G-T-A-C-C-A-A-T-T-A-A-C-C-C-C-C-A-C-A-A-A-C-C-C-C-A-A-A-A-C-C-C-C-A-A-A-A-C-C-C-C-A-A-A-A-T-C-C-C-A-A-A-T-G-G-T-G-A
  • the modified cDNA library was subsequently used as a template for synthesis of cRNA (copy RNA) by incubation with T3 RNA polymerase.
  • each of the cRNA preparations was processed in a three-step fashion.
  • an aliquot of cRNA was used for synthesis of first-strand cDNA using the 5' RT primer (G-A-G-C-T-C-C-A-C-C-G-C-G-G-G-T, (SEQ ID NO:349).
  • step two the cDNA product was used as a DNA template in four separate PCR reactions with each of the four 5 ' PCR primers of the form C-C-T-C-G- A-G-G-T-C-G- A-C-G-G-T- A-T-C- G-G-N (SEQ ID NO: 350) if Mspl was used as the restriction enzyme earlier in the process), each paired with a "universal" 3' PCR primer G-A-G-C-T-C-C-A-C-C-G-C- G-G-T (SEQ ID NO:351) to yield four sets of PCR reaction products ("Nl reaction products").
  • the cDNA product was used as a DNA template in four separate PCR reactions with each of the four 5' PCR primers of the form C-T-C-G-A-A-G-C-T-C-T-G-T-G-G-T-G-A-G-G-A- T-C-N (SEQ ID NO:355) each paired with a "universal" 3' PCR primer G-A-G-C-T-C- C-A-C-C-G-C-G-G-G-T (SEQ ID NO:351) to yield four sets of PCR reaction products ("Nl reaction products").
  • step three the product of each subpool was further divided into 64 subsubpools (2ng in 20 ⁇ l) for the second PCR reaction.
  • This PCR reaction comprised adding 100 ng of the fluoresceinated "universal" 3' PCR primer (SEQ ID NO: 351) conjugated to 6-FAM and 100 ng of the appropriate 5' PCR primer of the form C-G-A- C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO: 352) if the restriction enzyme used was Mspl, and using a program that included an annealing step at a temperature X slightly above the Tm of each 5' PCR primer to minimize artifactual mispriming and promote high fidelity copying.
  • Each polymerase chain reaction step was performed in the presence of TaqStart antibody (Clonetech). If the restriction enzyme used was Sau3AI, 100 ng of the 5' PCR primer of the form C-T-G-T-G-G-T-G-A-G-G-A-T-C-N-N-N-N (SEQ ED NO: 356) was used in this second PCR reaction with 100 ng of the fluoresceinated "universal" 3' PCR primer (SEQ ID NO: 351).
  • N4 reaction products The products (“N4 reaction products”) from the final polymerase chain reaction step for each of the samples were resolved on a series of denaturing DNA sequencing gels using the automated ABI Prizm 377 sequencer. Data were collected using the GeneScan software package (ABI) and normalized for amplitude and migration. In some experiments, the products were resolved by capillary electrophoresis on MegaBACE 1000 instruments (Amersham). Data from the MegaBACE 1000 were collected and processed with custom software (Digital Gene Technologies), which included size calibration and amplitude and migration normalization.
  • the PCR product was isolated, cloned into a TOPO vector (Invitrogen) and sequenced on both strands.
  • a DST cloned in this manner is EDD2_36 (SEQ ID NO: 172).
  • the extended TOGA ® assay was performed for each DST (see below).
  • PCR products were sequenced using a modification of a direct sequencing methodology (Innis et al., Proc. Nat'l. Acad. Sci., 85: 9436-9440 (1988).
  • PCR products corresponding to DSTs were gel purified and PCR amplified to incorporate sequencing primers at the 5'- and 3'- ends.
  • the sequence addition was accomplished through 5' and 3' ds-primers containing Ml 3 sequencing primer sequences (Ml 3 forward and Ml 3 reverse respectively) at their 5' ends, followed by a linker sequence and a sequence complementary to the DST ends.
  • a master mix containing all components except the gel purified PCR product template was prepared, which contained sterile H 2 O, 10X PCR II buffer, lOmM dNTP, 25 mM MgCl 2 , AmpliTaq/ Antibody mix (1.1 ⁇ g/ ⁇ l Taq antibody, 5 U/ml AmpliTaq), 100 ng/ ⁇ l of 5' ds-primer (5' TCC CAG TCA CGA CGT TGT AAA ACG ACG GCT CAT ATG AAT TAG GTG ACC GAC GGT ATC GG 3', SEQ ID NO:357), and 100 ng ⁇ l of 3' ds-primer (5' CAG CGG ATA ACA ATT TCA CAC AGG GAG CTC CAC CGC GGT GGC GGC C 3', SEQ ID NO:358).
  • 5' ds-primer 5' TCC CAG TCA CGA CGT TGT AAA ACG ACG GCT CAT ATG AAT T
  • PCR was performed using the following program: 94°C, 4 minutes and 25 cycles of 94°C, 20 seconds; 65°C, 20 seconds; 72°C, 20 seconds; and 72°C 4 minutes.
  • the resulting amplified PCR product was gel purified.
  • the purified PCR product was sequenced using a standard protocol for ABI
  • the 3' sequencing primer was the sequence 5' GGT GGC GGC CGC AGG AAT TTT TTT TTT TTT TT 3', (SEQ ID NO:361), PCR was performed using the following thermal cycling program: 96°C, 2 minutes and 29 cycles of 96°C, 15 seconds; 50°C, 15 seconds; 60°C, 4 minutes. (6) Verification Using the Extended TOGA ® Method
  • PCR primers (“Extended TOGA ® primers") were designed using one of three methods: (1) in suitable cases, the PCR product was isolated, cloned into a TOPO vector (Invitrogen) and sequenced on both strands; (2) in other cases, the TOGA ® PCR product was sequenced using a modification of a direct sequencing methodology (Innis et al., Proc. Nat'l. Acad.
  • PCR was performed using the Extended TOGA ® primers and the Nl PCR reaction products as a substrate. If the DST was generated with the restriction enzyme Mspl, oligonucleotides were synthesized with the sequence G-A-T-C-G-A-A-T-C extended at the 3' end with a partial Mspl site (C-G-G), and an additional 18 adjacent nucleotides from the determined sequence of the DST.
  • the 5' PCR primer was G-A-T-C-G-A-A- T-C-C-G-G-A-G-G-A-T-T-G-G-G-G-C-G-G-C-A-A-T-T (SEQ ID NO:364).
  • This 5' PCR primer was paired with the fluorescence labeled universal 3' PCR primer (SEQ ID NO:351) in a PCR reaction using the PCR Nl reaction product as substrate.
  • oligonucleotides were synthesized with the sequence G-A-T-C-G-A-A-T-C-C extended at the 3' end with the Sau3AI site G-A-T-C and an additional 18 nucleotides from the determined sequence of the DST.
  • the 5' PCR primer was G-A-T-C-G-A-A-T-C-C-G-A-T- C-C-C-C-T-C-T-G-A-G-T-C-C-T-C-T-G-C-C-C (SEQ ID NO:365).
  • This 5' PCR primer was paired with the fluorescence labeled universal 3' PCR primer (SEQ ID NO:351) in a PCR reaction using the PCR Nl reaction product as substrate.
  • the size of the PCR product generated with the Extended TOGA ® primer was compared to the size of the original PCR product that was produced in the TOGA ® reaction to insure that the PCR product produced using the Extended TOGA ® primer is the same length as the original PCR product. (7) DST Prioritization Process
  • Quantitative PCR using the ABI PRISM 7700 Sequence Detection System (PE Biosystems) that combines PCR, cycle-by-cycle fluorescence detection and analysis software for high-throughput quantitation of nucleic acid sequences. Reactions are characterized by the point in time when amplification of a PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles. The higher the expression level of the nucleic acid target, the earlier a significant increase in fluorescence is observed. Relative quantitation of the amount of target in the sample is accomplished by measuring the cycle number at which a significant amount of product is produced. The entire process is performed by the integrated software of the 7700 system.
  • PE Biosystems ABI PRISM 7700 Sequence Detection System
  • Primers for Real-Time Quantitative PCR validation are selected by the integrated software package accompanying the ABI PRISM 7700.
  • the templates for Real-Time PCR were cDNAs made from the RNA samples used in TOGA ® . Standard "housekeeping" genes were chosen for normalizing the quantitation of gene levels.
  • the normalization standard chosen for human samples was human GAPDH and was based on the similarity of expression across all human sample templates.
  • Beta-2 microglobulin was chosen for mouse samples based on its similarity across all mouse samples.
