WO2010129710A1 - Use of rankl to induce differentiation of microfold cells (m cells) - Google Patents

Abstract

The differentiation of microfold (M) cells is important in mucosal immunity. The differentiation of M cells is desirable, such as in situations in which a host response to an antigen is sub-optimal, or wherein increased uptake of a compound is desired. Methods are provided herein for increasing a mucosal immune response. The methods include selecting a subject in need of an increased mucosal immune response and administering to a subject a therapeutically effective amount of RANKL or another agonist of RANK, wherein the administration of RANKL or administration of an agonist of RANK results in the differentiation of M cells in the intestine or in another mucosal epithelium. The antigen can be a vaccine antigen. Methods are also provided for increasing drug delivery to a mucosa. The methods include administering to a subject a therapeutically effective amount of RANKL or an agonist of RANK and a therapeutically effective amount of a drug. In some examples, the drug is included in a particle or a bacteria, such as a Lactococcus.

Description

USE OF RANKL TO INDUCE DIFFERENTIATION OF MICROFOLD

CELLS (M CELLS)

CROSS REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Patent Application

No. 61/176,045 filed on May 6, 2009, and hereby is incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT This invention was made with United States government support pursuant to grant DK064730, from the National Institute of Diabetes, Digestive, and Kidney Diseases; the United States government has certain rights in the invention.

FIELD This application relates to the field of immunology, specifically to microfold

(M) cell differentiation.

BACKGROUND

The organized lymphoid tissues of the intestine are inductive sites for both the generation of secretory IgA and the generation of T cell tolerance to antigens present in the intestinal lumen, including those derived from food and the commensal flora (Fagarasan and Honjo, 2004; Iweala and Nagler, 2006). The Peyer' s patches (PP) in the distal small intestine (ileum) are an example of an organized lymphoid tissue. The follicle-associated epithelium (FAE) that covers the lymphoid follicles of both PP and isolated lymphoid follicles (ILF) contains specialized epithelial cells known as microfold cells (M cells) that provide a portal for efficient sampling of particulate antigens from the lumen (Kraehenbuhl and Neutra, 2000; Pabst et al., 2007). Antigens acquired through this major pathway for antigen sampling in the intestine are delivered into intraepithelial pockets within the M cells that lymphocytes and APC access from the subepithelial dome region. The M cell-mediated antigen acquisition pathway is involved in the development of immune responses to both pathogenic bacteria and commensal bacteria. Production of protective fecal IgA in mice after oral infection with invasive Salmonella species requires the presence of PP with M cells (Hashizume et al., 2008; Martinoli et al., 2007).

In addition, some commensal bacteria internalized through M cells are passed into dendritic cells that travel with their cargo to the draining mesenteric lymph node, leading to both IgA antibody production and establishment of T cell tolerance (Macpherson and Uhr, 2004). M cells also promote the development of T cell tolerance to antigens acquired through the gastrointestinal tract. Targeting ovalbumin (OVA) to mouse M cells via the reovirus sigma 1 protein resulted in enhanced development of oral tolerance in CD4⁺ T cells (Suzuki et al., 2008).

While most M cells in the small intestine of wild type mice are localized to the FAE of PP and ILF, occasional villi contain clusters of cells known as villous M cells that exhibit all the major defining characteristics of PP M cells including reactivity with the UEA-I lectin recognizing αl-2 fucose, stubby surface microvilli, presence of an intraepithelial pocket, and the capacity to ingest and transcytose particles the size of bacteria (Jang et al., 2004). Dense and diffuse patterns of distribution of villous M cells were distinguished by the density of M cells. About 40 to 50 dense villous M cell clusters were detected in the mouse small intestine. While the basic functional and ultrastructural features of M cells were initially described over 30 years ago (Owen and Jones, 1974), many basic questions about M cell differentiation and function remain unsolved.

SUMMARY

The differentiation of microfold (M) cells is important in mucosal immunity. The differentiation of M cells is desirable, such as in situations in which a host response to an antigen is sub-optimal, or wherein increased uptake of a compound is desired.

Methods are provided herein for increasing a mucosal immune response, for example, in the Peyer's patches. The methods include selecting a subject in need of an increased mucosal immune response and administering to a subject a therapeutically effective amount of RANKL or an agonist of RANK, wherein the administration of RANKL or an agonist of RANK results in the differentiation of M cells in the intestine. The methods also include administering to the subject a therapeutically effective amount of an antigen of interest. The therapeutically effective amount of RANKL or the agonist of RANK is administered sufficiently prior to administration of the antigen of interest to allow M cells to differentiate. In other embodiments, RANKL or an agonist of RANK is administered orally in formulations designed to selectively target delivery of RANKL or an agonist of RANK to the small intestine for the purpose of inducing M cell differentiation. Thus, in one embodiment, the methods disclosed herein are of use in generating a mucosal immune response to an antigen. In one specific, non-limiting example, the antigen is an antigen of a pathogen, such as an infectious agent. In a further embodiment, the methods disclosed herein are of use in augmenting an immune response to a vaccine. In yet another embodiment, the methods disclosed herein are of use in generating an immune response to a tumor antigen.

Methods are also provided for increasing delivery of an agent, such as a drug, to a mucosa that has M cells, such as to the gastrointestinal mucosa, for example the mucosa of the small intestine, such as the ileum. Methods are also provided for increasing delivery of an agent to the nasal mucosa. The methods include administering to a subject a therapeutically effective amount of RANKL or an agonist of RANK and a therapeutically effective amount of a drug. The therapeutically effective amount of RANKL or an agonist of RANK is administered sufficiently prior to the drug to allow M cells to differentiate and increase drug uptake, thereby increasing the delivery of the drug to the mucosa. In some examples, the drug is included in a particle or a bacteria, such as a Lactococcus.

The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-1D are a set of digital images and a graph showing that Peyer's patches (PP) of RANKL^"7" mice contain very few M cells. (A) UEA-I staining reveals far fewer M cells in a representative follicle from a RANKL^"7" PP compared to a wild type control PP. The number of M cells counted in each imaged follicle is indicated in the lower left hand corner. The arrowhead points to a rare residual UEA-I⁺ cell in the RANKL-/- follicle. The follicles shown are from the middle portion of the small intestine. Scale bar, 200 μm. (B) PP from RANKL^"7" mice also displayed loss of cells reactive with NKM 16-2-4. The large arrowheads point to cells that are dual staining for NKM 16-2-4 and UEA-I. The small arrowhead indicates a cell positive for UEA-I, but negative for NKM 16-2-4. The follicles shown are from the middle portion of the small intestine. Scale bar, 100 μm. (C) FAE of RANKL^"7" mice showed a lack of characteristic M cell features by transmission electron microscopy. The large arrowheads indicate intraepithelial pockets within the M cells. The small arrowheads point to the shorter microvilli found on the apical surface of M cells. Scale bars, 50 μm. (D) Scatter plot summarizing frequency of UEA-I⁺ M cells in individual PP follicles from RANKL^"7" and control mice (n=5 mice for both groups). All PP examined were assigned to 1 of 5 groups based on proximal to distal position. **, p < 0.001 (compared to control mice by ANOVA).

Figures 2A-2D are a sets of digital images and graphs showing administration of rRANKL to RANKL^"7" mice restores PP M cells. (A) RANKL^"7" mice were treated i.p. for 7 days with 250 μg/day of GST-RANKL or GST as a control. UEA-I staining of representative follicles from the distal small intestine shows restoration of the normal number and pattern of UEA-I⁺ M cells by GST- RANKL, but not by GST. Scale bar, 200 μm. (B) Reconstitution of UEA-I⁺ M cells requires 5 days of treatment with 250 μg/day GST-RANKL. The results based on 3 to 6 mice at each time point and include data from all PP except the most distal PP. (C) Uptake of 200 nm diameter fluorescent beads from isolated small intestinal loops into PP of RANKL^"7" mice 90 minutes after bead injection is restored to near wild type levels by prior treatment with GST-RANKL for 5 days. Frozen sections of PP were stained with rhodamine-UEA-I and DAPI. The inset shows clusters of fluorescent beads (indicated by arrowheads) within 2 adjacent UEA-I⁺ M cells on the surface of the FAE. Scale bar, 100 μm. (D) Summary scatter plot showing that GST-RANKL treatment reconstitutes both numbers of UEA-I⁺ M cells and uptake of fluorescent beads as assessed by image analysis of the percentage of pixels containing green fluorescent beads within the area of the PP follicles. * in B and D indicates p < 0.01 compared to untreated mice by ANOVA.

Figures 3A-3E are digital images and a graph showing administration of rRANKL induces a massive expansion in villous M cells. (A,B) Whole mount staining of villous M cells in untreated BALB/c mice with rhodamine-UEA-I and DAPI. Villous M cells in diffuse (A) and dense (B) patterns are found on occasional villi; inset in the lower right of B shows the pattern of M cell distribution on a single villus at higher magnification. Scale bar, 200 μm in A and 500 μm in B. (C,D) BALB/c mice were given 100 μg of GST-RANKL i.p. every 12 hours for 3 days, leading to an increased fraction of villi with M cells and an increase in the number of M cells per villus. Both diffuse (C) and dense (D) patterns of villous M cell distribution were observed. Scale bar, 200 μm in C and 500 μm in D. (E) Summary graph showing kinetics of induction of villous M cells in the diffuse and dense patterns of distribution following GST-RANKL administration.

Figures 4A-4D are digital images and graphs showing treatment of wild type mice with neutralizing anti-RANKL leads to loss of PP M cells. (A,B) BALB/c mice were treated i.p. with 250 μg of IKK22-5 mAb on days 0, 2, 4, and 6. On day 8, isolated bowel loops containing PP were injected with fluorescent beads and the mice euthanized after 90 minutes. Anti-RANKL treatment led to loss of UEA-I⁺ M cells detected by whole mount staining (A) and a decrease in the uptake of fluorescent beads detected on frozen sections of PP from the bead-injected loops (B). Scale bar, 200 μm in A and 100 μm in B. (C) Summary of data from all PP analyzed in A and B for UEA-I⁺ cells and fluorescent bead uptake. (D) Anti-

RANKL-induced loss of UEA-I⁺ M cells detected by whole mount staining begins by 4 days after start of antibody treatment. * in C and D indicates p < 0.001 compared to untreated mice by t test (C) or ANOVA (D).

Figure 5 is a set of digital images showing intestinal epithelial cells express RANK. Serial frozen sections of a PP from a wild type BALB/c mouse were stained with rat mAbs to mouse RANK, mouse RANKL, or an isotype control rat IgG2a antibody, followed by a biotinylated secondary antibody, streptavidin-peroxidase, and FITC-tyramide plus DAPI as a counterstain. RANK expression is localized to epithelial cells in the FAE and on the adjacent villi. Reticular stromal cells concentrated immediately beneath the epithelial layer are the only cells on which RANKL is detected. Scale bar, 200 μm.

Figures 6A-6D are sets of digital images and graphs showing that PP from RANKL^"7" mice contain far fewer autofluorescent particles than PP from control mice. (A) Frozen sections of PP from control and RANKL^"7" mice (n=4 per group) were stained with anti-CD68 followed by Alexa546-goat-anti-rat IgG plus DAPI. Autofluorescent particles detected in the FITC channel are much more prominent in the subepithelial dome area of control PP compared to RANKL^"7" PP. Merges of the autofluorescence with CD68 staining show that many of the autofluorescent particles in both groups are found in CD68⁺ macrophages (examples indicated by arrowheads). The insets show the boxed areas at higher magnification that contain CD68⁺ cells harboring autofluorescent particles. (B) The degree of autofluorescence in the PP follicle area was quantitated by threshold analysis. (C) Unstained whole mount preparations of PP from control and RANKL^"7" mice (n= 4 per group) were examined for autofluorescence using a FITC filter set. The dashed lines in each image outline a single B cell containing follicle. Autofluorescent particles were normally present and concentrated in the central portion of each dome in PP from control mice. The intensity of autofluorescence was substantially less in PP from RANKL^"7" mice and the autofluorescent particles were not selectively concentrated in the center of the dome as in control mice. Scale bar, 1 mm. (D) The degree of autofluorescence in the PP follicles was quantitated by threshold analysis. **, p <0.001 (compared to control mice by ANOVA). Figures 7A-7B are digital images showing that in vivo treatment of mice with polyclonal antibodies to RANK induces villous M cell development. BALB/c mice were treated 35 micrograms of affinity-purified polyclonal anti-mouse RANK antibodies on days 0 and 2. On day 4, the small intestinal tissue from a treated mice and an untreated control mouse were analyzed by microscopy for the presence of villous M cells by staining with rhodamine-UEA-I. Increased numbers of single villous M cells and clusters of villous M cells developed after the anti-RANK treatment. Figure 8 is a graph showing the effect of pretreatment with GST-RANKL on the production of IgM at day 10 following oral immunization of mice with fixed E. coli that produce ovalbumin (OVA).

Figure 9 is a graph showing the synergistic effect of administering RANKL in combination with another adjuvant (LPS) on the production of IgM antibodies to an antigen.

SEQUENCE LISTING

The nucleic and amino acid sequences are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

The Sequence Listing is submitted as an ASCII text file, Annex C/St. 25 text file, recreated on May 4, 2010, 9.21 KB, which is incorporated by reference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is an exemplary amino acid sequence of wild-type human RANKL.

SEQ ID NO: 2 is an exemplary amino acid sequence of wild-type mouse RANKL. SEQ ID NOs: 3 and 4 are additional exemplary mouse RANKL proteins, wherein the sequence begins at amino acid 137.

SEQ ID NO: 5 and 6 are primer sequences. DETAILED DESCRIPTION

Specific factors released from the lymphoid microenvironment immediately beneath the FAE have the potential to elicit M cell differentiation in the FAE and promote the function of M cells, but specific signaling mediators with such activity have not been identified to date (Kerneis et al., 1997; Mach et al., 2005). The most promising in vitro systems developed to date to study M cell function rely on the induction of M cell characteristics in cultured Caco-2 cells co-cultured with Peyer's patch lymphocytes or a B lymphoblastoid cell line (des Rieux et al., 2007; Kerneis et al., 1997; Kerneis et al., 2000).