  • NAD+ isocitrate dehydrogenase was chosen for for mouse samples based on its similarity of expression across all monkey samples.
  • Table 7 is a listing of 5' and 3' primers used in Real-Time PCR to validate expression among the EDD1 DSTs that were successfully validated.
  • Table 8 lists the 5' and 3' primers used in Real-Time PCR studies of the EDD1S DSTs
  • Table 9 lists the 5' and 3' primers used in Real-Time PCR studies of the EDD2/3 DSTs
  • Table 10 lists the 5' and 3' primers used in Real-Time PCR studies of the EDD4 DSTs
  • Table 11 lists the 5' and 3' primers used in Real-Time PCR studies of the EDD3/4S DSTs
  • Table 12 lists the 5' and 3' primers used in Real-Time PCR studies of the EDD5 DSTs.
  • DSTs Digital Sequence Tags
  • GenBank expressed sequences
  • Statistically significant sequence matches with greater than 95% nucleotide sequence matches across the overlap region can be used to generate a contiguous sequence ("contig") and serial searches with the accumulated contig sequence can be used to assemble extended sequence associated with the DST.
  • the polypeptide encoded by the expressed mRNA can be predicted.
  • extended sequence can also be generated by making a probe containing the DST sequence. The probe would then be used to select cDNA clones by hybridization methods known in the art. These cDNA clones may be selected from libraries of cDNA clones developed from the original RNA sample, from other RNA samples, from fractionated mRNA samples, or from other widely available cDNA libraries, including those available from commercial sources. Sequences from the selected cDNA clones can be assembled into contigs in the same manner described for database sequences.
  • the cDNA molecules can also be isolated directly from the mRNA by the rapid analysis of cDNA ends (RACE) and long range PCR. This method can be used to isolate the entire full-length cDNA or the intact 5' and 3' ends of the cDNA.
  • RACE rapid analysis of cDNA ends
  • sequence includes nucleotide and amino acid sequences.
  • the query sequence can be either protein or nucleic acid or any combination therein.
  • BLAST is a statistically driven search method that finds regions of similarity between your query and database sequences.
  • segment pairs consist of gapless alignments of any part of two sequences. Within these aligned regions, the sum of the scoring matrix values of their constituent symbol pairs is higher than some level that you would expect to occur by chance alone.
  • the scores assigned in any given BLAST search can be interpreted by those skilled in the Art to determine real relationships versus random similarities.
  • the BLAST program supports four different search mechanisms: • Nucleotide Query Searching a Nucleotide Database- Each database sequence is compared to the query in a separate nucleotide-nucleotide pairwise comparison. • Protein Query Searching a Protein Database- Each database sequence is compared to the query in a separate protein-protein pairwise comparison.
  • the BLAST program was used to search for matches between a sequence of the present invention and sequences in GenBank and EST databases. In this manner, it was possible to assign either a gene identity or an EST identity to most of the DSTs identified in the present invention. "Identity" per se has an art-recognized meaning and can be calculated as described in the definitions section above. In cases where the scores obtained in a BLAST search were interpreted by the experienced investigator as a random similarity, the DST sequence was referred to as novel. Novel sequences are listed in Table 20.
  • Extended sequences for human and mouse DSTs were generated in two ways. The most common method was to use the original DST sequence to do BLAST searches in the published database, and select those sequences with nearly 100%. sequence matches. An alignment between the DST and BLAST match sequences was generated, and the 5 '-most sequence was used for additional rounds of BLAST searching. Alignments between successive BLAST match sequences were used to compile a single consensus contiguous sequence ("contig"). Extended sequences generated in this manner are listed in Table 19, marked by the superscript 2.
  • PCR products corresponding to extended mouse sequences homologous to DSTs were gel purified, cloned into the TOPO plasmid vector (Invitrogen) and sequenced on both strands. Nucleotide sequences were determined by standard techniques. In order to verify that the cloned PCR product corresponded to the sequence of interest, sequences were aligned and assembled into contigs using the DNA alignment program SEQMAN (DNASTAR).
  • primers corresponding to human sequences found within the GenBank databases were used to amplify PCR products from cDNA preparations made from Caco-2 cells, co-culture (Caco-2/RajiB) cells, human intestine or human whole brain.
  • extended human cDNA sequences were identified using the Gene Trapper cDNA Positive Selection System (Invitrogen/GibcoBRL). PCR products corresponding to extended human sequences homologous to DSTs were gel purified, cloned into the TOPO plasmid vector (Invitrogen) and sequenced on both strands. Verification of the correct PCR product was performed as described for mouse sequences.
  • mice normal male and female mice (strain C57BL/6) were obtained from a commercial source (Charles River Laboratories). Mice were sacrificed and the Peyer's Patches were immediately frozen in OCT compound (Sakura Finetechnical Co.). For studies of regional expression, Peyer's Patches were isolated from the duodenum, the jejunum and the ileum (proximal to the stomach, midway between the stomach and cecum, and distal to the stomach, respectively). For studies of human Peyer's Patches, fresh-frozen adult ileal biopsy tissues containing normal-appearing small intestinal tissue including Peyer's Patches were obtained from Tissueinformatics.
  • the riboprobes were detected using an alkaline phosphatase conjugated anti-digoxigenin secondary antibody and incubation in an alkaline phosphatase substrate solution containing NBT/BCIP as described in Current Protocols in Molecular Biology, supra p.16.
  • RNAse digestion was removed by RNAse digestion and subsequent 55° C washes in (1) 50% formamide/2XSSC, (2) 2XSSC, (3) 1XSSC, and (4) O.IXSSC.
  • the riboprobes were detected using peroxidase conjugated anti- digoxigenin (Roche) followed by TSA-FITC amplification (NEN TSA-Plus Kit - FITC).
  • the M cells were identified using the murine M cell specific lectin, Ulex Europaeus agglutinin 1 (UEA), conjugated to Rhodamine (Vector) during the antibody incubation.
  • In situ hybridization signal (FITC, green) and UEA-1 lectin (TRITC, red) were visualized using a Zeiss LSM 5 Pascal confocal microscope using a 63 X Plan Apo objective and LSM 5 software.
  • tissue sections were incubated with rat anti-mouse Igd conjugated to biotin (Zymed) and UEA-TRITC (Vector) for 1 hour. This was followed by a 1 hour incubation with Avidin-FITC (Vector). Signal of tissue sections labeled with anti-Claudin 4 mAb (FITC, green) and UEA-1 lectin (TRITC, red) was visualized using a Zeiss LSM 5 Pascal confocal microscope with a 63X Plan Apo objective and LSM 5 software.
  • Table 1 is a summary of the expression levels of mRNAs determined from cDNA through the TOGA ® Method in EDD1. Table 1 describes the relative gene expression values for duplicate sets of each of the listed DSTs in the Caco-2, Caraj co-culture, and RajiB cell lines.
  • These cDNA molecules are identified by their digital address, that is, a partial 5' terminus nucleotide sequence coupled with the length of the molecule. The 5' terminus partial nucleotide sequence is determined by the recognition site for Mspl (CCGG) and the nucleotide sequence of the parsing bases of the 5' PCR primer used in the final PCR step.
  • the digital address length of the fragment was determined by interpolation on a standard curve and, as such, may vary +/- 1-2 b.p. from the actual length.
  • the entry in Table 1 that describes a DNA molecule identified by the TOGA ® address AGGA114 (also referred to as the "digital address" Mspl AGGA114) is further characterized as having a 5' terminus partial nucleotide sequence of CGGAGGA and a digital address length of 114 base pairs.
  • the DNA molecule identified by the TOGA ® address AGGA114 is further described as being associated with intestinal epithelium and M Cells.
  • EDD1 6 corresponds to the TOGA ® address described above) and a corresponding SEQ ID NO, (e.g. SEQ ID NO:4 identifies the complete DST sequence identified by the TOGA ® address AGGA114 described above).
  • SEQ ID NO:4 identifies the complete DST sequence identified by the TOGA ® address AGGA114 described above.
  • Table 2 is a summary of the expression levels of mRNAs determined from cDNA through the TOGA ® Method in EDD1 S. Table 2 describes the relative gene expression values for duplicate sets of each of the listed DSTs in the Caco-2, Caraj co-culture, and RajiB cell lines. These cDNA molecules are identified by their digital address, that is, a partial 5' terminus nucleotide sequence coupled with the length of the molecule.
  • the 5' terminus partial nucleotide sequence is determined by the recognition site for Sau3AI (GATC) and the nucleotide sequence of the parsing bases of the 5' PCR primer used in the final PCR step.
  • the digital address length of the fragment was determined as described for EDD1.