RANKL (receptor activator of NF-KB ligand) is a member of the TNF superfamily (Bachmann et al., 1999), also referred to as TNF-related activation induced cytokine (TRANCE) and TNFSFl 1. Like TNF-α, RANKL is initially synthesized as a transmembrane protein that can be released from the cell surface following cleavage by one of several metalloproteases (Hikita et al., 2006; Lum et al., 1999). RANKL signals through its receptor RANK and a downstream pathway that involves TRAF6 and the activation of NF- KB (Galibert et al., 1998; Wong et al., 1998). Osteoprotegerin (OPG) is a soluble decoy receptor for RANKL that allows for tight regulation of the circulating levels of RANKL (Simonet et al., 1997). A major breakthrough in establishing a biological role for RANKL-RANK interactions was the discovery that RANKL signaling through RANK is required for normal osteoclast function (Kim et al., 2000b; Kong et al., 1999). Mice deficient in either RANKL or RANK have osteopetrosis and severe skeletal abnormalities because they lack the number of osteoclasts needed to remodel bone normally. RANKL-RANK signaling is also involved in several other critical biological processes including development of lymph nodes, development of medullary thymic epithelial cells (mTEC), mammary gland lactation, and provision of survival signals to dendritic cells (Akiyama et al., 2008; Fata et al., 2000; Hikosaka et al., 2008; Kim et al., 2000b; Kong et al., 1999; Wong et al., 1997). The absence of all lymph nodes in RANKL-deficient mice demonstrates that

RANKL is an essential mediator in lymphoid organogenesis (Kim et al., 2000b; Kong et al., 1999). RANKL induces lymphotoxin (LT) αiβ₂ expression by lymphoid tissue inducer cells in the lymph node anlage (Yoshida et al., 2002). RANKL is not required for PP development, but the reduced size of PP reported in two independent lines of RANKL-deficient mice indicates that RANKL signaling is contributing to normal PP function (Kim et al., 2000b; Kong et al., 1999). More specific studies of PP and mucosal immune function were not reported as part of the initial characterization of these mice.

It is disclosed herein that RANKL is the critical factor controlling the differentiation of M cells from RANK-expressing intestinal precursor cells. Thus, RANKL is of use as for immunization, such as by administering RANKL (or a derivative or mimetic thereof), or administering an antibody that activates RANK to induce M cell differentiation. RANKL can be administered prior to the administration of an immunogen, such as a vaccine to enhance the immunogenic effect of the subsequently administered vaccine. In several embodiments, RANKL is administered prior to the administration of the immunogen, such as the vaccine. RANKL can also be used to increase the uptake of agents, such as chemical compounds, small molecules, proteins and nucleic acids to a mucosa, such as the intestinal mucosa or the nasal mucosa. Additional embodiments are disclosed below.

/. Abbreviations

DNA: deoxyribonucleic acid

FAE: follicle-associated epithelium (FAE)

GST: glutathione S-transferase

ILF: isolated lymphoid follicles M: Microfold mTEC: medullary thymic epithelial cells

OVA: ovalbumin

PBS: phosphate buffered saline

PCR: polyermase chain reaction PP: Peyer's Patch

RANK: receptor activator of NF- KB

RANKL: receptor activator of NF-KB ligand TNF: tumor necrosis factor

TRANCE: TNF-related activation induced cytokine, also known as RANKL

//. Terms Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Adjuvant: A vehicle used to enhance antigenicity; such as a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages) that is generally administered at the same time as the antigen. Immunstimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants (for example see U.S. Patent No. 6,194,388; U.S. Patent No. 6,207,646; U.S. Patent No. 6,214,806; U.S. Patent No. 6,218,371; U.S. Patent No. 6,239,116; U.S. Patent No. 6,339,068; U.S. Patent No. 6,406,705; and U.S. Patent No. 6,429,199). Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term "antigen" includes all related antigenic epitopes. "Epitope" or "antigenic determinant" refers to a site on an antigen to which B and/or T cells respond. In one embodiment, T cells respond to the epitope, when the epitope is presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance.

An antigen can be a tissue-specific antigen, or a disease-specific antigen. These terms are not exclusive, as a tissue- specific antigen can also be a disease specific antigen. A tissue-specific antigen is expressed in a limited number of tissues, such as a single tissue. Specific, non-limiting examples of a tissue specific antigen are a prostate specific antigen and/or a breast specific antigen. A tissue specific antigen may be expressed by more than one tissue, such as, but not limited to, an antigen that is expressed in both prostate and breast tissue. A disease- specific antigen is expressed coincidentally with a disease process. Specific non-limiting examples of a disease-specific antigen are an antigen whose expression correlates with, or is predictive of, tumor formation, such as prostate cancer and/or breast cancer. A disease- specific antigen can be an antigen recognized by T cells or B cells.

Amplification: Of a nucleic acid molecule (e.g., a DNA or RNA molecule) refers to use of a technique that increases the number of copies of a nucleic acid molecule in a specimen. An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of amplification can be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing using standard techniques. Other examples of amplification include strand displacement amplification, as disclosed in U.S. Patent No. 5,744,311; transcription-free isothermal amplification, as disclosed in U.S. Patent No. 6,033,881; repair chain reaction amplification, as disclosed in WO 90/01069; ligase chain reaction amplification, as disclosed in EP-A-320 308; gap filling ligase chain reaction amplification, as disclosed in U.S. Patent No. 5,427,930; and NASBA™ RNA transcription-free amplification, as disclosed in U.S. Patent No. 6,025,134.

Antibody: Immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. A naturally occurring antibody (e.g., IgG, IgM, IgD) includes four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. However, it has been shown that the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody. Thus, these antigen-binding fragments are also intended to be designated by the term "antibody." Specific, non-limiting examples of binding fragments encompassed within the term antibody include (i) a Fab fragment consisting of the V_L, V_H, C_L and C_HI domains; (ii) an F_d fragment consisting of the V_H and C_HI domains; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a dAb fragment (Ward et al., Nature 341:544-546, 1989) which consists of a V_H domain; (v) an isolated complementarity determining region (CDR); and (vi) a F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.

Immunoglobulins and certain variants thereof are known and many have been prepared in recombinant cell culture (e.g., see U.S. Patent No. 4,745,055; U.S. Patent No. 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP 125,023; Faoulkner et al., Nature 298:286, 1982; Morrison, J. Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239, 1984).

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects. Conservative variants: "Conservative" amino acid substitutions are those substitutions that do not substantially affect the biological activity of RANKL. Specific, non-limiting examples of a conservative substitution include the following examples: Original Residue Conservative Substitutions

Ala Ser

Arg Lys

Asn GIn, His

Asp GIu Cys Ser

GIn Asn

GIu Asp

His Asn; GIn

He Leu, VaI Leu He; VaI

Lys Arg; GIn; GIu

Met Leu; He

Phe Met; Leu; Tyr

Ser Thr Thr Ser

Trp Tyr

Tyr Trp; Phe

VaI He; Leu The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Non- conservative substitutions are those that reduce an activity or antigenicity. cDNA (complementary DNA): A piece of DNA lacking internal, non- coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.

Cancer: A malignant neoplasm that has undergone characteristic anaplasia with loss of differentiation, increased rate of growth, invasion of surrounding tissue, and is capable of metastasis. For example, prostate cancer is a malignant neoplasm that arises in or from prostate tissue, and breast cancer is a malignant neoplasm that arises in or from breast tissue (such as a ductal carcinoma). Residual cancer is cancer that remains in a subject after any form of treatment given to the subject to reduce or eradicate cancer. Metastatic cancer is a cancer at one or more sites in the body other than the site of origin of the original (primary) cancer from which the metastatic cancer is derived.

Dendritic cell (DC): Dendritic cells are the principle antigen presenting cells (APCs) involved in primary immune responses. Dendritic cells include plasmacytoid dendritic cells and myeloid dendritic cells. Their major function is to obtain antigen in tissues, migrate to lymphoid organs and present the antigen in order to activate T cells. Immature dendritic cells originate in the bone marrow and reside in the periphery as immature cells. Differentiation: The process by which cells become more specialized to perform biological functions, and differentiation is a property that is totally or partially lost by cells that have undergone malignant transformation. For example, dendritic cell precursors such as monocytes or plasmacytoid dendritic cells can differentiate into dendritic cells under the influence of certain cytokines and growth factors. It is disclosed herein that RANKL or an agonist of RANK can induce the differentiation of intestinal epithelial precursors into microfold cells.

Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, i.e. that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or 8 to 10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., "Epitope Mapping Protocols" in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). In one embodiment, an epitope binds an MHC molecule, such an HLA molecule or a DR molecule. These molecules bind polypeptides having the correct anchor amino acids separated by about eight to about ten amino acids, such as nine amino acids. Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter- dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see e.g., Bitter et al, Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide for transcription of the nucleic acid sequences.

Gut-associated lymphoid tissue (GALT): Tissue present in all vertebrates that includes small intestinal Peyer's patches as well as isolated lymphoid follicles. These structures consist of tightly packed follicles that are separated by small T-cell areas and contain 95% surface IgM-positive B-cells. Antigens and microorganisms in the intestinal lumen are separated from the lymphoid cells of Peyer's patches by follicle- associated epithelium (FAE) containing microfold (M)-cells. M-cells are clearly different from neighboring enterocytes and goblet cells and are specialized for transepithelial transport of antigens and microorganisms. The apical membranes of M-cells contain glycoconjugates that may mediate the binding of lectins and lectin-like microbial surface proteins. Lectin binding studies have identified fucosylated glycoconjugates unique to the apical surfaces and cytoplasmic contents of mouse M-cells. Microfold cells are scattered in the epithelial sheet covering lymphoid follicles of Peyer' s patches. M cells are responsible for transport of antigen, bacteria, viruses and microparticles to the antigen-presenting cells, within and under the epithelial barrier.

Host cells: Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen- specific response"). In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies.

Immunogenic peptide: A peptide which comprises an allele-specific motif or other sequence such that the peptide will bind an MHC molecule and induce a cytotoxic T lymphocyte ("CTL") response, or a B cell response (e.g. antibody production) against the antigen from which the immunogenic peptide is derived. In one embodiment, immunogenic peptides are identified using sequence motifs or other methods, such as neural net or polynomial determinations, known in the art. Typically, algorithms are used to determine the "binding threshold" of peptides to select those with scores that give them a high probability of binding at a certain affinity and will be immunogenic. The algorithms are based either on the effects on MHC binding of a particular amino acid at a particular position, the effects on antibody binding of a particular amino acid at a particular position, or the effects on binding of a particular substitution in a motif-containing peptide. Within the context of an immunogenic peptide, a "conserved residue" is one which appears in a significantly higher frequency than would be expected by random distribution at a particular position in a peptide. In one embodiment, a conserved residue is one where the MHC structure may provide a contact point with the immunogenic peptide.

Immunogenic peptides can also be identified by measuring their binding to a specific MHC protein (e.g. HLA-A02.01) and by their ability to stimulate CD4 and/or CD8 when presented in the context of the MHC protein. Immunogenic composition: A composition comprising an epitope of a polypeptide that induces a measurable T response against cells expressing the polypeptide, or induces a measurable B cell response (e.g., production of antibodies that specifically bind the antigen) against the polypeptide. It further refers to isolated nucleic acids encoding an immunogenic epitope of the polypeptide that can be used to express the epitope (and thus be used to elicit an immune response against this polypeptide). For in vitro use, the immunogenic composition can consist of the isolated nucleic acid, protein or peptide. For in vivo use, the immunogenic composition will typically comprise the nucleic acid, protein or peptide in pharmaceutically acceptable carriers, and/or other agents. A polypeptide, or nucleic acid encoding the polypeptide, can be readily tested for its ability to induce a T or a B cell response in various assays.

Inhibiting or treating a disease: Inhibiting a disease refers to inhibiting the full development of a disease. In several examples, inhibiting a disease refers to lessening symptoms of the disease, such as delaying the development of infection or decreasing the symptoms of a person who is known to be infected with a pathogen, or lessening a sign or symptom of the disease. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition related to the disease. "Prevention" refers to an intervention such that the signs and symptoms of the disease do not occur. Isolated: An "isolated" biological component (such as a nucleic acid or protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non- limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. Lymphocytes: A type of white blood cell that is involved in the immune defenses of the body. There are two main types of lymphocytes: B cells and T cells.

Mammal: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

Maturation: The process in which an immature cell, such as dendritic cell precursor or an intestinal epithelial cell precursor, changes in form or function to become a functionally mature dendritic cell (an antigen-presenting cell (APC)) or a microfold cell, respectively.

Nanospheres: Particles having a general average diameter in the range of about 50 to about 999 nanometers Open reading frame (ORF): A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. Peptide: A chain of amino acids of between 3 and 30 amino acids in length.

In one embodiment, a peptide is from about 7 to about 25 amino acids in length. In yet another embodiment, a peptide is from about 8 to about 10 amino acids in length. In yet another embodiment, a peptide is about 9 amino acids in length.

Peptide Modifications: RANKL includes synthetic embodiments, analogs (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules obtained starting with the disclosed peptide sequences) and variants

(homologs) of these proteins can be utilized in the methods described herein. Each polypeptide of this disclosure is comprised of a sequence of amino acids, which may be either L- and/or D- amino acids, naturally occurring and otherwise.

Peptides can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, can be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester, or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are each independently H or C₁-C_1O alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the peptide, whether amino-terminal or side chain, can be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or can be modified to C₁-C₁₆ alkyl or dialkyl amino or further converted to an amide.

Hydroxyl groups of the peptide side chains may be converted to C₁-C₁₆ alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C₂-C₄ alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the peptides of this invention to select and provide conformational constraints to the structure that result in enhanced stability. Peptidomimetic and organomimetic embodiments are envisioned, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains, resulting in such peptido- and organomimetics of RANKL having measurable or enhanced ability to generate an immune response. For computer modeling applications, a pharmacophore is an idealized three-dimensional definition of the structural requirements for biological activity. Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD). See Walters, "Computer-Assisted Modeling of Drugs," in Klegerman & Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo Grove, IL, pp. 165-174 and Principles of Pharmacology , Munson (ed.) 1995, Ch. 102, for descriptions of techniques used in CADD. Also included are mimetics prepared using such techniques. Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

A "therapeutically effective amount" is a quantity of a chemical composition or a cell to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit tumor growth or to measurably alter outward symptoms of the tumor. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in lymphocytes) that has been shown to achieve an in vitro effect. Polynucleotide: The term polynucleotide or nucleic acid sequence refers to a polymeric form of nucleotide at least 10 bases in length. A recombinant polynucleotide includes a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single- and double- stranded forms of DNA.

Polypeptide: Any chain of amino acids, regardless of length or post- translational modification (e.g., glycosylation or phosphorylation). In one embodiment, the polypeptide is RANKL.

Probes and primers: A probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Primers are short nucleic acids, preferably DNA oligonucleotides, of about 15 nucleotides or more in length. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, for example by polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art. One of skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 20 consecutive nucleotides will anneal to a target with a higher specificity than a corresponding primer of only 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers can be selected that comprise about 20, 25, 30, 35, 40, 50 or more consecutive nucleotides. Promoter: A promoter is an array of nucleic acid control sequences that directs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as in the case of a polymerase II type promoter (a TATA element). A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included (see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987).

Specific, non-limiting examples of promoters include promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used. A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.

The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.

Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified nucleic acid is one in which the nucleic acid is more enriched than the nucleic acid in its natural environment within a cell. Similarly, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell. In one embodiment, a preparation is purified such that the protein or peptide represents at least about 60% (such as, but not limited to, 70%, 80%, 90%, 95%, 98% or 99%) of the total peptide or protein content of the preparation.

RANK (receptor activator of NF-kappa-B) and RANKL (receptor activator of NF-kappa-B ligand): RANK is a member of the TNF receptor superfamily. The protein is also referred to as TNF receptor superfamily member 1 IA [TNFRSFl IA]. In the nomenclature of CD antigens this receptor has received the designation CD265. RANK and its ligand, RANKL, regulate interactions between T-cells (one of the cell types known to express RANKL) and dendritic cells (which express RANK and present antigens to T-cells). Interaction of RANK on T- cells with the ligand expressed on dendritic cells augments the ability of dendritic cells to stimulate naive T-cell proliferation in a mixed lymphocyte reaction and increases the survival of T-cells generated with IL4 and TGF-beta. RANK has been shown to mediate an essential signal for the formation of bone-resorbing osteoclasts.

RANK ligand (RANKL): A ligand for the RANK receptor, that specifically binds RANK and mediates biological activity through RANK. RANKL includes full length mammalian RANKL, including mouse and human RANKL, as well as biological fragments, such as soluble forms thereof that specifically bind RANK and activate RANK ligand-induced signaling through RANK. Exemplary RANKL polypeptides of use in the methods disclosed herein are described in U.S. Patent No. 6,419,929.

Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals.

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker gene and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

RANKL Polypeptides and Other Agonists of RANK Isolated RANKL polypeptides and analogs (or muteins) thereof having an activity exhibited by the native molecule (i.e. RANKL muteins that bind specifically to a RANK expressed on cells or immobilized on a surface or to RANKL- specific antibodies; soluble forms thereof that activate RANK ligand-induced signaling through RANK) are of use in the methods disclosed herein. RANKL polypeptides of use are described in U.S. Patent No. 6,419,929, which is incorporated herein by reference. An exemplary amino acid sequences for native human (wild-type) RANKL (GENBANK® Accession No. AAB86811, November 21, 1997) is: mrrasrdytk ylrgseemgg gpgaphegpl happppaphq ppaasrsmfv allglglgqv vcsvalffyf raqmdpnris edgthciyri lrlhenadfq dttlesqdtk lipdscrrik qafqgavqke lqhivgsqhi raekamvdgs wldlakrskl eaqpfahlti natdipsgsh kvslsswyhd rgwakisnmt fsngklivnq dgfyylyani cfrhhetsgd lateylqlmv yvtktsikip sshtlmkggs tkywsgnsef hfysinvggf fklrsgeeis ievsnpslld pdqdatyfga fkvrdid (SEQ ID NO: 1)

An exemplary amino acid sequence for wild-type mouse RANKL (GENBANK® Accession No. AAB86812, November 21, 1997) is: mrrasrdygk ylrsseemgs gpgvphegpl hpapsapapa pppaasrsmf lallglglgq vvcsialfly fraqmdpnri sedsthcfyr ilrlhenadl qdstlesedt lpdscrrmkq afqgavqkel qhivgpqrfs gapammegsw ldvaqrgkpe aqpfahltin aasipsgshk vtlsswyhdr gwakisnmtl sngklrvnqd gfyylyanic frhhetsgsv ptdylqlmvy vvktsikips shnlmkggst knwsgnsefh fysinvggff klrageeisi qvsnpslldp dqdatyfgaf kvqdid (SEQ ID NO: 2)

Such polypeptides are substantially free of contaminating endogenous materials and can be produced with or without associated native-pattern glycosylation. Derivatives of RANKL of use also include various structural forms of the primary proteins which retain biological activity. Due to the presence of ionizable amino and carboxyl groups, for example, a RANKL protein may be in the form of acidic or basic salts, or may be in neutral form. Individual amino acid residues may also be modified by oxidation or reduction. The primary amino acid structure may be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like, or by creating amino acid sequence mutants. Covalent derivatives are prepared by linking particular functional groups to amino acid side chains or at the N- or C- termini. Derivatives of RANKL may also be obtained by the action of cross-linking agents, such as M-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, at cysteine and lysine residues. RANKL polypeptides can also be covalently bound through reactive side groups to various insoluble substrates, such as cyanogen bromide- activated, bisoxirane-activated, carbonyldiimidazole-activated or tosyl- activated agarose structures, or by adsorbing to polyolefin surfaces (with or without glutaraldehyde cross -linking). Once bound to a substrate, the polypeptides may be used to selectively bind (for purposes of assay or purification) antibodies raised against the proteins or against other proteins which are similar to RANKL, as well as other proteins that bind RANKL or homologs thereof. Soluble forms of RANKL are also of use in the methods disclosed herein.

The nucleotide and predicted amino acid sequence of the RANKL is shown in SEQ ID NOs: 1 and 2 (murine and human, respectively). Computer analysis indicated that the RANKL is a Type 2 transmembrane protein; murine RANKL contains a predicted 48 amino acid intracellular domain, 21 amino acid transmembrane domain and 247 amino acid extracellular domain, and human RANKL contains a predicted 47 amino acid intracellular domain, 21 amino acid transmembrane domain and 249 amino acid extracellular domain (see U.S. Patent No. 6,419,929, incorporated herein by reference).

Soluble RANKL comprises a signal peptide and the extracellular domain or an active fragment thereof. Signal (or leader) peptides are well-known in the art, and include that of murine interleukin-7 or human growth hormone. RANKL is similar to other members of the TNF family in having a region of amino acids between the transmembrane domain and the receptor binding region that does not appear to be required for biological activity; this is referred to as a "spacer" region. Amino acid sequence alignment indicates that the receptor binding region is from about amino acid 162 of human RANKL to about amino acid 317 beginning with an Ala residue that is conserved among many members of the family. Fragments of the extracellular domain will also provide soluble forms of RANKL. Those skilled in the art will recognize that the actual receptor binding region may be different than that predicted by computer analysis. Thus, the N-terminal amino acid of a soluble RANKL is expected to be within about five amino acids on either side of the conserved Ala residue. Alternatively, all or a portion of the spacer region can be included at the N-terminus of a soluble RANKL, as can be all or a portion of the transmembrane and/or intracellular domains, provided that the resulting soluble RANKL is not membrane - associated. Those skilled in the art can prepare these and additional soluble forms through routine experimentation; exemplary soluble forms are disclosed, for example, in U.S. Patent No. 6,419,929, incorporated herein by reference.

Fragments can be prepared using known techniques to isolate a desired portion of the extracellular region, and can be prepared, for example, by comparing the extracellular region with those of other members of the TNF family (of which RANKL is a member) and selecting forms similar to those prepared for other family members. Alternatively, unique restriction sites or PCR techniques that are known in the art can be used to prepare numerous truncated forms which can be expressed and analyzed for activity, suitable fragments are also disclosed in U.S. Patent No. 6,419,929, incorporated herein by reference.

Exemplary RANKL proteins of use in the methods disclosed herein include about amino acid 137 to about amino acid 316 of mouse RANKL. Exemplary RANKL proteins of use in the methods disclosed herein include about amino acid 158 to about amino acid 316 of mouse RANKL. One of skill in the art can readily produce proteins that include from about amino acid 138, 139, 140, 141, 142, 143, 144, 145, 145, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, or 157 to about amino acid 314, 315, or 316 of mouse RANKL. In this context, "about refers to within one amino acid. Thus, this disclosure encompasses the use of amino acids 137 to 316 of mouse RANKL, amino acids 137 to 315 of mouse RANKL, amino acids 136 to 316 of mouse RANKL, amino acids 136 to 315 of mouse RANKL, amino acids 138 to 316 of mouse RANKL, and amino acids 138 to 315 of mouse RANKL. Exemplary RANKL proteins also include the use of amino acids 158 to 316 of mouse RANKL, amino acids 158 to 315 of mouse RANKL, amino acids 157 to 316 of mouse RANKL, amino acids 157 to 315 of mouse RANKL, amino acids

156 to 316 of mouse RANKL, and amino acids 156 to 315 of mouse RANKL.

Exemplary RANKL proteins of use in the methods disclosed herein include about amino acid 138 to about amino acid 317 of human RANKL. One of skill in the art can readily produce proteins that include from about amino acid 138, 139, 140, 141, 142, 143, 144, 145, 145, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,

157 or 158 to about amino acid 314, 315, 316, or 317 of human RANKL. In this context, "about refers to within one amino acid. Thus, this disclosure encompasses the use of amino acids 138 to 317 of human RANKL, amino acids 138 to 316 of human RANKL, amino acids 137 to 317 of human RANKL, amino acids 137 to 316 of human RANKL, amino acids 139 to 317 of human RANKL, and amino acids 139 to 316 of human RANKL. Exemplary RANKL proteins also include the use of amino acids 159 to 317 of human RANKL, amino acids 159 to 316 of human RANKL, amino acids 158 to 317 of human RANKL, amino acids 158 to 315 of human RANKL, amino acids 157 to 317 of human RANKL, and amino acids 157 to 316 of human RANKL. These human and mouse peptides are exemplary only.

Other forms of the RANKL polypeptides of use include covalent or aggregative conjugates of the proteins or their fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C- terminal fusions. For example, the conjugated peptide may be a signal (or leader) polypeptide sequence at the N-terminal region of the protein which co- translationally or post-translationally directs transfer of the protein from its site of synthesis to its site of function inside or outside of the cell membrane or wall (e.g., the yeast α-f actor leader).

Protein fusions can include peptides added to facilitate purification or identification of RANKL proteins and homologs (e.g., poly-His). The amino acid sequence of the inventive proteins can also be linked to an identification peptide such as that described by Hopp et al., Bio/Technology 6:1204 (1988). Such a highly antigenic peptide provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. The sequence of Hopp et al. is also specifically cleaved by bovine mucosal enterokinase, allowing removal of the peptide from the purified protein. Fusion proteins capped with such peptides may also be resistant to intracellular degradation in E. coli.

Fusion proteins also include the amino acid sequence of a RANKL linked to an immunoglobulin Fc region. Fragments of an Fc region may also be used, as can Fc muteins. For example, certain residues within the hinge region of an Fc region are critical for high affinity binding to FcγRI. Canfield and Morrison (J. Exp. Med. 173:1483; 1991) reported that Leu₂₃₄and Leu₂₃s were critical to high affinity binding of IgGγ3 to FcγRI present on U937 cells. Similar results were obtained by Lund et al. (J. Immunol. 147:2657, 1991; Molecular Immunol. 29:53, 1991). Such mutations, alone or in combination, can be made in an IgG₁ Fc region to decrease the affinity of IgG₁ for FcR. Depending on the portion of the Fc region used, a fusion protein can be expressed as a dimer, through formation of interchain disulfide bonds. If the fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a protein oligomer with as many as four RANKL regions.

In another embodiment, RANKL proteins further comprise an oligomerizing peptide such as a leucine zipper domain. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, 1988). Leucine zipper domain is a term used to refer to a conserved peptide domain present in these (and other) proteins, which is responsible for dimerization of the proteins. The leucine zipper domain (also referred to herein as an oligomerizing, or oligomer- forming, domain) includes a repetitive heptad repeat, with four or five leucine residues interspersed with other amino acids. Examples of leucine zipper domains are those found in the yeast transcription factor GCN4 and a heat-stable DNA- binding protein found in rat liver (C/EBP; Landschulz et al., Science 243:1681, 1989). Two nuclear transforming proteins, fos and jun, also exhibit leucine zipper domains, as does the gene product of the murine proto-oncogene, c-myc (Landschulz et al., Science 240:1759, 1988). The products of the nuclear oncogenes fos and jun comprise leucine zipper domains preferentially form a heterodimer (O'Shea et al., Science 245:646, 1989; Turner and Tjian, Science 243:1689, 1989). The leucine zipper domain is necessary for biological activity (DNA binding) in these proteins. The fusogenic proteins of several different viruses, including paramyxovirus, coronavirus, measles virus and many retroviruses, also possess leucine zipper domains (Buckland and Wild, Nature 338:547,1989; Britton, Nature 353:394, 1991; Delwart and Mosialos, AIDS Research and Human Retroviruses 6:703, 1990). The leucine zipper domains in these fusogenic viral proteins are near the transmembrane region of the proteins; it has been suggested that the leucine zipper domains could contribute to the oligomeric structure of the fusogenic proteins. Oligomerization of fusogenic viral proteins is involved in fusion pore formation (Spruce et al, Proc. Natl. Acad. Sci. U.S.A. 88:3523, 1991). Leucine zipper domains have also been recently reported to play a role in oligomerization of heat-shock transcription factors (Rabindran et al., Science 259:230, 1993).

Leucine zipper domains fold as short, parallel coiled coils. (O'Shea et al., Science 254:539; 1991) The general architecture of the parallel coiled coil has been well characterized, with a "knobs-into-holes" packing as proposed by Crick in 1953 (Acta Crystallogr. 6:689). The dimer formed by a leucine zipper domain is stabilized by the heptad repeat, designated (abcdefg)_n according to the notation of McLachlan and Stewart (J. MoI. Biol. 98:293; 1975), in which residues a and d are generally hydrophobic residues, with d being a leucine, which line up on the same face of a helix. Oppositely-charged residues commonly occur at positions g and e. Thus, in a parallel coiled coil formed from two helical leucine zipper domains, the "knobs" formed by the hydrophobic side chains of the first helix are packed into the "holes" formed between the side chains of the second helix. The leucine residues at position d contribute large hydrophobic stabilization energies, and are important for dimer formation (Krystek et al., Int. J. Peptide Res. 38:229, 1991). Lovejoy et al. synthesized a triple- stranded α-helical bundle in which the helices run up-up-down (Science 259:1288, 1993). RANKL can (or cannot) include associated native -pattern glycosylation.

Proteins expressed in yeast or mammalian expression systems, e.g., COS-7 cells, may be similar or slightly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system. Expression of DNAs encoding the inventive proteins in bacteria such as E. coli provides non- glycosylated molecules. Functional mutant analogs of RANKL protein having inactivated N-glycosylation sites can be produced by oligonucleotide synthesis and ligation or by site-specific mutagenesis techniques. These analog proteins can be produced in a homogeneous, reduced-carbohydrate form in good yield using yeast expression systems. N-glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet ASn-A₁ -Z, where A, is any amino acid except Pro, and Z is Ser or Thr. In this sequence, asparagine provides a side chain amino group for covalent attachment of carbohydrate. Such a site can be eliminated by substituting another amino acid for Asn or for residue Z, deleting Asn or Z, or inserting a non-Z amino acid between A.sub.l and Z, or an amino acid other than Asn between Asn and A₁.