  • the entry in Table 2 that describes a DNA molecule identified by the TOGA ® address CCCT257 (also referred to as the "digital address" Sau3AI CCCT257) is further characterized as having a 5' terminus partial nucleotide sequence of GATCCCCT and a digital address length of 257 base pairs.
  • the DNA molecule identified by the TOGA ® address CCCT257 is further described as being associated with intestinal epithelium and M Cells.
  • EDD1S_48 corresponds to the TOGA ® address described above) and a corresponding SEQ TD NO, (e.g. SEQ ID NO: 76 identifies the complete DST sequence identified by the TOGA ® address CCCT257 described above).
  • Table 3 sets forth the results obtained from TOGA ® in the EDD2/3 experiment. Again, these cDNA molecules are identified by their digital address, that is, a partial 5' terminus nucleotide sequence coupled with the length of the molecule. The 5' terminus partial nucleotide sequence is determined by the recognition site for Mspl (CCGG) and the nucleotide sequence of the parsing bases of the 5' PCR primer used in the final PCR step. For example, the entry in Table 3 that describes a DNA molecule identified by the TOGA ® address GGGT246 (also referred to as the "digital address" Mspl
  • GGGT246 is further characterized as having a 5' terminus partial nucleotide sequence of CGGGGGT and a digital address length of 246 base pairs.
  • the DNA molecule identified by the TOGA ® address GGGT246 is further described as being associated with the development of intestinal epithelium and M Cells.
  • Fluorescence intensities for each DST address identified in Table 3 are given for individual cDNA samples in each of the mouse Peyer's Patch and non-Peyer's Patch associated tissues assessed in EDD2/3 : whole Peyer's Patch (PPW), microdissected Peyer's Patch domes (PPD), whole non-Peyer's Patch associated tissue (NPPW), whole mesenteric lymph nodes (MLNW), follicle associated epithelium enriched for M cell specific RNA compared to whole Peyer's Patch (FAE), and non-Peyer's Patch associated epithelium (NFAE) as listed in the labeled columns.
  • the data was generated in duplicate (e.g.
  • DSTs were categorized according to their relative occurrence in each of the cultures as set forth in Table 3. Certain DSTs were selected for further study according to the criteria described above and were assigned a DST ED number (designated as EDD2_##, e.g. EDD2_13 corresponds to the TOGA ® address described above) and a corresponding SEQ ED NO, (e.g. SEQ ID NO: 158 identifies the DST identified by the TOGA ® address GGGT246 described above).
  • EDD2_ DST ED number
  • SEQ ID NO: 158 identifies the DST identified by the TOGA ® address GGGT246 described above.
  • Table 4 sets forth the results obtained from the EDD4 experiment. Again, these cDNA molecules are identified by their digital address, that is, a partial 5' terminus nucleotide sequence coupled with the length of the molecule. The 5' terminus partial nucleotide sequence is determined by the recognition site for Mspl (CCGG) and the nucleotide sequence of the parsing bases of the 5' PCR primer used in the final PCR step. The digital address length of the fragment was determined as for EDD1.
  • the entry in Table 4 that describes a DNA molecule identified by the TOGA ® address GATG236 (also referred to as the "digital address" Mspl GATG236) is further characterized as having a 5' terminus partial nucleotide sequence of CGGGATG and a digital address length of 236 base pairs.
  • the DNA molecule identified by the TOGA ® address GATG236 is further described as being associated with the development of intestinal epithelium and M Cells.
  • Fluorescence intensities for each DST address identified in Table 4 are given for individual cDNA samples in each of the tissue types isolated in EDD4 from Salmonella-infected mice: follicle associated epithelium (FAE) RNA from Peyer's Patch in these mice (PPST) and non-FAE RNA from Peyer's Patch in these mice (NPPST). For all of the samples, the data was generated in duplicate (e.g. PPST.1 and PPST.2) and the corresponding intensities are provided in a separate column.
  • Certain DSTs were selected for further study according to the criteria described above and were assigned a DST ED number (designated as EDD4_##, e.g. EDD4 17 corresponds to the TOGA ® address described above) and a corresponding SEQ ID NO, (e.g. SEQ ID NO: 192 identifies the DST identified by the TOGA ® address GATG236 described above).
  • Table 5 is a summary of the expression levels of mRNAs determined from cDNA through the TOGA ® Method in EDD3/4S. Again, these cDNA molecules are identified by their digital address, that is, a partial 5' terminus nucleotide sequence coupled with the length of the molecule. The 5' terminus partial nucleotide sequence is determined by the recognition site for Sau3AI (GATC) and the nucleotide sequence of the parsing bases of the 5' PCR primer used in the final PCR step. The digital address length of the fragment was determined as described for EDD1.
  • the entry in Table 5 that describes a DNA molecule identified by the TOGA ® address CCAA228 (also referred to as the "digital address" Sau3 Al CCAA228) is further characterized as having a 5' terminus partial nucleotide sequence of GATCCCAA and a digital address length of 228 base pairs.
  • the DNA molecule identified by the TOGA ® address CCAA228 is further described as being associated with intestinal epithelium and M Cells.
  • Fluorescence intensities for each DST address identified in Table 5 are given for individual cDNA samples in each of the tissue types isolated in EDD3/4S: from normal mice, follicle associated epithelium (FAE) RNA from Peyer's Patches non-FAE RNA (NFAE); from Salmonella infected mice, FAE (PPST) RNA from Peyer's Patches and NFAE (NPPST) RNA.
  • FAE follicle associated epithelium
  • NFAE non-FAE RNA
  • PPST FAE
  • NPPST NFAE
  • Table 6 sets forth the results obtained from the EDD5 experiment. Again, these cDNA molecules are identified by their digital address, that is, a partial 5' terminus nucleotide sequence coupled with the length of the molecule. The 5' terminus partial nucleotide sequence is determined by the recognition site for Mspl (CCGG) and the nucleotide sequence of the parsing bases of the 5' PCR primer used in the final PCR step. The digital address length of the fragment was determined as for EDD1.
  • the entry in Table 6 that describes a DNA molecule identified by the TOGA ® address AATC244 (also referred to as the "digital address" Mspl AATC244) is further characterized as having a 5' terminus partial nucleotide sequence of CGGAATC244 and a digital address length of 244 base pairs.
  • the DNA molecule identified by the TOGA ® address AATC244 is further described as being associated with the development of intestinal epithelium and M Cells.
  • Fluorescence intensities for each DST address identified in Table 6 are given for individual cDNA samples in each of the tissue types isolated in EDD5 from cynomolgus monkeys: follicle associated epithelium (FAE) RNA from Peyer's Patch and non-FAE RNA. For all of the samples, the data was generated in duplicate (e.g. FAE.1 and FAE.2) and the corresponding intensities are provided in a separate column.
  • Certain DSTs were selected for further study according to the criteria described above and were assigned a DST ID number (designated as EDD5_##, e.g. EDD5_ 1 corresponds to the TOGA ® address described above) and a corresponding SEQ ID NO, (e.g. SEQ ID NO: 224 identifies the DST identified by the TOGA ® address AATC244 described above).
  • TOGA ® data examples are shown in Figures 1 and 5.
  • the TOGA ® data shown in Figure 1 were generated with a 5'-PCR primer (C-G- A-C-G-G-T- A-T-C-G- G-A-G-G-A (SEQ ID NO:362) paired with the "universal" 3' primer (SEQ ID NO:351) labeled with 6-carboxyfluorescein (6FAM, ABI) at the 5' terminus.
  • PCR reaction products were resolved by gel electrophoresis on 4.5% acrylamide gels and fluorescence data acquired on ABI377 automated sequencers. Data were analyzed using GeneScan software (Perkin-Elmer). The sequences of the PCR products were determined using standard techniques.
  • Figure 1 shows the PCR products produced from mRNA extracted from Caco-2, Caraj co-culture and RajiB cells.
  • the horizontal (x) index line indicates the fragment size in base pairs.
  • the vertical (y) index indicates the relative fluorescence of each peak which corresponds to the relative expression of the RNA in the cells.
  • the vertical line drawn through the graph indicates a PCR product of approximately 114 b.p. that is associated with the M-cell like phenotype of the Caraj co-culture cells (SEQ ID NO:4; DST EDD1 6).
  • the sequences of the PCR products were determined using standard techniques.
  • FIG. 5 The results of TOGA ® analysis using a 5' PCR primer with parsing bases CCCT (SEQ ID NO: 363) and the universal 3' primer (SEQ ID NO:351) are shown in Figure 5.
  • This figure shows the PCR products produced from mRNA extracted from Caco-2, Caraj co-culture and RajiB cells.
  • the horizontal (x) index line indicates the fragment size in base pairs.