RANKL polypeptide derivatives can also be obtained by mutations of the native RANKL or subunits thereof. A RANKL mutated protein, as referred to herein, is a polypeptide homologous , such as 90%, 95%, 96%, 97%, 98%, or 99% homologous to a native RANKL protein, but which has an amino acid sequence different from the native protein because of one or a plurality of deletions, insertions or substitutions. The effect of any mutation made in a DNA encoding a mutated peptide may be easily determined by analyzing the ability of the mutated peptide to bind its counterstructure(such as a receptor) in a specific manner. Moreover, activity of RANKL analogs, muteins or derivatives can be determined by a biological assay (for example, induction of NF-κB activation).

Analogs of RANKL may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues can be deleted or replaced with other amino acids to prevent formation of incorrect intramolecular disulfide bridges upon renaturation. Other approaches to mutagenesis involve modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present.

Subunits of RANKL can be constructed by deleting terminal or internal residues or sequences. Soluble forms of RANKL can be readily prepared and tested for their ability to induce NF-κB activation. Polypeptides corresponding to the cytoplasmic regions, and fragments thereof (for example, a death domain) can be prepared by similar techniques. Generally, substitutions of use are conservative substitutions. Substitutions can be made of those amino acids are those which do not affect the biological activity of RANKL (i.e., ability of RANKL to bind antibodies specific for the corresponding native protein in substantially equivalent a manner, the ability to bind the RANK receptor in substantially the same manner as the native protein, the ability to induce a RANKL signal, or ability to induce NFKB activation).

Examples of substitutions of use include substitution of amino acids outside of the binding domain(s) (either ligand/receptor or antibody binding areas for the extracellular domain, or regions that interact with other, intracellular proteins for the cytoplasmic domain), and substitution of amino acids that do not alter the secondary and/or tertiary structure of the native protein. Additional examples include substituting one aliphatic residue for another, substitutions of one polar residue for another. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are also of use. In one embodiment, the solubility of the RANKL protein is not changed or increased, and/or the biological activity of RANKL is unaffected. In some embodiments, the solubility and/or biological activity is within about 20%, within about 15%, within about 10%, within about 5% or within about 1-5% of the wild type parent RANKL protein, such as a wild type human or mouse RANKL protein. Thus, the solubility and/or biological activity of the altered RANKL protein can be within 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of wild-type RANKL. Thus, the solubility and/or biological activity is unchanged from the wild-type. In other examples, the solubility and/or biological activity is significantly increased as compared to wild-type RANKL. In some embodiments, the solubility and/or biological activity is increased about 40%, increased about 50%, increased about 75%, or increased about 100% or within about 100-500% of the wild type RANKL protein, such as a wild-type mouse or human RANKL protein. Thus, the solubility and/or biological activity can increased at least 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or 500% of wild-type RANKL. In this context "about" refers to within 1%.

In one example, a RANKL polypeptide of use is a murine RANKL wherein a serine is substituted for a cysteine at position 220 in wild-type mouse RANKL (C220S). In other example, a RANKL polypeptide of use is a murine RANKL wherein an arginine is substituted for an isoleucine at position 246 in wild-type mouse RANKL (I246R). In some examples, the RANKL polypeptide of use is murine RANKL wherein a serine is substituted for a cysteine at position 220 and an arginine is substituted for an isoleucine at position 246 of wild- type murine RANKL (C220S/I246R). In additional examples, a RANKL polypeptide of use includes a murine RANKL wherein an arginine is substituted for an alanine at position 171 in wild type mouse RANKL (A171R). Thus, the RANKL polypeptide of use is murine RANKL wherein a serine is substituted for a cysteine at position 220, an arginine is substituted for an isoleucine at position 246, and an arginine is substituted for an alanine at position 171 in wild type mouse RANKL (A171R/C220S/A246R). These soluble proteins can be fused to tags consisting of GST and/or six histidine residues to assist with isolation and purification of these proteins.

Exemplary amino acid sequences are provided below, wherein the first 136 nucleotide sequences are from murine RANKL (indicated by " — ") and wherein each sequence shown below begins at position 137.

C220S/I246R Mouse RANKL Amino Acid Sequence qrfs gapammegsw ldvaqrgkpe aqpfahltin Aasipsgshk vtlsswyhdr gwakisnmtl sngklrvnqd gfyylyaniS frhhetsgsv ptdylqlmvy vvktsRkips shnlmkggst knwsgnsefh fysinvggff klrageeisi qvsnpslldp dqdatyfgaf kvqdid (SEQ ID NO: 3) A171R/C220S/I246R Mouse RANKL Amino Acid Sequence qrfs gapammegsw ldvaqrgkpe aqpfahltin Rasipsgshk vtlsswyhdr gwakisnmtl sngklrvnqd gfyylyaniS frhhetsgsv ptdylqlmvy vvktsRkips shnlmkggst knwsgnsefh fysinvggff klrageeisi qvsnpslldp dqdatyfgaf kvqdid (SEQ ID NO: 4)

The amino acid residues that are changed from the wild-type RANKL are indicated with capital letters and underlining.

In one example, a RANKL polypeptide of use is a human RANKL wherein a serine is substituted for a cysteine at position 221 in wild-type human RANKL (C221S). In other example, a RANKL polypeptide of use is a human RANKL wherein an arginine is substituted for an isoleucine at position 247 in wild-type human RANKL (I247R). In some examples, the RANKL polypeptide of use is human RANKL wherein a serine is substituted for a cysteine at position 221 and an arginine is substituted for an isoleucine at position 247 of wild- type human RANKL (C221S/I247R). In additional examples, a RANKL polypeptide of use includes a human RANKL wherein an arginine is substituted for an alanine at position 172 in wild type human RANKL (A172R). Thus, the RANKL polypeptide of use is human RANKL wherein a serine is substituted for a cysteine at position 221, an arginine is substituted for an isoleucine at position 247, and an arginine is substituted for an alanine at position 172 in wild type human RANKL (A172R/C221S/1247R). These soluble proteins can be fused to tags consisting of GST and/or six histidine residues to assist with isolation and purification of these proteins. Additional forms of human RANKL, such as human RANKL with increased solubility, are described in U. S Patent 7,399,829, which is incorporated by reference herein.

The biological activity of RANKL analogs or muteins can be determined by testing the ability of the analogs or variants to induce a signal through RANK, for example, activation of transcription or inducing differentiation of macrophage-like cells into osteoclasts. . Suitable assays also include, for example, assays that measure the ability of a RANKL peptide or mutein to bind cells expressing RANK, and/or the biological effects thereon (see U.S. Patent No. 6,419,929, incorporated herein by reference). Polynucleotides encoding the RANKL can also be utilized. These polynucleotides include DNA, cDNA and RNA sequences which encode the polypeptide of interest.

A nucleic acid encoding RANKL can be cloned or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self- sustained sequence replication system (3SR) and the Qβ replicase amplification system (QB). For example, a polynucleotide encoding the protein can be isolated by polymerase chain reaction of cDNA using primers based on the DNA sequence of the molecule. A wide variety of cloning and in vitro amplification methodologies are well known to persons skilled in the art. PCR methods are described in, for example, U.S. Patent No. 4,683,195; Mullis et al, Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). Polynucleotides also can be isolated by screening genomic or cDNA libraries with probes selected from the sequences of the desired polynucleotide under stringent hybridization conditions. The polynucleotides encoding RANKL include a recombinant DNA which is incorporated into a vector into an autonomously replicating plasmid or virus or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences. The nucleotides of the invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double forms of DNA.

DNA sequences encoding RANKL polypeptide can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.

A polynucleotide sequences encoding RANKL can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.

The polynucleotide sequences encoding RANKL can be inserted into an expression vector including, but not limited to, a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.

Transformation of a host cell with recombinant DNA can be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl₂ method using procedures well known in the art. Alternatively, MgCl₂ or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors can be used. Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding RANKL, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

Additional agonists of RANK are of use in the methods that are disclosed herein. These agonists include chemical compounds, small molecules and antibodies that bind RANK and activate the receptor. In one example, polyclonal antibodies raised to mouse RANK activate osteoclast formation from precursor cells in the spleen (see Nakagawa et al., Biochem. Biophys. Res. Commun. 53:395-400, 1998.

Method of Increasing the Differentiation of M cells and Methods for Increasing Delivery of an Agent

Methods for increasing a mucosal immune response are provided herein. These methods include selecting a subject in need of an increased mucosal immune response and administering to a subject a therapeutically effective amount of RANKL or an agonist of RANK. The RANKL or agonist of RANK administration results in the differentiation of microfold cells in the intestine. The therapeutically effective amount of RANKL or the agonist of RANK is administered such that M cells are induced to differentiate.

Following the RANKL administration, the subject is administered a therapeutically effective amount of an antigen of interest, or a nucleic acid encoding the antigen of interest, thereby increasing the mucosal immune response to the antigen. The antigen or nucleic acid encoding the antigen can be part of a vaccine.

An antigenic polypeptide can be utilized that is a short peptide sequence including a single epitope. Alternatively, heat killed vaccines or attenuated vaccines can be administered. When an antigenic polypeptide is utilized, the antigenic polypeptide can be a sequence of amino acids as short as eight or nine amino acids, sufficient in length to provide an antigenic epitope in the context of presentation by a cellular antigen presenting complex, such as the major histocompatibility complex (MHC). Larger peptides, in excess of 10 amino acids, 20 amino acids or 30 amino acids are also suitable antigenic polypeptides, as are much larger polypeptides provided that the antigenic polypeptide does not disrupt the structure. Exemplary embodiments ranging from short peptides (for example, less than 20 amino acids in length) to large polypeptides (for example, greater than 150 amino acids), including multiple antigenic epitopes and having a complex secondary structure, are described in the examples herein. In one specific, non-limiting example an antigenic formulation includes about 0.1 μg to about 1,000 μg, or about 1 to about 100 μg of a selected antigen. An antigen preparation can also contain buffers, excipients, and preservatives, amongst other ingredients. The antigen can be administered in PLGA poly(lactic-co-glycolic acid), or any other a biocompatible polymer used for protein encapsulation. The antigen can be administered as a controlled release formulation.

In various embodiments, the antigenic polypeptide is that of a pathogenic organism, such as a virus or bacterial agent that can produce undesirable symptoms in a subject following exposure to the organism. Nucleic acids encoding antigenic polypeptides are also of use.

In additional embodiments, the antigen is administered with a mucosal adjuvant, such as cholera toxin. The antigen can also be administered together with other immuno stimulatory adjuvants after RANKL pretreatment to induce M cell differentiation. These adjuvants include ligands for Toll-like receptors (TLR) including lipopolysaccharide (LPS) and monophosphoryl lipid A (TLR4 agonists), flagellin (TLR5 agonist), imiquimod and resiquimod (TLR7 and TLR8 agonists), CpG immuno stimulatory oligonucleotides (TLR9 agonist), including D and K type oligodeoxynucleotides (see U.S. Patent No. 6,977,245, herein incorporated by reference). The antigen can be from any pathogen, including bacterial, fungal and viral pathogents. Exemplary bacterial disease organisms include: Group A streptococci, Group B streptococci, Streptococcus faecalis, Staphylococcus aureus, Listeria monocytogenes, Helicobacter pylori, Bacillus anthracis, Brucella abortus, Brucella melitensis, Neisseria gonorrhoeae, Neisseria meningitidis, Hemoplilus influenzae, Mycobacterium tuberculosis, Bordetella pertussis, Vibrio cholerae, Salmonella typhi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Escherichia coli 0157:H7, Escherichia coli, Escherichia coli (bovine scouring strains), Chlamydia pneumoniae and Chlamydia trachomatis. Exemplary bacterial toxins and microorganisms include: A/B bacterial toxins, such as Shiga toxin- Shigella, Shiga-like toxins -Enterohemorrhagic E. Coli, Diptheria toxin- Corynebacterium diptheriae, Botulinum toxin-Clostridium botulinum, Tetanus toxin-Clostridium tetani, Cholera toxin- Vibrio cholerae, A toxin-Pseudomonas aeruginosa, LT-ETEC-Escherica coli; Dick (Erythrogenic) toxin-Streptococcus pyrogenes; Lethal toxin-Bacillus anthracis; Alpha toxin-Staphylococcus aureus; and Plague toxin- Yersinia pestis. Exempalry fungal diseases include: Candida albicas; Aspergillus fumigatus; Cryptococcus neoformans; Coccidioides immitis; and Histoplasma capsulatum. Exemplary viral diseases and causative agents include: Rhinoviruses-polio, cold viruses; Alphaviruses-yellow fever, encephalitis; Lyssavirus-rabies; Calcivirus-norwalk virus; Prthopox virus-smallpox; Papillomavirus-warts; HIV; HPV; Herpesvirus-genital herpes, simplex, shingles, chickenpox; Bunyavirus-hentavirus; Coronavirus-respiratory infections; Mobillivirus-mumps, measles; Reovirus-respiratory infections; Enterovirus- intestinal infections; Influenzavirus-influenza. Exemplary spirochetal diseases and organisms include Treponema pallidum (syphilis) and Borrelia recurrentis (Recurring fever). Exemplary protozoan diseases and causative agents include: Entamoeba histolytica; Giardia lamblia; Taxoplamsa gondii; Plasmodium species (Plasmodium); Trypanosoma cruzi; Trypanosoma gambiiense; Leishmaniasis donovani; Pneumocystis carinii, Cryptosporidium, Trichomonas vaginalis; Schistosoma mansoni and Tritrichomonas faetus.