  • the vertical (y) index indicates the relative fluorescence of each peak which corresponds to the relative expression of the RNA in the cells.
  • the vertical line drawn through the graph indicates a PCR product of approximately 257 b.p. that is associated with the M-cell like phenotype of the Caraj co-culture cells (SEQ ID NO:76; DST EDD1S_48).
  • an Extended TOGA ® assay was performed for each DST.
  • the length of the PCR product generated with the Extended TOGA ® primer was compared to the length of the original PCR product that was produced in the TOGA ® reaction.
  • the results for SEQ ID NO:4 are shown in Figure 2.
  • the upper panel shows the PCR product generated with the Extended TOGA ® primer designed based on the directly sequenced sequence (SEQ ID NO: 364) and the fluorescence labeled universal 3' PCR primer (SEQ ID NO:351).
  • the middle panel of Figure 2 shows the PCR products produced in the original TOGA ® reaction using a 5' PCR primer (SEQ ID NO: 362) and the fluorescence labeled universal 3'
  • PCR primer (SEQ ED NO:351).
  • the traces from the top and middle panels are overlaid, demonstrating that the PCR product produced using the Extended TOGA ® primer based on the directly sequenced sequence is the same length as the original PCR product.
  • Figure 6 shows similar results for SEQ ID NO: 76. The length of the PCR product generated with an Extended TOGA ® primer generated from a database match was compared to the length of the original PCR product that was produced in the TOGA ® reaction.
  • the upper panel shows the PCR product generated with the Extended TOGA ® primer designed based on the database-matched sequence (SEQ ID NO: 365) and the fluorescence labeled universal 3' PCR primer (SEQ ID NO:351).
  • the middle panel of Figure 6 shows the PCR products produced in the original TOGA ® reaction using a 5' PCR primer (SEQ ID NO: 363) and the fluorescence labeled universal 3' PCR primer (SEQ ID NO:351).
  • the traces from the top and middle panels are overlaid, demonstrating that the PCR product produced using the Extended TOGA ® primer based on the database-matched sequence is the same length as the original PCR product.
  • EDD4 includes the Real-Time PCR assay results for EDD4 DSTs validated on normal murine FAE and NFAE. Examples of Real-Time PCR validation are shown in Figures 3 and 7.
  • Figure 3 compares the results of Real Time PCR to duplicate runs of TOGA ® . As the chart in Figure 3 illustrates, the DST EDD1 6 (SEQ ID NO:4) is found in a much greater proportion in the Caraj co-culture than in the Caco-2 and RajiB cultures in both TOGA ® runs and Real Time PCR.
  • Figure 7 compares the results of Real Time PCR to duplicate runs of TOGA ® for SEQ ID NO: 76.
  • the DST EDD1S_48 (SEQ ID NO: 76) is found in a greater proportion in the Caraj co- culture than in the Caco-2 and RajiB cultures in both TOGA ® runs and Real Time PCR.
  • the relative abundance is measured as compared to Caco-2.
  • differential gene expression identified by TOGA was confirmed by Real- Time PCR assays for several DSTs identified in EDDl, EDD1S, EDD2/3, EDD4, EDD3/4 and EDD5. Assignment of Identities to DSTs
  • the BLAST program was used to search for matches between a DST sequence of the present invention and sequences in GenBank and EST databases. In most cases, it was possible to assign either a gene identity or an EST identity to each DST. For example, EDD1S 71 (SEQ ID NO: 94) corresponds to the human laminin beta 3 subunit. In cases where the scores obtained in the BLAST search were interpreted by the experienced investigator as a random similarity, the DST sequence was referred to as novel. Table 20 lists the novel sequences identified in the present invention. In some cases, extended sequences of novel DSTs were identified by the Gene Trapper cDNA Positive Selection System (Invitrogen/GibcoBRL). Such novel extended sequences are listed in Table 20.
  • EDD1S_71 SEQ ID NO: 94
  • EDD2_13 SEQ ID NO: 1528 correspond to the human (EDD1S_71) and mouse (EDD2_13) laminin beta 3 subunit.
  • in situ hybridization to mouse Peyer's Patch tissue using an antisense probe based on the entire nucleotide sequence corresponding to EDD1_6 identified cells positive for signal in the FAE and the follicle-associated crypts Table 21 summarizes the results of in situ hybridization studies performed on mouse Peyer's Patches using probes corresponding to DSTs from EDDl, EDD1S, EDD2/3, EDD4, and EDD3/4S
  • more than 30 DSTs were expressed in the FAE, in some cases exclusively (for example, EDD1S_42, probe generated based on extended sequence SEQ ID NO 295), or in other cases, with signal also localizing to other tissue sites (for example, EDDl S I 05, probe generated based on SEQ ID NO 309)
  • EDDl S I 05 probe generated based on SEQ ID NO 309
  • the expression pattern of DSTs identified by TOGA ® should ideally be localized to the FAE of Peyer's Patches Using Peyer's Patches from human small intestinal biopsy samples, n situ hybridization studies localized the expression of EDD1S_48 (probe generated based on extended sequence SEQ ID NO 298) and EDDl S I 05 (probe generated based on extended sequence SEQ ID NO 308) to the FAE, as indicated by the arrows in Figure 10 and Figure 18 This confirms that TOGA ® studies of the Caraj co-culture model (EDDl and EDD1S) successfully identified mRNA transcripts specific to the FAE of human Peyer's Patches
  • PGRP-S belongs to the PGRP family of proteins that recognize peptidoglycan, a component of bacterial cell walls.
  • PGRP-S is an extracellular protein that is expressed at high levels in bone marrow (See Reference 23).
  • a related protein, PGRP-L is a transmembrane cell surface protein expressed at high levels in the liver.
  • murine PGRP-L also called TAGL-alpha; in the present invention, the sequence is referred to as EDD4 PGRP-L, SEQ ID NO: 332
  • EDD4 PGRP-L SEQ ID NO: 332
  • Figure 16 is an example of colocalization studies of PGRP-L (SEQ DD NO: 332) immunofluorescence in situ hybridization signal and fluorescently-labeled UEA signal. Unlike EDD4 17, PGRP-L expression does not colocalize with UEA signal, indicating that this gene is expressed by FAE cells distinct from M cells. Thus, identification of an M cell specific DST, EDD4_17 (SEQ ID NO: 192), led to the identification of a protein expressed in FAE but not within M cells.
  • EDDl S 48 is the human Clostridium perfringens enterotoxin receptor, claudin-4.
  • Claudin-4 is a member of a family of transmembrane tissue-specific proteins, the claudins, that are essential components of intercellular tight junctions.
  • claudin-4 was detected by immunofluorescent labeling using an antibody to murine claudin-4.
  • Fluorescently-labeled UEA identified M cells (bottom panel, arrows point to two examples of M cells).
  • the anti-claudin-4 staining colocalized with M cells. This result establishes that TOGA ® analysis of the Caraj human coculture model led to the identification of a protein expressed by murine M cells.
  • the present invention also relates to the genes corresponding to SEQ DD NOs: 1- 345, and translations of SEQ DD NOs: 1-345.
  • the corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material. Methods of determining the corresponding genes are described further in subsection (9) above. Homologs, Paralogs and Orthologs
  • homologs of the polynucleotides, polypeptides and genes of the invention and regions thereof including paralogous genes and orthologous genes are also provided in the present invention.
  • Nucleic acid homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for the desired homolog.
  • Studies of gene and protein evolution often involve the comparison of homologs—sequences that have common origins but may or may not have common activity. Sequences that share an arbitrary level of similarity determined by alignment of matching bases are called homologous.
  • homologous There are many cases in which genes have duplicated, assumed somewhat different functions and been moved to other regions of the genome (e.g. alpha and beta globin).
  • paralogs e.g Lundin, 1993, who refers to Fitch, 1976 for this distinction. They must be distinguished from orthologs (homologous genes in different species, such as beta globin in human and mouse) if any sensible comparisons are to be made.
  • orthologs homologous genes in different species, such as beta globin in human and mouse
  • Parenter genes are genes within the same species produced by gene duplication in the course of evolution. They may be arranged in clusters or distributed on different chromosomes, an arrangement which is usually conserved in a wide range of vertebrates. "Orthologous genes” describes homologous genes in different species which are descended from the same gene in the nearest common ancestor. Orthologs tend to have similar function.
  • the Comparative Gene Mapping Committee recommended explicit criteria for establishing homology between genes mapped in different species, as well as urging inclusion of specific criteria in comparative gene mapping publications (O'Brien and Graves, 1991). The evidence for gene homology might also be recorded in The Comparative Animal Genome database (TCAGdb). Revised criteria for determining homology can include any of the following (the most stringent are asterixed):
  • Preferred embodiments of the present invention include homologs, paralogs and orthologs of the polynucleotides, polypeptides and genes of the invention and regions thereof.
  • polypeptides of the invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art. See, e.g., Curr. Prot. Mol. Bio., supra p.16, Chapter 16.
  • the polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification (such as multiple histidine residues), or an additional sequence for stability during recombinant production.
  • polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
  • a recombinantly produced version of a polypeptide, including the secreted polypeptide can be substantially purified by the one-step method described in Smith & Johnson (Gene, 67:31-40, 1988).
  • Polypeptides of the invention also can be purified from natural or recombinant sources using antibodies of the invention raised against the secreted protein in methods which are well known in the art.
  • polypeptides of the invention also can be expressed by the target cells such as M cells or intestinal epithelium of the gastrointestinal tract following transfection of the same cells and provide their function in these cells following expression therein.
  • the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called Signal P (Nielsen et al., Protein Engineering, 10: 1-6 (1997), which predicts the cellular location of a protein based on the amino acid sequence.
  • Signal P Neelsen et al., Protein Engineering, 10: 1-6 (1997), which predicts the cellular location of a protein based on the amino acid sequence.
  • McGeoch and von Heinje are inco ⁇ orated.
  • the present invention provides secreted polypeptides having a sequence corresponding to the translations of SEQ DD NOs: 1-345 and their corresponding genes which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point.
  • secreted polypeptides having a sequence corresponding to the translations of SEQ DD NOs: 1-345 and their corresponding genes which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point.
  • cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species.
  • the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence.
  • the naturally occurring signal sequence may be further upstream from the predicted signal sequence.
  • the predicted signal sequence will be capable of directing the secreted protein to the ER.
  • Polynucleotide, Polypeptide and Gene Variants Polynucleotide, Polypeptide or gene variants differ from the polynucleotides, polypeptides and genes of the present invention, but retain essential properties thereof. In general, variants have close similarity overall and are identical in many regions to the polynucleotide or polypeptide of the present invention.
  • polynucleotides having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity will encode a polypeptide identical or nearly identical to an amino acid sequence contained in the translations of SEQ DD NOs: 1-345 or their corresponding genes.
  • Further embodiments of the present invention include genes having at least 80% identity, more preferably at least 90% identity, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to genes corresponding to a sequence contained in SEQ DD NOs: 1-345.
  • genes having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity respectively to genes of the invention genes hybridizable to genes of the invention, genes with sequential nucleotide deletions from either the 5' terminus or the 3' terminus, or a species homolog of genes of the invention will encode a polypeptide identical or nearly identical to an amino acid sequence contained in the translations of genes of the invention.
  • the above polypeptides should exhibit at least one biological activity of the protein.
  • polypeptides of the present invention include polypeptides having at least 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98%, or 99% similarity to an amino acid sequence contained in translations of SEQ DD NOs: 1-345 or their corresponding genes.
  • Methods for aligning polynucleotides or polypeptides are codified in computer programs, including the GCG program package (Devereux et al., Nuc. Acids Res. 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul et al., J. Molec. Biol. 215:403 (1990)), and Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl 53711) which uses the local homology algorithm of Smith and Waterman (Adv. in AppMath., 2:482-489 (1981)).
  • the parameters are set such that the percentage of identity is calculated over the full length of the reference polynucleotide or gene or region thereof and that gaps in identity of up to 5% of the total number of nucleotides in the reference polynucleotide are allowed.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci., 6:237-245 (1990)).
  • sequence includes nucleotide and amino acid sequences.
  • sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences.
  • the result of said global sequence alignment is presented in terms of percent identity.
  • a polynucleotide having a nucleotide sequence of at least 95% "identity" to a sequence contained in SEQ DD NOs: 1-345 means that the polynucleotide is identical to a sequence contained in SEQ DD NOs: 1-345 or the cDNA except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the total length (not just within a given 100 nucleotide stretch).
  • a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference polypeptide means that the amino acid sequence of the polypeptide is identical to the reference polypeptide except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the total length of the reference polypeptide.
  • a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the variants may contain alterations in the coding regions, non-coding regions, or both.
  • polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide are preferred.
  • Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred.
  • variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred.
  • Polynucleotide variants can be produced for a variety of reasons. For instance, a polynucleotide variant may be produced to optimize codon expression for a particular host (i.e., codons in the human mRNA may be changed to those preferred by a bacterial host, such as E. coli).
  • Variants may also arise by the process of ribosomal frameshifting, by translational read-through at naturally occuring stop codons, and by decoding of in-frame translational stop codons UGA through insertion of selanocysteine (See The RNA World, 2 nd edition, ed: Gesteland, R.F., Cech, T.R., & Atkins, J.F.; Cold Spring Harbor Laboratory Press, 1999).
  • the variants may be allelic variants.
  • Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (Lewin, Ed., Genes II, John Wiley & Sons, New York (1985)).
  • allelic variants can vary at either the polynucleotide and/or polypeptide level.
  • non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis. See, e.g., Curr. Prot. Mol. Bio., Chapter 8.
  • variants may be generated to improve or alter the characteristics of the polypeptides of the present invention.
  • polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as decreased aggregation.
  • aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity (see, e.g., Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes, 36: 838-845 (1987); Cleland et al., Crit. Rev. Therap.
  • C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained.
  • the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus.
  • Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.
  • the invention further includes polypeptide variants which show substantial biological activity.
  • Such variants include deletions, insertions, inversions, repeats, frameshifting, read-through translational variants, and substitutions selected according to general rules known in the art so as have little effect on activity.
  • Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science, 247: 1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
  • the first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, the amino acid positions which have been conserved between species can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions in which substitutions have been tolerated by natural selection indicate positions which are not critical for protein function. Thus, positions tolerating amino acid substitution may be modified while still maintaining biological activity of the protein.
  • the second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site-directed mutagenesis or alanine-scanning mutagenesis (the introduction of single alanine mutations at every residue in the molecule) can be used (Cunningham et al., Science, 244:1081-1085 (1989)). The resulting mutant molecules can then be tested for biological activity.
  • tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin; replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and T ⁇ ; and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
  • variants of the present invention include: (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code; (ii) substitution with one or more of amino acid residues having a substituent group; (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (e.g., polyethylene glycol); (iv) fusion of the polypeptide with additional amino acids, such as an IgG Fc fusion region peptide, a leader or secretory sequence, or a sequence facilitating purification.
  • substitutions with one or more of the non-conserved amino acid residues where the substituted amino acid residues may or may not be one encoded by the genetic code
  • substitution with one or more of amino acid residues having a substituent group such as a compound to increase the stability and/or solubility of the polypeptide (e.g., poly
  • polynucleotide fragment refers to a short polynucleotide having a nucleic acid sequence contained in that shown in SEQ DD
  • the short nucleotide fragments are preferably at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length.
  • a fragment "at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in that shown in SEQ ED NOs: 1- 345 or their corresponding genes. These nucleotide fragments are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, and greater than 150 nucleotides) are preferred.
  • polynucleotide fragments of the invention include, for example, fragments having a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401- 450, to the end of SEQ DD NOs: 1-345 or their corresponding genes.
  • “about” includes the particularly recited ranges, larger or smaller by several nucleotides (i.e., 5, 4, 3, 2, or 1 nt) at either terminus or at both termini.
  • these fragments encode a polypeptide which has biological activity.
  • polypeptide fragment refers to a short amino acid sequence contained in the translations of SEQ DD NOs: 1-345 and their corresponding genes. Protein fragments may be "free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, or 61 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, or 60 amino acids in length. In this context "about” includes the particularly recited ranges, larger or smaller by several amino acids (5, 4, 3, 2, or 1) at either extreme or at both extremes.
  • Preferred polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids ranging from 1- 60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred.
  • polypeptide fragments encoding these polypeptide fragments are also preferred.
  • polypeptide, gene, and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix-forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • Polypeptide fragments of the translations of SEQ DD NOs: 1-345 or their corresponding genes falling within conserved domains are specifically contemplated by the present invention.
  • polynucleotide fragments and gene fragments encoding these domains are also contemplated.
  • Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention.
  • the biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
  • antigenic epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA, 82:5131-5135 (1985), further described in U.S. Patent No. 4,631,211).
  • antigenic epitopes preferably contain a sequence of at least seven, more preferably at least nine, and most preferably between about 15 to about 30 amino acids. Antigenic epitopes are useful to raise antibodies, including monoclonal antibodies, that specifically bind the epitope.