Exemplary antigens to be included in whole or in part as suitable immunogens, or to be encoded by the a nucleic acid, and the diseases with which they are associated include, but are not limited to: tuberculosis (e.g., BCG antigen: Kumar et al., Immunology (1999) 97(3):515-521), leprosy (e.g., antigen 85 complex: Naito et al., Vaccine (1999) 18(9-10):795-798), malaria (e.g., surface antigen MSA-2: Pye et al., Vaccine (1997) 15(9):1017-1023), diphtheria (e.g., diphtheria toxoid: U.S. Pat. No. 4,691,006 ), tetanus (e.g., tetanus toxin: Fairweather et al., Infect Immun (1987) 55(l l):2541-2545), leishmania (e.g., Leishmania major promastigotes: Lasri et al., Vet Res (1999) 30(5):441-449), salmonella (e.g., covalently bound capsular polysaccharide (Vi) with porin, both isolated from S. typhi.: Singh et al., Microbiol Immunol (1999) 43(6):535-542), schistomiasis (e.g., major antigen of Schistosoma mansoni (Sm28 GST): Auriault et al., Pept Res (1991) 4(1):6-1 1), measles (e.g., the surface glycoprotein and fusion protein of measles virus: Machamer et al., Infect Immun (1980) 27(3):817-825), mumps (e.g., hemagglutinin-neuraminidase (HN) viral gene product: Brown et al., J Infect Dis (1996) 174(3):619-622), herpes (e.g., HSV-2 surface glycoproteins (gB2 and gD2): Corey et al., JAMA (1999) 282(4):331-340), AIDS (e.g., gpl60: Pontesilli et al., Lancet (1999) 354(9182):948-949), influenza (e.g., immunodominant peptide from hemagglutinin: Novak et al., J Clin Invest (1999) 104(12):R63-67), Group A streptococcus: (extracellular cysteine protease, Lukomski, S. et al., Infect. Immun. 1999. 67(4): 179-1788, Streptococcal inhibitor of complement (Sic) (Lukomski, S., et al., Infect. Immun. 2000. 68(2): 535-542, Hyaluronic acid capsule, Schrager, H. et al., J. Clin. Invest. 1998. 101: 1708-1716), Group B streptococcus (capsular polysaccharide, Type I, II, II, IV and V, Pincus, S. H, et al., J. Immunol. 1998. 160: 293-298.), Shigella species (Lipopolysaccharide (0 somatic antigen) Phalipon, A., et al., Eur. J. Immunol. 1997. 27: (10), 2620-2625), Brucella abortus (Lipopolysaccharide (antigen A) Montaraz, J., et al., Immun. 1986. 51: 961-963), Escherichia coli (EPEC) (intimin,and/or Hp90 protein, Hartland, et al., MoI. Microbiol. 1999. 32 (1): 151-158, Kenny, B., et al., Cell. 1997. 91: 511-520),

Escherichia coli (EHEC) 0157-H7 (lipopolysaccharide (LPS), Konadu, E., et al., Infect. Immun. 67:6191-6193), Salmonella typhi (Vi capsular polysaccharide, Singh, et al., Microbiol. Immunol. 1999. 43(6): 535-542), Vibrio cholerae (cholera toxin B subunit, Liljeqvist, S., et al., Appl. Environ. Micro. 1997. 63(7): 2481-2488), Helicobacter pylori (Urease A and B, Lee, C. et al. J. Infect. Dis. 1995. 172: 161- 172, Le .sup.b binding adhesin, Iver, D. et al., Science, 1998. 279: 373-377), Bordetella pertussis (Filamentous hemagglutinin (FHA), Brennan, M. and R. Shahin. Am. J. Respir. Crit. Care Med. 1996, 154:145-149), Haemophilus influenze (HMW1/HMW2 adhesin, St. Geme, J. The Finnish Med. Soc. DUODECIM. Ann. Med. 1996, HifE pilus (adhesin), Hia adhesin, Barenkamp, S and J. St. Geme, MoI. Microbiol. 1996. 19: 1215-1223), Chlamydia peumoniae (Major outer membrane protein (MOMP), Peterson, E., et al., Infect. Immun., 1996. 64(8): 3354-3359), HIV (Fusion-dependent immunogen, LaCasse, R. A., et al., Science. 1999. 283: 357-362, 5-Helix protein, Root, M. et al., Sciencexpress Report, Jan. 11, 2001), Poliovirus (M cell ligand, Frey, A. et al., Behring Inst. Mitt. 1997. 98: 376-389), Measles virus (surface glycoprotein, fusion protein, Machamer et al., Infect. Immun. 1980. 27(3): 817-825), Cryptococcus neoformans (Capsular polysaccharide- glucuronoxylomannan, Blackstock, R. and A. Casadevall. 1997. Immunol. 92:334- 339), and Schistosoma mansoni (9B antigen peptides, Arnon, R. et al., Immunology. 101(4): 555-562).

The present method can also be used to increase M cell differentiation to induce tumor immunity. Exemplary tumor specific antigens may be derived from cancers including: leukemia-lymphocytic, granulocytic, monocytic or myelocytic; Lymphomas; basal cell carcinoma; squamous cell carcinoma; breast, colon, endometrial, pancrecatic, lung, etc. carcinoma; and uterine, vaginal, prostatic, testis, ostogenic or pulmonary sarcoma (see Wang R F., J MoI Med (1999) 77(9):640-655). Tumor antigens according to the invention include 707-AP (707 alanine proline), AFP (alpha (.alpha.)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T cells 4), BAGE (B antigen), .beta.-catenin/m (.beta.-catenin/mutated), Bcr-abl (breakpoint cluster region- Abelson), CAMEL (CTL-recognized antigen on melanoma), CAP-I (carcinoembryonic antigen pep tide- 1), CASP-8 (caspase-8), CDC27m (cell division-cycle 27 mutated), CDK4/m (eye line-dependent kinase 4 mutated) CEA (carcinoembryonic antigen), CT (cancer/testis antigen), Cyp-B (cyclophilin B), DAM ((differentiation antigen melanoma) (the epitopes of DAM-6 and DAM-IO are equivalent, but the gene sequences are different; DAM-6 is also called MAGE-B2 and DAM-IO is also called MAGE-Bl), ELF2M (elongation factor 2 mutated), ETV6-AML1 (Ets variant gene 6/acute myeloid leukemia 1 gene ETS), G250 (glycoprotein 250), GAGE (G antigen), GnT-V (N- acetylglucosaminyltransferase V), GpIOO (glycoprotein 100 kD), HAGE (helicose antigen), HER 2/neu (human epidermal receptor- 2/neurological), HLA-A*0201- R170I (arginine (R) to isoleucine (I) exchange at residue 170 of the .alpha.-helix of the .alpha.2-domain in the HLA- A2 gene), HPV-E7 (human papilloma virus E7), HSP70-2M (heat shock protein 70-2 mutated), HST-2 (human signet ring tumor-2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxyl esterase KIAA0205 (name of the gene as it appears in databases), LAGE (L antigen), LDLR/FUT (low density lipid receptor/GDP-L-fucose: .beta.-D- galactosidase 2-.alpha.-L-fucosyltransferase), MAGE (melanoma antigen), MART- 1/Melan-A (melanoma antigen recognized by T cells- I/Melanoma antigen A), MClR (melanocortin 1 receptor), Myosin/m (myosin mutated), MUCl (mucin 1), MUM-I , -2, -3 (melanoma ubiquitous mutated 1, 2, 3), NA88-A (NA cDNA clone of patient M88), NY-ESO- l=New York-esophageous 1), P15 (protein 15),pl90 minor bcr-abl (protein of 190 KD bcr-abl), Pml/RAR. alpha, (promyelocytic leukaemia/retinoic acid receptor .alpha.), PRAME (preferentially expressed antigen of melanoma), PSA (prostate-specific antigen), PSM (prostate-specific membrane antigen), RAGE (renal antigen), RUl or RU2 (renal ubiquitous 1 or 2), SAGE (sarcoma antigen), SART-I or SART-3 (squamous antigen rejecting tumor 1 or 3), TEL/ AMLl (translocation Ets-family leukemia/acute myeloid leukemia 1), TPI/m (triosephosphate isomerase mutated), TRP-I (tyrosinase related protein 1, or gp75), TRP-2 (tyrosinase related protein 2), TRP-2/INT2 (TRP-2/intron 2), WTl (Wilms¹ tumor gene). These antigens are disclosed in references that are cited in Cancer Immunology Immunotherapy 50:3-15 (2001), which is herein incorporated by reference. The cited references may be consulted for methods of isolating the specific antigens or genes encoding the specific antigens for use in the vaccines of the invention.

In general, appropriate DNA conjugate or immunogen complex (or other delivery vector) to deliver the DNA to a target M cell that improves host immune responses against a specific pathogen or other immunogen. For example, such a vaccine may be comprised of a polybasic conjugate/DNA complex by incorporating an M cell ligand. Thus, for any given immunogen encoded by a nucleic acid or mimicked by a peptide encoded by a nucleic acid that can be used for eliciting a host response, such a response can be enhanced through effective targeting mediated by M cell ligands. n additional embodiments, an M cell specific ligand conjugated or complexed to immunogen via an appropriate linker. Immunogens in this instance would include a variety of macromolecules such as peptides, proteins, lipoproteins, lipids, glycoproteins, polysaccharides, carbohydrates, some nucleic acids, and certain of the teichoic acids, or any other molecule or gene from a pathogen or tumor cell that could be used to generate a protective immune response. Such immunogens may be conjugated or complexed with the M cell specific ligand using any means known in the art. For instance, immunogens may be conjugated to an M cell specific ligand using an appropriate crosslinker. Cross-linking may be performed with either homo- or heterobifunctional agents, i.e., SPDP, DSS, SIAB. Alternatively, immunogens may be complexed with an M cell specific ligand using an appropriate complexing agent. Complexes may be formed between a 6 X Histidine tag on one molecule and a nitrilotriacetic acid-metal ion complex on the other molecule. Methods for cross-linking are disclosed in PCT/DKOO/0053 1 (see PCT Publication No. WO 01/22995) which is herein incorporated by reference. Alternatively, conjugates and complexes can comprise the following scenarios: polypeptides with attached immunogens may be conjugated to M cell specific ligands; liposomes can replace the polypeptide, wherein the M cell specific ligand may be conjugated to a liposome containing the immunogens, or conjugated to a liposome with one or several copies of an immunogen or different immunogens attached/displayed to its surface; and peptide and protein immunogens may be expressed as fusion proteins operably linked to the M cell specific ligand.

Examples of an M cell specific ligand are of the protein σl of a reovirus, or an adhesin of Salmonella or a polio virus. Additional examples include an antibody that specifically binds M cells (see for example, Nochi et al., J. Exp. Med. 204: 2789-2796, 2007, incorporated herein by reference. "M cell specific ligand" refers to a molecule that selectively binds to a receptor available on the surface of follicle associated epithelial cell subpopulations, and an M cell specific physiologic effect accompanies that binding (e.g., uptake of pathogen). For example, the enteric adhesin, protein σl of reovirus, is an M cell specific ligand, as would be any M tropic portion or fragment of σl that retains the ability to selectively bind to follicle associated epithelial cell subpopulations. Bassel-Duby et al. characterized the amino acid sequence of protein σl and defined a carboxy terminal portion of the protein as being responsible for receptor interaction (Nature, May- June 1985, 315(6018): 421-3). Similarly, by characterizing deletion mutants of protein σl, Nagata et al. defined the receptor binding domain as being localized to two restriction fragment- generated domains in the carboxy terminus of the protein (Virology, September 1987, 160(1): 162-8). Nibert and colleagues found that there were two separate domains that contributed to receptor binding, one in the amino terminus and one in the carboxy terminus of protein σl (J. Virol., August 1995, 69(8): 5057-67). Therefore, M cell-tropic variants of protein σl would also include variants with internal deletions but retaining both the amino and carboxy terminus. An M cell ligand of the invention would also include a tetramer or trimer of protein σl or variants of protein σl, as σl has been reported to form tetramers and dimers in binding to cells (see Banerha et al., Virol. 167: 601-12 (1988); see also Strong et al., Virol. 184(l):23-32 (1991)). In some embodiments, RANKL or the agonist of RANK is administered prior to the amount of antigen. The administration of RANKL or the agonist of RANK allows M cells to differentiate prior to the administration of the antigen or the nucleic acid encoding the antigen. For example, RANKL or the agonist of RANK can be administered at least one, at least two, at least three, at least four, at least five, at least six or at least seven days prior to administering the antigen or nucleic acid encoding the antigen. For example, the RANKL or the agonist of RANK can be administered about one day to about one week, about two to about six days, about three to about five days, or about four days prior to the administration of the antigen or the nucleic acid encoding the antigen. The RANKL or the agonist of RANK and/or the antigen can be administered intra-nasally or orally. Suitable formulations and routes of administration are described below. The agonist of RANK can be an antibody that specifically binds RANK. Controlled release formulations can be utilized, as described below. In addition, the method can be performed multiple times, so that multiple administrations are achieved.

In some embodiments, the number of microfold cells is determined following administration of RANKL or the agonist of RANK. The number of differentiated M cells can also be determined. Methods for detecting M cells are described in the Examples section below. Methods are also disclosed herein for increasing drug delivery to a mucosa.

The mucosa can be any mucosa of interest, including the small intestine, colon or nasal mucosa. These methods include administering to a subject a therapeutically effective amount of RANKL or an agonist of RANK, as described above, and administering to the subject a therapeutically effective amount of an agent. For example, the therapeutically effective amount of RANKL or the agonist of RANK can be administered two to five days prior to the agent to allow time for induction of M cells in the epithelium of the small intestine. Longer periods of pretreatment (up to 10 days) may be required to fully induce M cell development at other mucosal sites. Thus, the delivery of the drug to the mucosa is increased. In one embodiment, RANKL or the agonist of RANK, the agent, or both are administered in a microparticle, such as a lactobacillus or a liposome. For example, the microparticle has a diameter of 100 nanometers to 10 microns. In one example, the agent is a therapeutic protein is administered in Lactococcus lacti.

In some embodiments, RANKL and/or an agent (such as an antigen or vaccine) is administered intranasally and wherein the mucosa is nasal mucosa. In other embodiments, the RANKL and/or the agent (such as an antigen or a vaccine) is administered orally, and wherein the mucosa is the intestinal mucosa. The agent can be any agent of interest, including antigens, therapeutic proteins, vaccines, antibodies, compounds, or small molecules. The agent can conjugated to a microfold (M) cell ligand, as described above. In several examples, the M cell ligand is a reovirus attachment protein or an antibody that specifically binds M cells.