  • immunogenic epitopes can be used to induce antibodies or to select binding partners according to methods well known in the art. (See, e.g., Sutcliffe et al., (1983) Supra; Wilson et al., (1984) Supra; Chow et al., Proc. Natl. Acad. Sci., USA, 82:910-914; and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985)).
  • a preferred immunogenic epitope includes the secreted protein.
  • the immunogenic epitope may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse).
  • a carrier protein such as an albumin
  • the immunogenic epitope may be prescribed without a carrier, if the sequence is of sufficient length (at least about 25 amino acids).
  • immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting.)
  • antibody As used herein, the term "antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody
  • antibodies of the present invention include chimeric, single chain, and human and humanized antibodies.
  • the antibodies may be chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies.
  • Such humanized antibodies may be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. Co et al. (Nature, 351:501-2, 1991).
  • a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody.
  • a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody.
  • Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature, 332:323, 1988), Liu et al. (PNAS, 84:3439, 1987), Larrick et al.
  • One method for producing a human antibody comprises immunizing a non- human animal, such as a transgenic mouse, with a polypeptide translated from a nucleotide sequence chosen from SEQ DD NOs: 1-345 or the corresponding genes, whereby antibodies directed against the polypeptide translated from a nucleotide sequence chosen from SEQ DD NOs: 1-345 or the corresponding genes are generated in said animal.
  • Procedures have been developed for generating human antibodies in non- human animals.
  • the antibodies may be partially human, or preferably completely human.
  • mice have been prepared in which one or more endogenous immunoglobulin genes are inactivated by various means and human immunoglobulin genes are introduced into the mice to replace the inactivated mouse genes.
  • transgenic mice may be genetically altered in a variety of ways.
  • the genetic manipulation may result in human immunoglobulin polypeptide chains replacing endogenous immunoglobulin chains in at least some (preferably virtually all) antibodies produced by the animal upon immunization. Examples of techniques for production and use of such transgenic animals are described in U.S. Patent Nos. 5,814,318, 5,569,825, and 5,545,806, which are inco ⁇ orated by reference herein.
  • Antibodies produced by immunizing transgenic animals with a polypeptide translated from a nucleotide sequence chosen from SEQ DD NOs: 1-345 or the corresponding genes are provided herein.
  • Monoclonal antibodies may be produced by conventional procedures, e.g., by immortalizing spleen cells harvested from the transgenic animal after completion of the immunization schedule.
  • the spleen cells may be fused with myeloma cells to produce hybridomas by conventional procedures. Examples of such techniques are described in U.S. Patent No. 4,196,265, which is inco ⁇ orated by reference herein.
  • a method for producing a hybridoma cell line comprises immunizing such a transgenic animal with an immunogen comprising at least seven contiguous amino acid residues of a polypeptide translated from a nucleotide sequence chosen from SEQ DD NOs: 1-345 or the corresponding genes; harvesting spleen cells from the immunized animal; fusing the harvested spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds a polypeptide translated from a nucleotide sequence chosen from SEQ DD NOs: 1-345 or the corresponding genes.
  • hybridoma cell lines and monoclonal antibodies produced therefrom, are encompassed by the present invention.
  • Monoclonal antibodies secreted by the hybridoma cell line are purified by conventional techniques. Examples of such techniques are described in U.S. Patent No. 4,469,630 and U.S. Patent No. 4,361,549.
  • Antibodies are only one example of binding partners to epitopes or receptor molecules. Other examples include, but are not limited to, synthetic peptides, which can be selected as a binding partner to an epitope or receptor molecule.
  • the peptide may be selected from a peptide library as described by Appel, JR, Philippe, B, Houghten, RA, and Pinilla, C (Biotechniques, 13, 901-905); and Dooley CT., Ny, P, Bidlack, JM, and Houghten, RA (J. Biol. Chem 273, 18848-18856, 1998).
  • Binding assays can select for those binding partners (antibody, synthetic peptide, or other molecule) with highest affinity for the epitope or receptor molecule, using methods known in the art. Such assays may be done by immobilizing the epitope or receptor on a solid support, allow binding of the library of antibodies or other molecules, and wash away those molecules with little or no affinity. Those binding partners or antibodies with highest affinity for the epitope or receptor will remain bound to the solid support. Alternatively, arrays of candidate binding partners may be immobilized, and a labeled soluble receptor molecule is allowed to interact with the array, followed by washing unbound receptors. High affinity binding would be detectable by the presence of bound label.
  • Antibodies or other binding partners may be employed in an in vitro procedure, or administered in vivo to inhibit biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ DD NOs: 1-345 or the corresponding genes.
  • a therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective for reducing a biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-345 or the corresponding genes.
  • administration of antibodies specific for the identified polynucleotides could be used to modulate the transport properties of M cells or in the conversion of intestinal epithelium cells to an M cell phenotype through loss of function of the target protein.
  • Antibodies which have an inhibitory effect on the transport function of M cells can be used to effectively block the pathogenesis of microorganisms that exploit the M cell as a mechanism for invasion (See References 4- 6). Administration of these inhibitory antibodies could be used in cases where potential contact with these microorganisms is anticipated.
  • Antibodies or binding partners to receptors or polypeptides found on M cells can also be attached to drug-loaded particles, antigens, DNA vaccines, immune modulators, other peptides and proteins for specific binding to the M cells for enhanced delivery of the drug-loaded particles, antigens, DNA vaccines, immune modulators, other peptides and polypeptides to these cells for enhanced mucosal immunity pu ⁇ oses or outcome.
  • Exemplary vaccines, drugs, and therapeutic genes include but not limited to the following: Vaccines: H.pylori, C. difficile, rotavirus, influenza, diptheria, tetanus, pertussis, Hepatitis A,B and C, conjugate vaccines including S. pneumonia; Drugs: peptides including insulin, LHRH, buserelin, vasopressin, recombinant interleukins including rL-2 and JL-12; Genes delivered by vectors such as adeno-associated virus, liposomes, PLGA, canarypox virus, adenovirus, retrovirus,: eg. EL-1 antagonist, GM-CSF antagonists, etc.
  • conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to an antibody directed against a polypeptide translated from a nucleotide sequence chosen from SEQ DD NOs: 1-345 or the corresponding genes.
  • detectable or therapeutic agent attached to an antibody directed against a polypeptide translated from a nucleotide sequence chosen from SEQ DD NOs: 1-345 or the corresponding genes.
  • agents include but are not limited to diagnostic radionucleotides, therapeutic radionucleotides, and cytotoxic drugs. See, e.g., Thrush et. al (Annu.Rev.Immunol., 14:49-71, 1996, p. 41).
  • the conjugates find use in in vitro or in vivo procedures. Fusion Proteins
  • Fusion proteins may be used for targeting certain molecules or particles to the M cells, as one end may be for targeting, while the other end may be used to attach to the delivery vehicle.
  • Any polypeptide of the present invention can be used to generate fusion proteins.
  • the polypeptide of the present invention when fused to a second protein, can be used as an antigenic tag.
  • Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide.
  • secreted proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.
  • domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions.
  • the fusion does not necessarily need to be direct, but may occur through linker sequences.
  • fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
  • polypeptides of the present invention can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • EP A 0 464 533 (Canadian counte ⁇ art 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (see, e.g., EP A 0 232 262).
  • deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hTL-5
  • Fc portions for the pu ⁇ ose of high-throughput screening assays to identify antagonists of hTL-5
  • polypeptides of the present invention can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein (Proc. Natl. Acad. Sci. USA 86:821-824 (1989)).
  • Another peptide tag useful for purification, the "HA" tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell, 37:767 (1984)).
  • Other fusion proteins may use the ability of the polypeptides of the present invention to target the delivery of a biologically active peptide. This might include focused delivery of a toxin to tumor cells, or a growth factor to stem cells.
  • any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention. See, e.g., Curr. Prot. Mol. Bio., Chapter 9.6.
  • Vectors, Host Cells, and Protein Production The present invention also relates to vectors containing a polynucleotide or gene of the present invention or regions thereof, host cells, and the production of polypeptides by recombinant techniques.
  • the vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • Polynucleotides or genes of the present invention or regions thereof can be inserted in phage display vectors to select molecule ligands for binding to M cells and assays can be developed to test for the effectiveness of the ligand in promoting active and specific transport of the vector across M cells. See O'Mahony, U.S. Patent No. 6117632.
  • the polynucleotides or genes or regions thereof may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid.
  • the vector may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. See, e.g., Curr. Prot. Mol. Bio., Chapters 9.9, 16.15.
  • the polynucleotide or gene or gene region insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells, and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria include pQ ⁇ 70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, PNH16A, PNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc.
  • eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may, in fact, be expressed by a host cell lacking a recombinant vector.
  • a polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • polypeptides of the present invention may be glycosylated or may be non- glycosylated.
  • polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
  • Polypeptides of the present invention can also be recovered from products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.
  • the expression of the polynucleotide, gene or gene region or the level of the intact polypeptide product can be increased in order to increase the rate of transport of the drug delivery vehicle, or decreased in order to decrease the rate of transport of such vehicle.
  • a polynucleotide or gene or region thereof of the invention can be administered alone or with other polynucleotides to a mammalian subject by a recombinant expression vector comprising the polynucleotide or gene or regions thereof.
  • a mammalian subject can be a human, baboon, chimpanzee, macaque, cow, horse, sheep, pig, dog, cat, rabbit, guinea pig, rat or mouse.
  • the recombinant vector comprises a polynucleotide shown in SEQ DD NOs: 1-345 or their corresponding genes or region thereof or a polynucleotide which is at least 98% identical to a nucleic acid sequence shown in SEQ DD NOs: 1-345 or their corresponding genes or region thero
  • the recombinant vector comprises a variant polynucleotide that is at least 80%, 90%, or 95% identical to a polynucleotide comprising SEQ DD NOs: 1-345 or their corresponding genes or region thereof or it may constitute just a fragment of the polypeptides that retains the desired activity or characteristics.
  • a polynucleotide, gene, region therof or recombinant expression vector of the invention or a combination thereof with an antigen, epitope, or with a DNA vaccine can be used to express a polynucleotide in said subject for the treatment of, for example, inflammatory bowel disease, glutenenteropathy, infectious diseases or any other disease treatable by an oral vaccine (including, without limitation, vaccines for H.pylori, C. difficile, rotavirus, influenza, diptheria, tetanus, pertussis, Hepatitis A, B, and C, conjugate vaccines including S. pneumonia, etc. and DNA vaccines for the same ).
  • an oral vaccine including, without limitation, vaccines for H.pylori, C. difficile, rotavirus, influenza, diptheria, tetanus, pertussis, Hepatitis A, B, and C, conjugate vaccines including S. pneumonia, etc. and DNA vaccines for the same ).
  • a polynucleotide, gene, or region thereof in target cells would effect greater production of the encoded polypeptide.
  • the regulation of other genes may be secondarily up- or down-regulated.
  • transcription factors specifically required for the induction of M cell differentiation may be sufficient to induce intestinal epithelium to acquire M cell function by upregulating those genes necessary to provide trans-epithelial transport machinery.
  • a naked polynucleotide, gene or region thereof can be administered to target cells.
  • Polynucleotides, genes, or a region thereof and recombinant expression vectors of the invention can be administered as a pharmaceutical composition (including, without limitation, genes delivered by vectors such as adeno-associated virus, lipsomes, PLGA, canarypox virus, adenovirus, retrovimses including IL-1 and GM-CSF antagonists).
  • Such a composition comprises an effective amount of a polynucleotide, gene or region thereof or recombinant expression vector, and a pharmaceutically acceptable formulation agent selected for suitability with the mode of administration.
  • Suitable formulation materials preferably are non-toxic to recipients at the concentrations employed and can modify, maintain, or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adso ⁇ tion, or penetration of the composition. See Remington 's Pharmaceutical Sciences (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990).
  • the cells of a mammalian subject may be transfected in vivo, ex vivo, or in vitro.
  • Administration of a polynucleotide, gene or region thereof or a recombinant vector containing a polynucleotide, gene or region thereof to a target cell in vivo may be accomplished using any of a variety of techniques well known to those skilled in the art.
  • U.S. Patent No. 5,672,344 describes an in vivo viral-mediated gene transfer system involving a recombinant neurotrophic HSV-1 vector.
  • compositions of polynucleotides and recombinant vectors can be transfected in vivo by oral, buccal, parenteral, rectal, or topical administration as well as by inhalation spray.
  • parenteral as used herein includes, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitoneally.
  • nucleic acids and/or vectors of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more vectors of the invention or other agents.
  • the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
  • Non-viral delivery system Another delivery system for polynucleotides or genes of the invention or regions thereof is a "non-viral" delivery system.
  • Techniques that have been used or proposed for gene therapy include DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO 4 precipitation, gene gun techniques, electroporation, lipofection, and colloidal dispersion (Mulligan, R., (1993) Science, 260 (5110): 926-32). Any of these methods are widely available to one skilled in the art and would be suitable for use in the present invention. Other suitable methods are available to one skilled in the art, and it is to be understood that the present invention may be accomplished using any of the available methods of transfection.
  • Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation.
  • an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used.
  • Antisense oligonucleotide molecules can be provided in a DNA constmct and introduced into a cell as described above to decrease the level of gene products of the invention in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates,'' acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
  • Modifications of gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of a gene of the invention. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred.
  • triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons.
  • Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al., in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N Y., 1994).
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent nucleotides, can provide sufficient targeting specificity for mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a polynucleotide or gene of the invention or region thereof. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al., (1992) Trends Biotechnol, 10:152-158; Uhlmann et al., (1990) Chem. Rev., 90:543-584; Uhlmann et al., (1987) Tetrahedron. Lett., 215:3539-3542.
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, (1987) Science, 236:1532-1539; Cech, (1990) ⁇ WJ. Rev. Biochem., 59:543-568; Cech, (1992) Curr. Opin. Struct. Biol., 2:605-609; Couture & Stinchcomb, (1996) Trends Genet., 12:510-515. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Patent 5,641,673).
  • ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a polynucleotide of the invention can be used to generate ribozymes which will specifically bind to mRNA transcribed from the polynucleotide or gene or region thereof.
  • Methods of designing and constructing ribozymes which can cleave RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. (1988) Nature, 334:585-591).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, e.g., Gerlach et al., EP 321,201).
  • Specific ribozyme cleavage sites within a RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences' GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary stmctural features which may render the target inoperable.
  • Suitability of candidate RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • the nucleotide sequences shown in SEQ DD NOs: 1-345 inclusive and their complements and their corresponding genes and regions thereof provide sources of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target.
  • the hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA constmct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA constmct into cells in which it is desired to decrease polynucleotide expression.
  • the constmct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA constmct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destmction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • Polypeptides of the present invention can be used to modulate M cell function by providing competition for ligands within the intestinal lumen.
  • patients can be administered a polypeptide of the present invention in an effort to reduce the uptake of allergens present in food, or reduce the immune response to infectious agents present within the intestine.
  • antibodies directed to a polypeptide of the present invention can also be used to alter M cell function.
  • administration of an antibody directed to a polypeptide of the present invention can bind and reduce ove ⁇ roduction of the polypeptide.
  • administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
  • Polypeptides can be used as antigens to trigger immune responses.
  • antibodies against the peptide may cross-link these molecules on the surface of M cells, and may up-regulate the function or activity of M cells or may induce other intestinal epithelial cells to acquire an active M cell functional phenotype.
  • a mammalian subject preferably a human
  • delivery of the polypeptide or antibody may be in the form of injection or transplantation of cells or tissues containing an expression vector such that a recombinant form of the polypeptide will be secreted by the cells or tissues, as described above for transfected cells.
  • the frequency and dosage of the administration of the polypeptides or antibodies will be determined by factors such as the biological activity of the pharmacological preparation and the goals in modulating or modifying intestinal epithelial or M cell function. In the case of antibody deliveries, the frequency of dosage will also depend on the ability of the antibody to bind and neutralize the target molecules in the target tissues.
  • Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. See, e.g., Curr. Prot. Mol. Bio., Chapterl 1.15.
  • the polypeptides of the present invention can be used to test the following biological activities. Using molecules encoded by polynucleotides or genes of the present invention or drug and oral vaccine delivery
  • Effective delivery of dmgs or oral vaccines across the intestinal epithelium depends on identifying a target receptor on intestinal epithelium, so that a specific ligand for that receptor can be used to bind that receptor.
  • the ligand ideally would be linked physically to the dmg to be delivered, whether it is an encapsulated form of dmg, a synthetic vaccine antigen, or other formulation of a compound requiring delivery across the intestinal epithelium.
  • the binding of the ligand-dmg complex to the receptor would then induce specific transport across the epithelium, permitting release of the dmg, antigen or other compound within the body.
  • targeting of antigens to M cell specific receptors would enable focused delivery across the FAE to the associated Peyer's Patch lymphoid tissue, triggering specific immune responses.
  • the polynucleotides, genes and corresponding polypeptides of the present invention comprising M cell or intestinal epithelium specific receptors, could be used first to identify ligands with high binding affinity for the target receptors.