By using agents in a nanosphere ("NS" ! formulation the agent can be delivered more efficiently to target organs and tissues such as the small intestinal epithelium, preventing or substantially reducing potential effects on other organs and tissues, Generally, nanosphere-^ized particles have a general average size in the range of about 50 to about 999 nanometers. Nanospheres are also capable of releasing the drug in a controlled manner, thereby minimizing the need for frequent drug administration, These nanospheres can be effectively used to lranstect cells due to the nanosize of the encapsulated drag. These nanospheres due to their small size are capable of targeting and delivering the vaccine material to the Peyεr's patches in the intestine, without any degradation in the harsh acidic environment of the stomach due to an effective enteric coating. Also, because of their small size they are capable of penetrating into a tumor easily, RANKL can be encapsulated in a nanosphere,

NanospbereΛ can be prepared using a process using a mini-spray dryer without appreciable denaturation of the bioactive material, such as RANRL or a RANK agonist, In one aspect of the present disclosure, a polymer matrix is pre- cross-linked with glutaraldehyde, followed by neutralization of the excess glυtaraldehyde with sodium bi-sυlfite and then adding RANKL or a RANK agonist to the pre-cross-!inked and neutralized matrix. After adding the RANfKL or the RANK agonist, the crosslinked polymer matrix containing RANKL or the RANKL agonist is spray dried. Various parameters for Hie spray dryer, such as, but not limited to, inlet temperature, pump flow, aspiration rate and air pressure have been optirnized for obtaining nanospheres, see PCT Publication No, WO 2010/037142. which is incorporated herein by reference. In one example, albumin can be used as a matrix and glυtaraldehyde can be used as a cross-linking agent. In another embodiment, nanospberεs can be prepared using beta-cyclodextrin {instead of albumin) as the polymer matrix to encapsulate RANKL or a RANK agonist. In a further embodiment, the nanospheres can include an aery late polymer, a cellulose polymer, or both, for example, the nanospheres can include about 25% to about 30% f weight/weight) of an acrylate polymer and a cellulose polymer, such as about 28% i, weight/weight) of acrylate polymer and cellulose polymer. The nanospheres can also include enitosan, such as about 2 to about i ϋ% (weight/weight) cbitosan. such as about 5% (weight/weight) chitosan. In this context, about refers to within 0,5 %. Thus, nanospheres can be produced by encapsulating RANKL or a RANK agonist in a ρre-cross- linked and neutralized polymer matrix. However, nanospheres can also be produced and used wherein the polymer matrix is not eross- linked, In some examples, the polymer matrix is not cross-linked with gluteraldehyde. In additional examples, spray drying can be used to produce to nanospheres, such as without the use of a cross-linking agent.

The nanospheres can be prepared in a large scale aseptic manufacturing processes on an industrial scale on a cost effective basis, With the present processes the RANfKL> or RAN-K agonist is directly converted from the solution formulation into the final nanosphere form, thus eliminating the need for a separate step to remove the solvent from the particles after they are formed. Thus, particles are directly converted to a dry powder form. Since the RAJN KL or RANK agonist is converted to the dry powder form, it is very stable and thus would be expected to have a longer half life when compared to a solution formulation. Furthermore, by controlling the extent of cross-linking of the albumin polymer matrix, the release of the RAN KL or the RANK agonist can be effectively controlled. Greater cross- linking of the albumin polymer matrix, results in slower release of the RANKL or the RANfK agonist from the polymer matrix. Orally administered D-L lactide/glycolide acid copolymer microspheres are also absorbed in the terminal ileum and can release an encapsulated protein (see Haneda et al., J. Gastrointestin. Surg. 11(5): 568-577, 2007, incorporated by reference herein). These microspheres also can be used to deliver RANKL and/or a RANK agonist and other agents.

Pharmaceutical Compositions

RANKL and the polypeptide antigens, nucleic acids, or any agents including chemical compounds and small molecules of use in the method described herein may be formulated in a variety of ways. Pharmaceutical compositions are thus provided for both local (e.g. inhalational) use and for systemic use. Therefore, the disclosure includes within its scope pharmaceutical compositions for use in human or veterinary medicine. While RANKL or an agonist of RANK will typically be used to treat human subjects, related molecules may also be used to treat similar or identical diseases in other vertebrates, such other primates, dogs, cats, horses, and cows. Pharmaceutical compositions that include at least RANKL or an agonist of

RANK, or including an antigen of interest or a nucleic acid encoding the antigen, or that include an additional agent as an active ingredient (such as an adjuvant), may be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen. Additional active ingredients include, for example, anti-infective agents (such as to prevent secondary infections), anti-inflammatory agents, bronchodilators, enzymes, expectorants, leukotriene antagonists, leukotriene formation inhibitors, and mast cell stabilizers. A suitable administration format may best be determined by a medical practitioner for each subject individually. Various pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, e.g., Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42: 2S, 1988.

The dosage form of the pharmaceutical composition will be determined by the mode of administration chosen. For instance, in addition to injectable fluids, inhalational and oral formulations can be employed. Inhalational preparations can include aerosols, particulates, and the like. In general, the goal for particle size for inhalation is about lμm or less in order that the pharmaceutical reach the alveolar region of the lung for absorption. Oral formulations may be liquid (e.g., syrups, solutions, or suspensions), or solid (e.g., powders, pills, tablets, or capsules). For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those of ordinary skill in the art. For oral preparations, RANKL or a RANK agonist can be included in a nanosphere or microsphere, see for example, PCT Publication No. WO 2010/037142, which is incorporated by reference herein.

The compositions or pharmaceutical compositions also can be administered by any route, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intraperitoneal, intrasternal, or intraarticular injection or infusion, or by sublingual, oral, topical, intranasal, or transmucosal administration, or by pulmonary inhalation. When molecules are provided as parenteral compositions, e.g. for injection or infusion, they are generally suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful buffers include sodium citrate-citric acid and sodium phosphate- phosphoric acid, and sodium acetate-acetic acid buffers. A form of repository or "depot" slow release preparation may be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection or delivery.

Compositions are also suitably administered by sustained-release systems. Suitable examples of sustained-release forms include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, microcapsules or nanospheres), suitable hydrophobic materials (such as, for example, an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt). Sustained-release formulations may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray.

Preparations for administration can be suitably formulated to give controlled release of active ingredients. For example, the pharmaceutical compositions may be in the form of particles comprising a biodegradable polymer and/or a polysaccharide jellifying and/or bioadhesive polymer, an amphiphilic polymer, an agent modifying the interface properties of the particles and a pharmacologically active substance. These compositions exhibit certain biocompatibility features which allow a controlled release of the active substance. See U.S. Patent No. 5,700,486.

Additionally, microparticles or nanospheres can be designed for controlled release. For oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (for example, pre gelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (for example, potato starch or sodium starch glycolate); or wetting agents (for example, sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. For administration by inhalation, the compounds can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Pharaiaceutical compositions that comprise active ingredients as described herein as an active ingredient will normally be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen. The pharmaceutically acceptable carriers and excipients useful in this invention are conventional. For instance, parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. Excipients that can be included are, for instance, proteins, such as human serum albumin or plasma preparations. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. For example, for parenteral administration, agents can be formulated generally by mixing them at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. A pharmaceutically acceptable carrier is a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

Generally, the formulations are prepared by contacting the active ingredients each uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Optionally, the carrier is a parenteral carrier, and in some embodiments it is a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Nonaqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. The pharmaceutical compositions that comprise RANKL, an agonist of

RANKL, an antigen of interest, or another agent of interest, in some embodiments, will be formulated in unit dosage form, suitable for individual administration of precise dosages. The amount of active compound(s) administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated.

The therapeutically effective amount of any active ingredient will be dependent on RANKL or the agonist of RANK utilized, the subject being treated, the severity and type of the affliction, and the manner of administration. For example, a therapeutically effective dose is readily determined by one of skill in the art based on the potency of the specific compound, the age, weight, sex and physiological condition of the subject.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

Example 1 Material and Methods Mice: Mice carrying a RANKL null mutation on a C57BL/6 background

(Kim et al, RANCE. J. Exp. Med. 192, 1467-1478, 2000a) were used to establish a breeding colony. RANKL null mice lack teeth, so weanling null mice born in this colony are routinely given powdered mouse chow. Mice heterozygous for the RANKL null mutation were also backcrossed to BALB/c mice (Taconic) for a total of 4 generations. Fl progeny of the first backcross were intercrossed to produce RANKL null mice and littermate controls on a mixed C57BL/6 and BALB/c background. RANKL null mice with the mixed genetic background are closer in weight to their heterozygous and wild type littermates and less likely to die prematurely. Experiments using RANKL null mice were done with either the C57BL/6 background mice or the mice with a mixed background. BALB/c mice

(Taconic) were used for experiments examining the effects of anti-RANKL mAb on PP M cells. Two models of B cell deficient mice were used: μMT mice and J₁₁ ⁷ mice (Taconic). CCR6 deficient mice were from a colony of homozygous Ccr6^tml(EGFP)Irw mice maintained on a C57BL/6 background (Kucharzik et al, Eur. J. Immunol. 32, 104-112, 2002).

Recombinant mouse RANKL: A bacterial expression construct encoding a glutathione S-transferase (GST) fusion protein containing amino acids 137-316 of mouse RANKL was assembled in the pGEX-5X-l vector (GE Healthcare) using a modification of a previously described method (Kubota et al., J. Bone Miner. Res. 17, 257-265, 2002). Specifically, the primers 5'- CACCCCCGGGTCAGCGCTTCTCAGGAGCT-S' (SEQ ID NO: 5) and 5'- CTCGAGTCAGTCTATGTCCTGAAC-3' (SEQ ID NO: 6) were used to PCR amplify a cDNA clone for mouse RANKL (Open Biosystems). After the PCR product was cloned into the pENTR-D-TOPO cloning vector (Invitrogen) and sequenced, the Smal-Xhol fragment was subcloned into pGEX-5X-l. The construct was transformed into the BL21 E. coli strain (Stratagene) for fusion protein expression. The cultures were induced with 20 μM IPTG for 16 hours at 20 ⁰C and the GST-RANKL purified from bacterial lysate by affinity chromatography on a GSTrap FF column (GE Healthcare) followed by dialysis against multiple changes of PBS. Recombinant GST prepared from empty pGEX-5X-l vector was used as a control for GST-RANKL. Biological activity of the GST-RANKL fusion protein was confirmed by its ability to induce differentiation of the RAW264.7 macrophage line (American Type Culture Collection) into multinucleate osteoclasts positive for tartrate resistant acid phosphatase. The GST-RANKL fusion protein was administered to RANKL null mice by daily i.p. injections of 250 μg per day for up to 7 days.

Antibodies and lectins: Monoclonal and polyclonal antibodies were purchased from eBioscience, unless otherwise stated. The mAbs used for immunofluorescence staining of frozen sections were anti-RANKL (IK22-5), anti- RANK (R12-31), anti-RANK (LOB 14-8; GeneTex), PE-conjugated anti-B220 (RA3-6B2), biotinylated GL7 (for detection of activated germinal center B cells), APC-conjugated anti-Thyl.2 (53-2.1; BD Biosciences), and anti-CD68 (FA-I l; AbD Serotec). The rat mAb NKM 16-2-4 specific for mouse M cells was purified from hybridoma supernatant and labeled with FITC (Terahara et al., J. Immunol. 180, 7840-7846, 2008). A purified rat IgG2a isotype control mAb (BD Biosciences) was used as a control for staining of frozen tissue sections with the rat IgG2a anti- RANKL and anti-RANK mAbs. Biotinylated polyclonal goat anti-rat IgG (BD

Biosciences) was used as a secondary reagent for detection of most unconjugated rat primary antibodies. Alexa 546-conjugated goat anti-rat IgG (Invitrogen) was used for the detection of the anti-CD68 mAb. Rhodamine-UEA-I was purchased from Vector Labs. The anti-RANKL antibody (IK22-5) used for in vivo RANKL neutralization experiments was prepared as described previously (Kamijo et al., Biochem. Biophys. Res. Commun. 347, 124-132, 2006). Mice were treated with 250 μg of antibody i.p. every 2 days.

ELlSA for measurement of fecal IgA: Fecal pellet samples were collected and extracted by making a 1:10 suspension (w/v) with PBS. After the suspension was vortexed and spun for 10 min at 12,000g, the supernatant was stored at -70⁰C. Polyclonal goat anti-mouse IgA antibody (Southern Biotechnology) was used as a capture antibody. The bound mouse IgA was detected with peroxidase-labeled goat anti-mouse IgA antibody (Southern Biotechnology) using TMB (BD Biosciences) as the peroxidase substrate. A mouse IgA,κ isotype control mAb (BD Biosciences) was used to establish a standard curve.

Immunofluorescence staining of frozen sections: Frozen sections of PP and adjacent intestinal tissue were cut on a cryostat and prepared for antibody staining experiments as previously described (Taylor et al., J. Immunol. 178, 5659-5667,

2007). The sections were washed in PBS and blocked in TNB buffer (PerkinElmer Life Sciences). Antibodies diluted in TNB buffer were applied for one hour at room temperature or overnight at 4⁰C. Biotinylated primary mAbs were detected using streptavidin-conjugated peroxidase followed by FITC-tyramide from a tyramide signal amplification kit (PerkinElmer Life Sciences). Unconjugated primary rat mAbs were detected by a combination of biotinylated polyclonal goat-anti-rat IgG (BD Biosciences) followed by streptavidin-peroxidase and FITC-tyramide. DAPI (Sigma-Aldrich) at 10 ng/ml was used as a nuclear counterstain. The slides were mounted in Pro Long antifade reagent (Invitrogen). Images were acquired using a Nikon 8Oi fluorescence microscope and edited with Photoshop (Adobe Systems).

Transmission electron microscopy: Mice were perfusion fixed using 2.5% glutaraldehyde solution in cacodylate buffer. Individual PP were isolated, bisected through the center of the domes, and embedded in Epon resin. Thin sections from the PP of control and RANKL null mice were examined using an H-7500 Hitachi electron microscope.

Whole mount staining ofPeyer's patches for detection of UEA-I⁺ M cells and intrinsic autofluorescence: For detection of M cells in PP, individual PP were excised and vortex mixed in 0.5% Tween20-PBS followed by a shaking incubation with 100 μg/ml DNase for 20 minutes at 37⁰C to promote dissociation of mucus from the epithelial layer. The PP were blocked with TNB buffer for 15 minutes at 4⁰C, and stained with rhodamine-UEA-I in TNB for 40 minutes at 4° C. Each stained PP was mounted under a 20 mm X 20 mm coverslip in 100 μl PBS. A count of UEA-I⁺ M cells was done for the PP follicle with the most M cells. This method resulted in some degree of underestimation of full extent of M cell depletion in RANKL null mice because often only one of several PP follicles had any M cells in the mutant mice, while all the follicles in wild type PP typically had a comparable number of M cells. Because the outlines of individual UEA-I⁺ M cells were best delineated in images acquired using a 6Ox objective, composite images representing single follicles were assembled by stitching of up to 4 overlapping individual 6Ox images using PhotoStitch 3.0 (Canon). To examine small intestine tissue for the presence of villous M cells, thin strips of tissue were cut and stained with rhodamine UEA-I as described above for PP. Villi with M cells on their surface were classified as showing a dense or diffuse pattern of villous M cells using specific criteria (Jang et al, Proc. Natl. Acad. Sci. U.S.A. 101, 6110-6115, 2004). Specifically, villi with one or more clusters of M cells in which 75% or more of the area within the cluster was occupied with M cells were considered to have a dense distribution of villous M cells. Villi with at least one characteristic UEA-I⁺ M cell on the surface, but not meeting the dense distribition criteria, were considered to have a diffuse distribution. For detection of the intrinsic autofluorescence, PP were washed as described above and then mounted immediately without any staining reagents. Images of autofluorescence were acquired using the same filter set normally used for FITC fluorescence with a constant manual exposure lasting 650 milliseconds for PP for all strains examined. The relative intensity of the autofluorescence signals was determined by threshold analysis using ImageJ v 1.36b software (available on the Internet). The images were saved as 8-bit grayscale images and then converted to binary images by thresholding at a grayscale cutoff point of 60 out of 255. The percentage of the pixels with an autofluorescence signal intensity that exceeded this threshold was calculated for the area occupied by each PP.