  • These ligands may be polypeptide ligands, including antibodies, or they may be small organic compounds, and may be identified by any of several various binding assays, as discussed above. In some cases, they may be similar to naturally occurring polypeptides, carbohydrate moieties, or glycolipids produced by microorganisms present in the gut.
  • These ligands may be chemically linked to a delivery vehicle (e.g., encapsulated dmg), to a polypeptide antigen, or to a compound of interest.
  • Oral delivery of the ligand and its attached parcel would be able to bind with high specificity to the receptor present on M cells or other intestinal epithelial cell. Binding would induce transepithelial transport, and effective and efficient delivery across the intestinal epithelium to the region of intestine desired.
  • the ligands to the receptors encoded by the polynucleotides and genes of the present invention can be used for the development of oral vaccines (including, without limitation, vaccines for H. pylori, C. difficile, rotavirus, influenza, diphtheria, tetanus, pertussis, Hepatitis A, B, and C, conjugate vaccines including S. pneumonia, etc. and DNA vaccines for the same) by fusing a vaccine to the ligand or fusing a particulate system such as a liposome which is loaded with the vaccine to the targeting ligand. Once the fusion vaccine is transported across the epithelium, it becomes bioavailable, inducing an immune response for protecting the host from infection.
  • oral vaccines including, without limitation, vaccines for H. pylori, C. difficile, rotavirus, influenza, diphtheria, tetanus, pertussis, Hepatitis A, B, and C, conjugate vaccines including S. pneumonia,
  • oral delivery of excess amounts of ligand or soluble forms of receptor encoded by polynucelotides of the present invention may be used to interfere with productive binding to receptors on intestinal epithelium, and prevent transepithelial transport of naturally occurring molecules or other similar molecules.
  • titrations of ligands and soluble receptors in mixtures may be used to produce a controlled rate of delivery of dmg or antigen for transepithelial transport.
  • polynucleotides, polypeptides, and genes of the present invention and regions thereof can be used in assays to test for one or more biological activities. If these polynucleotides, polypeptides, genes and regions thereof do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides, polypeptides, genes and regions thereof could be used to treat the associated disease.
  • a polypeptide, polynucleotide, gene or region thereof of the present invention may be useful in treating deficiencies or disorders of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Since M cell transport of intestinal antigens appears to be important in triggering immune responses to food antigens and enteric infectious organisms, modification of M cell activity by polypeptides, polynucleotides, or genes of the present invention or regions thereof may be used to alter the immune response to specific antigens.
  • a polynucleotide, polypeptide or gene of the present invention or region thereof may be useful in the treatment, diagnosis, or detection of autoimmune disorders.
  • Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destmction of the host tissue. Therefore, the administration of a polypeptide, polynucleotide or gene of the present invention or region thereof that inhibits an immune response in the Peyer's Patch, particularly the proliferation, differentiation, or chemotaxis of T-cells, or in some way results in the induction of tolerance, may be an effective therapy in preventing autoimmune disorders.
  • the molecules targeted for dmg delivery to M cells can be used to transport antigens for modifying immune responses to self tissues.
  • autoimmune disorders examples include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Pu ⁇ ura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye disease.
  • allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated by a polypeptide or polynucleotide of the present invention, or delivery targeted to these molecules may be used to transport allergens for the pu ⁇ ose of inducing immunological tolerance to the allergens.
  • these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
  • a polypeptide, polynucleotide, or gene of the present invention or a region thereof can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated.
  • the immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Delivery of antigens to Peyer's Patches by targeting molecules of the invention may be used to trigger immune responses to infectious agents.
  • the polypeptide, polynucleotide or gene of the present invention or a region thereof may also directly inhibit immune responses to the infectious agent.
  • a polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds.
  • the binding of the polypeptide and the molecule may activate (i.e., an agonist), increase, inhibit (i.e., an antagonist), or decrease activity of the polypeptide or the molecule bound.
  • Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
  • the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a stmctural or functional mimetic (see, Coligan et al., Current Protocols in Immunology 1(2),
  • the molecule can be closely related to the natural receptor to which the polypeptide binds or, at least, related to a fragment of the receptor capable of being bound by the polypeptide (e.g., an active site). In either case, the molecule can be rationally designed using known techniques.
  • the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane.
  • Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.
  • the assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.
  • the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures.
  • the assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.
  • an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody.
  • the antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate. Therefore, the invention includes a method of identifying compounds which bind to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound or a library of compounds such as a combinatorial library with a polypeptide of the invention; and (b) determining if binding has occurred.
  • the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound or a library of compounds such as a combinatorial library with a polypeptide of the invention, (b) assaying a biological activity, and (c) determining if a biological activity of the polypeptide has been altered.
  • Compounds that are identified by their ability to bind to polypeptides of the invention can then be used as targeting ligands to deliver molecules or particles to M cells for the pu ⁇ ose of delivering dmg formulations (including, without limitation, peptides including insulin, LHRH, buserelin, vasopressin, recombinant interleukins including IL-2, EL- 12, etc., genes delivered by vectors such as adeno-associated vims, lipsomes, PLGA, canarypox vims, adenovirus, retrovimses including D -1 and GM-CSF antagonists) or a vaccine (including, without limitation, vaccines for H.pylori, C.
  • dmg formulations including, without limitation, peptides including insulin, LHRH, buserelin, vasopressin, recombinant interleukins including IL-2, EL- 12, etc., genes delivered by vectors such as adeno-associated vims
  • polynucleotides and polypeptides of the present invention may be embodied in other forms without departing from the teachings or essential characteristics of the invention.
  • the described embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore to be embraced therein.

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Abstract

Selon cette invention, on a identifié et revendiqué des polynucléotides, des polypeptides, des anticorps, des partenaires de liaison et des gènes associés à l'épithélium intestinal ou au développement, à la différenciation ou à la fonction des cellules M, à l'immunité des muqueuses, à l'administration de médicaments et au diagnostic et au traitement de maladies.
EP02763973A 2001-04-04 2002-04-04 Genes exprimes dans l'epithelium intestinal et les cellules m des plaques de peyer Withdrawn EP1383929A4 (fr)

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US281416P 2001-04-04
PCT/US2002/010873 WO2002080852A2 (fr) 2001-04-04 2002-04-04 Genes exprimes dans l'epithelium intestinal et les cellules m des plaques de peyer

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1463523A2 (fr) * 2001-08-20 2004-10-06 Genentech, Inc. Proteine et acide nucleique du gene 1 induit par l'acide retinoique de type gpcr

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EP1419252A2 (fr) * 2001-04-04 2004-05-19 Merrion Research I Limited Analyse genetique de plaques de peyer et de cellules m, procedes et compositions ciblant les plaques de peyer et les recepteurs de cellules m
AU2003243151A1 (en) 2002-08-16 2004-03-03 Agensys, Inc. Nucleic acid and corresponding protein entitled 251p5g2 useful in treatment and detection of cancer
JPWO2008056705A1 (ja) * 2006-11-10 2010-02-25 国立大学法人 東京大学 Ｍ細胞特異的抗体
WO2019018410A1 (fr) * 2017-07-17 2019-01-24 The General Hospital Corporation Compositions et procédés de traitement des maladies inflammatoires de l'intestin
CN108763197B (zh) * 2018-05-10 2021-11-09 上海依智医疗技术有限公司 一种医疗术语库的形成方法和装置

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US6225290B1 (en) * 1996-09-19 2001-05-01 The Regents Of The University Of California Systemic gene therapy by intestinal cell transformation
US5910403A (en) * 1997-05-15 1999-06-08 The Regents Of University Of California Methods for measuring cellular proliferation and destruction rates in vitro and in vivo
EP1000084A4 (fr) * 1997-07-08 2003-03-26 Human Genome Sciences Inc 123 proteines humaines secretees

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Title
DATABASE EMBL [Online] 27 April 1998 (1998-04-27), "on86e12.s1 Soares_NFL_T_GBC_S1 Homo sapiens cDNA clone IMAGE:1563598 3', mRNA sequence." XP002365227 retrieved from EBI accession no. EM_PRO:AA928150 Database accession no. AA928150 *
See also references of WO02080852A2 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1463523A2 (fr) * 2001-08-20 2004-10-06 Genentech, Inc. Proteine et acide nucleique du gene 1 induit par l'acide retinoique de type gpcr
EP1463523A4 (fr) * 2001-08-20 2005-08-10 Genentech Inc Proteine et acide nucleique du gene 1 induit par l'acide retinoique de type gpcr

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AU2002338345A1 (en) 2002-10-21
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EP1383929A4 (fr) 2006-06-07

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