Assessment of fluorescent nanoparticle uptake in isolated small intestinal loops: The uptake of 200 nm diameter fluorescent polystyrene latex nanoparticles (Fluoresbrite YG; Polysciences) by M cells in the FAE of individual PP was assessed using a modification of previously described isolated small intestinal loop models (Chabot et al., J. Immunol. 176, 4275-4283, 2006; Pappo and Ermak, Clin. Exp. Immunol. 76, 144-148, 1989). Mice were anesthetized using an isoflurane vaporizer. After opening the peritoneum through a longitudinal midline incision, 2 or 3 segments of small intestine measuring 1-2 cm in length and containing a single PP were tied off with nylon filament. The loops were injected with 200 μl of a suspension of 200 nm nanoparticles diluted in PBS to a concentration of IxIO¹² beads/ml and returned to the peritoneal cavity. The mice were euthanized 90 minutes after the injection of beads, and the individual PP were excised, washed in 0.5% Tween 20-PBS, fixed in 4% paraformaldehyde in PBS for 15 minutes, and embedded in OCT. Frozen sections cut from these PP were examined by microscopy after counterstaining with DAPI, leaving out a cold acetone fixation step because it dissolved the polystyrene Fluoresbrite beads, preventing their visualization. Quantitative analysis of the degree of bead uptake into PP follicles was done by threshold analysis using ImageJ as described above for the analysis of autofluorescence, except that a grayscale cutoff point of 75 was used. Statistical analysis: Differences between the mean values for groups were analyzed by either ANOVA with Tukey correction (for multiple groups) or Student's t test as calculated using PRISM® (GraphPad Software). A p value of less than 0.01 was considered significant.

Example 2 UEA-I⁺ M cells are dramatically decreased in the FAE of PP from RANKL null mice M cells in mouse PP can be detected using the UEA-I lectin specific for α(l,2)-fucose linkages. In wild type mice, whole mount microscopy of PP follicles revealed an average of over 100 radially arranged UEA-I⁺ M cells that extended from the edges of the follicles towards the central subepithelial dome area. In contrast, UEA-I⁺ M cells were rare in PP from RANKL null mice and sometimes completely absent from individual follicles (Figure IA). The loss of M cells in PP from RANKL null mice was confirmed by staining PP sections with a recently described rat mAb (NKM 16-2-4) that is more selective than UEA-I for the specific α(l,2)-fucose moiety characteristically displayed by mouse M cells (Terahara et al., 2008). The FAE in RANKL null mice had a substantial decrease in the number of cells reactive with NKM16-2-4 compared to controls (Figure IB). Cells with the defining ultrastructural features of M cells by transmission electron microscopy (i.e. presence of intraepithelial pockets and blunting of the apical microvilli in comparison to normal enterocytes) were readily apparent in the FAE from control mice, but absent from the FAE of RANKL null mice (Figure 1C). While the number of UEA-I⁺ M cells was significantly decreased in all PP examined from RANKL null mice, a proximal to distal gradient in the number of UEA-I⁺ cells per dome was observed in RANKL null mice that was not seen in wild type mice (Figure ID). UEA-I⁺ M cells were almost completely absent in the most proximal PP from RANKL null mice, and progressively increased in more distal PP. In RANKL null mice, the most UEA-I⁺ M cells were consistently detected in the most distal ileal PP, although even in this PP the average number of M cells per dome was still less than a third of the wild type level. The calculated 73-fold depletion of UEA-I⁺ M cells in RANKL null mice (Table 1) was substantially greater than the reduction in M cells observed in both μMT B cell deficient mice and CCR6 deficient mice, strains of mutant mice previously shown to have a significant reduction in the number of M cells (Golovkina et al, Science 286, 1965-1968, 1999; Lugering et al, Am. J. Pathol. 166, 1647-1654, 2005). Table 1. Extent of M cell depletion seen in three mutant mouse models

Relative Fold

Number of PP Domes per PP M Cells per Dome Number Decrease

Mice Actual (Normalized) ^a Actual (Normalized) Actual (Normalized)*⁵ of M Cells' in M Cells^d

Wild Type 8.2 (1.00) 5.64 (1.00) 116 (1.00) 1.000 1

CCR6^"7" 5.5 (0.67) 3.4 (0.61) 53 (0.45) 0.189 5 μMT 5.7 (0.70) 3.4 (0.60) 28 (0.24) 0.101 10

RANKL^"7" 5.3 (0.64) 2.2 (0.40) 6 (0.05) 0.014 73

a The number of PP was counted in RANKL^"'^" mice (n=8), μMT B cell deficient mice (n=5), CCR6^"'^" mice (n=5), and wild type mice (n=10). The results are normalized to wild type levels set at 1.00. 0 ^b The number of UEA-I⁺ M cells were counted dome in the follicle with the most M cells in each PP examined. Scatter plots of these results are presented in Figure 2C (RANKL -^"/-^" mice) and Figure S 1

(μMT and CCR6 r-^"l'-^" mice). c The three normalized fractions (number of PP, domes, M cells) were multiplied to yield the fraction of M cells relative to wild type mice, an approach modeled on that described previously by 5 Golovkina et al. (Golovkina et al., Science 286, 1965-1968, 1999). d Ratio of total number of M cells in wild type mice to total number in the mutant strain.

Example 2

UEA-I⁺ M cells can be restored in RANKL null mice by treatment with 0 exogenous RANKL

To determine if the deficiency of M cells in the FAE of RANKL null PP could be restored by replacement of RANKL, RANKL null mice were injected i.p. for 7 consecutive days with 250 μg per day of either recombinant GST-RANKL fusion protein or recombinant GST as a control. On day 7, the PP follicles of 5 RANKL null mice treated with GST-RANKL had a near normal number of UEA-I⁺ M cells distributed in a normal pattern, while GST-treated mice remained profoundly M cell deficient (Figure 2A). Daily treatment of RANKL null mice with rGST-RANKL for shorter intervals demonstrated that day 5 was the first time point at which the number of UEA-I⁺ M cells was significantly increased over untreated RANKL null mice (Figure 2B).

Example 3 RANKL null mice have a defect in the uptake of 200 nm fluorescent beads into

PP follicles that is corrected by administration of RANKL While UEA-I is a useful immunohistochemical marker of mouse M cells, this method of identification does not detect M cells based on their specialized ability to take up particulate antigens from the lumen and transport them to meet APC in the intraepithelial pockets. Measuring uptake of fluorescent nanoparticles injected into loops of small intestine is a method that directly assesses M cell function in the FAE of PP (Chabot et al, J. Immunol. 176, 4275-4283, 2006; Pappo and Ermak, Clin. Exp. Immunol. 76, 144-148, 1989). Frozen sections of PP in isolated loops of small intestine from RANKL null mice and RANKL null mice treated with GST-RANKL or GST (as a control) were compared at 90 minutes after injection of fluorescent 200 nm nanoparticles into the loops. In the GST-RANKL- treated mice more UEA-I⁺ M cells were present and some of these cells contained multiple fluorescent beads (Figure 2C). Beads that had already passed through the epithelial layer to reach the PP follicle were observed in APC in the vicinity of the subepithelial dome or deeper in the B cell follicle. Image analysis was used to quantify the magnitude of bead uptake in the GST-RANKL reconstituted mice and controls (Figure 2D). Untreated RANKL null mice or those treated with GST had over 10-fold less uptake of beads than control wild type mice. GST-RANKL treatment for 7 days restored bead uptake to near wild type levels.

Example 4

Systemic administration of RANKL also leads to widespread induction of villous M cells

In the course of treating RANKL null mice with GST-RANKL and evaluating the reconstitution of M cells in PP, it was noted that the number of UEA- I⁺ cells present on small intestinal villi was also increased. This effect of RANKL treatment was further evaluated in BALB/c mice, in which less than 10% of small intestinal villi have any villous M cells at baseline with most of these villous M cells present in a diffuse pattern (Figure 3A,B). Treatment with systemic GST-RANKL i.p. for 4 consecutive days induced large numbers of UEA-I⁺ cells with the features of M cells on the surface of the villi (Figure 3C,D). Induction of an increased number of villous M cells began by 24 hours after the first injection of GST- RANKL; 4 days after the start of RANKL treatment all small intestinal villi had at least some UEA-I⁺ cells present, with 70% of villi showing a diffuse pattern and the remaining 30% exhibiting a dense pattern (Figure 3E).

Example 5

Neutralizing antibody to RANKL reproduces the M cell deficiency observed in

RANKL null mice Some of the developmental defects in RANKL null mice, such as the total absence of lymph nodes, cannot be corrected by simply injecting the mice with the absent cytokine as adults. This raises the issue of whether the M cell deficit observed in PP from RANKL null mice might be a byproduct of early developmental alterations in the PP of these mice. To address this issue, wild type BALB/c mice were treated i.p. with a neutralizing anti-RANKL antibody to determine if acute blockade of RANK/RANKL signaling would lead to loss of PP M cells. Mice were treated i.p. with 250 μg of the IK22-5 rat anti-mouse RANKL mAb every 2 days, a dose previously shown to block the activity of RANKL in vivo (Kamijo et al., Biochem. Biophys. Res. Commun. 347, 124-132, 2006). The number of M cells in the PP follicles was evaluated after various lengths of treatment by both UEA-I staining and by uptake of fluorescent 200 nm beads from isolated small intestinal loops. After 8 days of antibody treatment, the number of M cells present in the PP and the degree of uptake of fluorescent beads by PP in isolated loops were both dramatically decreased (Figure 4A-C). Analysis of the kinetics of the anti- RANKL effects showed that the number of UEA-I⁺ M cells dropped precipitously between 2 and 4 days, and declined further between 4 and 8 days (Figure 4D). Example 6 Epithelial cells in the small intestine express RANK

RANK is known to be expressed by multiple cell types including osteoclasts, dendritic cells, mammary epithelial cells, and thymic epithelial cells. Since the above-described experiments with RANKL null mice and neutralizing anti-RANKL antibody showed that RANKL is essential for normal M cell development within the FAE, immunohistochemical staining was used with anti-RANK antibodies to determine what cells in the vicinity of PP expressed the RANK. Staining for RANK was observed on epithelial cells in the FAE and was also detected on villous and crypt epithelial cells (Figure 5). The same epithelial distribution of RANK staining was observed with the R12-31 mAb was also observed with the LOB 14-8 anti- RANK mAb. Serial sections of the same PP showed that RANKL expression was restricted to stromal cells concentrated beneath the FAE as previously shown (Taylor et al., J. Immunol. 178, 5659-5667, 2007). These results suggest that RANKL exerts its effects on M cell differentiation through short-range delivery from the stromal cells to the FAE on the other side of the basement membrane.

Example 7 RANKL null mice have fewer autofluorescent particles in their Peyer's patches Pigmented particulate material has been described in macrophages in murine and human PP by several techniques, including direct visualization, routine histology, and electron microscopy (Powell et al., Gut 38, 390-395, 1996; Shepherd et al., Hum. Pathol. 18, 50-54, 1987; Thoree et al., Inflamm. Res. 57, 374-378, 2008; Urbanski et al., Mod. Pathol. 2, 222-226, 1989). In the course of doing immunofluorescence microscopy on frozen sections of PP from wild type and

RANKL null mice, autofluorescent particles were observed in the follicles of wild type PP concentrated in the subepithelial dome area and at the base of the PP (Figure 6A). Many of these autofluorescent particles were located within the cytoplasm of CD68⁺ macrophages. RANKL null mice had a much lower density of these autofluorescent particles within CD68⁺ cells. Image analysis was done on fluorescence images acquired using identical camera settings (Figure 6B), revealing a highly significant decrease in the degree of autofluorescence. Based on these observations on frozen sections, whole mount images taken from the mucosal aspect of the small intestine were evaluated for the presence of auto fluorescent particles. PP follicles from wild type mice displayed a higher concentration of autofluorescent particles in the center of the PP follicles than anywhere else in the small intestine, with a lower density of particles in the interfollicular areas (Figure 6C). Strikingly, far fewer autofluorescent particles were observed by the same technique in PP from RANKL null mice. The pattern of distribution of the particles in RANKL null mice was also changed, with loss of the characteristic central aggregates of autofluorescent particles. Image analysis of whole mount images acquired revealed the fraction of the follicular area occupied by autofluorescent particles was much less in RANKL null mice than in controls (Figure 6D). This result indicates that the frequency of these autofluorescent particles is highly correlated with the number of UEA-I⁺ M cells and provides a way to assess the chronic ongoing activity of PP M cells.

Example 8 RANKL null mice exhibit decreased PP germinal center formation and fecal

IgA production PP were previously reported to be smaller than normal in two independently derived strains of RANKL null mice (Kim et al., Proc. Natl. Acad. Sci. U.S.A. 97, 10905-10910, 2000b; Kong et al., Nature 397, 315-323, 1999), but other aspects of PP function were not examined. It was asked whether the loss of M cell function in RANKL null mice was associated with impaired B cell responses to antigens internalized from the intestinal lumen. The frequency and extent of germinal center development in PP from RANKL null mice and littermate controls was compared using an antibody (GL7) that preferentially binds activated germinal center B cells. Compared to PP from controls, PP from RANKL null mice at 10 to 12 weeks of age exhibited a smaller percentage of germinal centers containing GL7⁺ cells in the B cell zones and a relative expansion of the T cell zones (Figure S2A). This finding suggested that the production of secretory IgA might also be impaired in RANKL null mice. Fecal IgA concentrations in mice from 4 to 12 weeks of age were consistently decreased in RANKL null mice compared to littermate controls. Antigen sampling M cells have been described in both mammalian and avian species as part of the FAE covering the organized lymphoid structures of the respiratory and digestive tract (Kraehenbuhl and Neutra, Annu. Rev. Cell Dev. Biol. 16, 301-332, 2000; Kunisawa et al, Trends Immunol. 29, 505-513, 2008; Neutra et al., Nat. Immunol. 2, 1004-1009, 2001). However, the specific signals and signaling pathways that trigger the differentiation of these M cells from precursor cells located in the stem cell zone of the crypts or from the enterocytes on the surface of the FAE were not identified (Mach et al., Immunol. Rev. 206, 177-189, 2005). There are strains of mutant mice created by gene-targeting that retain PP but exhibit decreased numbers of M cells in these PP. Specifically, B cell deficient mice such as μMT mice exhibit significantly reduced numbers of M cells in PP (Golovkina et al., Science 286, 1965-1968, 1999). Additional support for a role of B cells in promoting M cell development has come from in vitro studies in which co-culture of freshly isolated B lymphocyte or B lymphocyte lines with model intestinal epithelial cell lines cultured on semipermeable supports promoted the development of M cell- like features by the epithelial cells, including transcytosis of particulate antigens (des Rieux et al., Eur. J. Pharm. Sci. 30, 380-391, 2007; Kerneis et al., Science 277, 949- 952, 1997). However, neither in vivo analysis of PP from B cell deficient mice or experiments based on the in vitro M cell differentiation system have elucidated the specific mechanism by which B cells promote differentiation of M cells in the FAE.

To determine if PP were functionally compromised in the absence of RANKL, the PP of RANKL null mice were characterized, as described above. Staining of PP from RANKL null mice with the UEA-I lectin reactive with murine M cells revealed a profound depletion in UEA-I⁺ cells compared to wild type mice. Taking into account all of the factors that contribute to the total number of M cells within small intestinal PP (i.e. number of PP, number of follicles per PP, number of M cells per follicle), RANKL null mice had less than 2% of the number of UEA-I⁺ M cells found in wild type mice. On a quantitative basis, this is a much more pervasive loss of M cells than that measured in B cell deficient μMT mice (with 13% of wild type levels) by the same methods. While the UEA-I lectin is the immunohistochemical reagent relied on most heavily to establish that RANKL null mice are deficient in M cells, several independent means of confirming this deficiency was used in M cells, including functional measurements of M cell activity using uptake of fluorescent nanoparticles, transmission electron microscopy, and immuno staining with the NKM 16-2-4 monoclonal antibody specific for mouse M cells. RANKL acting through its specific receptor (RANK) is known to play an important developmental role in multiple tissues. The most striking and best-studied of the deficits in RANKL null mice are the absence of any lymph nodes and the failure of osteoclast development, leading to osteopetrosis and a malformed skeleton. One potential explanation of the loss of M cells observed in PP from RANKL null mice in the experiments described above is an early developmental defect in PP development that permanently compromises the capacity of the FAE to generate conventional M cells. Two types of experiments were done to test this possibility. First, it was examined whether the M cell defect was reversible if a source of exogenous recombinant RANKL was provided. Daily injections of GST- RANKL given for 5 or more days provided a nearly complete reconstitution of the number of M cells per PP follicle. Second, neutralizing mAb to RANKL was used to test whether acute depletion of RANKL in adult wild type mice would also cause loss of M cells. After 4 days of anti-RANKL treatment to inhibit normal RANKL- RANK interactions, the number of UEA-I⁺ M cells in each PP follicle plunged to levels approaching those in the RANKL null mice. Thus, production of RANKL must be sustained in the adult PP to permit the continued production and/or survival of M cells.

RANK is expressed on multiple cell types including osteoclasts and their precursors, dendritic cells, endothelial cells, mTEC, and mammary epithelial cells. Without being bound by theory, the simplest model to explain the observed effects of RANKL on M cell differentiation is that RANKL derived from the subepithelial dome stromal cells in the PP acts in a paracrine fashion on the adjacent epithelial cells of the FAE. Because RANKL is a type II membrane protein that is synthesized in a transmembrane form, cleavage by metalloproteases is needed to generate a soluble form of the cytokine (Hikita et al, J. Biol. Chem. 281, 36846-36855, 2006; Lum et al., J. Biol. Chem. 274, 13613-13618, 1999). Without being bound by theory, RANKL can be acting directly through RANK on enterocytes because immunohistochemical staining of small intestinal tissue including a PP showed that the bulk of the RANK staining is localized to the epithelium, with roughly equivalent levels of RANK on the FAE and villous epithelium. Gene expression profiling studies comparing flow sorted PP M cells and villous enterocytes revealed that both of these intestinal epithelial cell types express mRNA for RANK (Terahara et al., J. Immunol. 180, 7840-7846, 2008) (gene expression data archived in NCBI Gene Expression Omnibus under accession number GSE7838).

The capacity of soluble recombinant RANKL injected systemically to induce a massive expansion in the number of M cells on all small intestinal villi provides further insights into the mechanism of action of RANKL in induction of M cells.

RANKL-mediated induction of villous M cells demonstrates that RANK-expressing epithelial precursor cells located in both dome- associated crypts next to PP follicles and in standard small intestinal crypts have the potential to differentiate into M cells if exposed to sufficient stimulation with RANKL. Under normal conditions, M cell development is primarily restricted (other than a small number of scattered villous M cells) to the organized lymphoid tissues of the small intestine (i.e. PP and ILF) because constitutive expression of RANKL is restricted to subepithelial stromal cells at these sites. When the spatial restriction of RANKL availability in the small intestine is bypassed by systemic injection, RANKL is able to trigger M cell differentiation in epithelial precursors in both dome-associated crypts adjacent to organized lymphoid tissues and normal crypts.

Accumulation of pigmented microparticles in the follicles of PP has been previously described. Pigmented areas of the terminal ileal mucosa in the vicinity of PP have been observed by colonoscopy, direct gross observation of surgical resection specimens, and analysis of tissue sections (Powell et al., Gut 38, 390-395, 1996; Shepherd et al., Hum. Pathol. 18, 50-54, 1987; Urbanski et al., Mod. Pathol. 2, 222-226, 1989). Subsequent studies localized this pigment to CD68⁺ macrophages and identified the composition of the pigmented material by chemical and spectroscopic studies as inorganic microparticles of titanium dioxide, aluminosilicates, or other silicates derived from the external environment and foods (Powell et al., Br. J. Nutr. 98 Suppl 1, S59-63, 2007; Thoree et al., Inflamm. Res. 57, 374-378, 2008). Localization of these microparticles within PP is attributed to uptake of these particles from the intestinal lumen by the M cells in PP. In the course of doing immunofluorescence microscopy on frozen sections of PP, it was discovered that the follicles of wild type PP harbor autofluorescent particles at a much greater density than the adjacent small intestine. The observations of autofluorescent particles with the same localization in PP suggest that a subset of these inorganic microparticles also exhibit autofluorescence. PP from RANKL null mice contained far fewer autofluorescent particles than wild type PP, with the reduction comparable in magnitude to the loss of UEA-I⁺ M cells. These findings suggest that the accumulated M cell-mediated uptake of these microparticles that takes place in normal PP is largely ablated in RANKL null mice by the loss of functional M cells.

The experimental results presented herein identify RANKL as a key cytokine signal involved in inducing the differentiation of M cells from precursors in the FAE. A small number of residual UEA-I⁺ M cells was consistently observed in the RANKL null mice. A model that postulates that additional signals besides RANKL are involved in promoting the development of M cells. The most distal PP in RANKL null mice was invariably the PP with the largest number of M cells per follicle, suggesting that an increased density of luminal commensal bacteria can accentuate the extent of M cell differentiation locally in situations in which loss of an M cell-inducing factor results in a global decrease in M cell differentiation. The most distal PP also consistently had the highest number of UEA-I⁺ M cells per follicle in B cell deficient mice and CCR6 deficient mice, indicating that this positional effect on M cell development in PP is not restricted to RANKL null mice. One of the other signals capable of promoting M cell development could be contributed by local B cells in the PP, since absence of mature B cells also leads to depletion of PP M cells (Golovkina et al, Science 286, 1965-1968, 1999), although not to the same degree as in RANKL null mice. Exogenous administration of GST- RANKL to B cell deficient J_H ^~'^~ mice does not increase the number of PP M cells, indicating that the contribution of B cells to the development of M cells does not involve simply providing RANKL.

A better understanding of the regulation of M cell differentiation has therapeutic applications. Specifically targeting orally administered antigens to M cells using either monoclonal antibodies to M cell surface receptors, lectins, or bacterial adhesins specific for M cells remains an active area in the development of vaccines for oral delivery (Foxwell et al., Hum. Vaccin. 3, 220-223, 2007). Combining antibody-mediated M cell targeting with a strong mucosal adjuvant (such as cholera toxin) already shows promise as a strategy for the establishment of both mucosal and systemic immunity to antigens administered for the purpose of vaccination (Nochi et al., J. Exp. Med. 204, 2789-2796, 2007). Local manipulation of the RANKL-RANK pathway can provide opportunities for further promoting the efficacy of these vaccines. The density of M cells in the FAE of PP differs between different species: the FAE of rabbit PP contains up to 50% M cells, while M cells typically make up only 5 to 10% of the mouse and human FAE (Davis and Owen, Springer Semin. Immunopathol. 18, 421-448, 1997). Systemic delivery or ideally local delivery of exogenous RANKL has the could increase the frequency/number of human M cells in both the PP FAE and the villous epithelium to supraphysiologic levels, thereby increasing the efficiency of delivery of M cell-targeted vaccines administered at mucosal surfaces.

Example 9 Use of Agonists of RANK to Induce M Cell Differentiation

Several types of antibodies raised to the RANK receptor have been shown to have agonist activity at the RANK receptor, specifically inducing the development of osteoclasts from macrophages and other osteoclast precursor cells. This type of agonist activity of an antibody against a receptor is commonly observed among members of the tumor necrosis factor receptor family and has been well documented for other members of this family including the lymphotoxin beta receptor (LTbetaR) and 4- IBB.

To determine if antibodies to mouse RANK share with RANKL the activity to induce M cell differentiation, a commercially available preparation of polyclonal affinity-purified goat anti-mouse RANK antibodies was obtained (R & D Systems, Inc., product number AF692). These antibodies are tested by the vendor for their ability to stimulate mouse RANK using a bioassay based on the development of mature osteoclasts from precursors. Wild type mice were given 35 micrograms of this antibody by intra-peritoneal (i.p.) injection on day 0 and day 2. On day 4, the small intestine tissue was examined by whole mount microscopy for the induction of increased numbers of villous M cells reactive with rhodamine-labeled UEA-I. In some areas of the distal small intestine, there was substantial induction of M cells on most of the villi. Figure 7A shows large numbers of individual villous M cells and clusters of villous M cells on villi from an anti-RANK-treated mouse. In contrast, villi from the same region in untreated mice show only rare single villous M cells. This finding shows that agents such as antibodies to RANK that share with RANKL the ability to activate the RANK receptor also have the capacity to induce M cell differentiation.

Example 10 Pre-treatment with RANKL Enhances an Antibody Response The results presented below demonstrate that administration of RANKL increases the immune response to an antigen. In addition, the results demonstrate a synergistic effect of combining the use of RANKL and another immuno stimulatory adjuvant.

Materials and Methods: An E. coli DH5α bacterial strain expressing the model antigen ovalbumin (E. coli-OYA) under the control of the anaerobically inducible nirB promoter was used as a source of antigen for oral immunizations of BALB/c mice. Bacterial cultures were grown overnight to saturation in tightly capped bottles to provide anaerobic conditions that induce activity of the nirB promoter. The bacteria were washed and fixed by a 30-minute incubation in 10% buffered formalin at room temperature, followed by multiple washes with PBS. Groups of mice were orally immunized with a single dose of 1 X 10⁹ fixed bacteria using a gavage needle. Some groups were pretreated with GST-RANKL fusion protein given by subcutaneous injection (40 μg per day for 4 consecutive days) to induce M cells and also given a final subcutaneous injection of 40 μg of GST- RANKL at the time of oral immunization. Some groups received a single i.p. injection of 10 μg E. coli LPS at the time of oral immunization as a systemic adjuvant.

The primary IgM antibody responses in serum were measured by ELISA on samples collected 7 to 10 days after the oral immunizations. Micro titer plates were coated with ovalbumin and antibody binding to the ovalbumin was detected by incubation of serial dilutions of serum followed by a secondary polyclonal goat anti- mouse IgM antibody coupled to horseradish peroxidase. TMB was used as a substrate to develop the plates. The absorbance of the wells at 605 nm were read using a microplate reader and the data plotted to allow for the calculation of the endpoint titer for each serum sample.

Results: A single oral immunization of mice with fixed E. coli-OYA after pretreatment for 4 days with subcutaneous injections of GST-RANKL to expand the number of intestinal M cells stimulated an IgM antibody response to OVA that was detectable in serum samples collected 10 days after the immunization. In mice pretreated with GST as a control protein (without treatment with RANKL), an IgM antibody response to OVA could not be detected above the background signal of the prebleed controls. Use of the endpoint titer method of analysis revealed that the RANKL-pretreated mice mounted an IgM response that was approximately 8-fold greater than that seen in mice pretreated with GST as a control protein (Figure 9).

To determine if the adjuvant effect of pretreatment with GST-RANKL showed synergy with the administration of a single i.p. injection of lipopolysaccharide (LPS) given at the time of oral immunization, groups of mice were treated with either GST-RANKL pretreatment, i.p. LPS, or a combination of both treatments. Control groups received GST instead of GST-RANKL. Both GST- RANKL alone and LPS alone increased the IgM anti-OVA response at 7 days over mice receiving just GST pretreatment. The combination of GST-RANKL and LPS gave the strongest IgM antibody response, revealing a synergistic effect between the two classes of adjuvants tested in the experiment (Figure 10). Example 11 Microparticle or Nanoparticle encapsulation of RANKL

This example documents that oral formulations were produced for the delivery of RANKL. These formulations induce the production of M cells. Small microparticles or nanoparticles can be used to encapsulate RANKL, and these microparticles or nanoparticles survive transit through the acidic environment in the stomach. These particles can release proteins in the small intestine, wherein the luminal pH is neutral.

Administration of this formulation of RANKL, such as GST-RANKL, results in lower levels of osteoclast activation as compared to subcutaneous injections of GST-RANKL, as judged by serum levels of osteoclast-specific protein TRAP5b.

RANKL was encapsulated in particles using a Buchi 191 spray dryer. Other methods can be used to encapsulate RANKL, see for example PCT Application No. PCT/US2009/058896, which is incoporated herein by reference.

The following particles were produced in a Biichi spray dryer are (all percentages listed are weight/weight):

Purified GST-RANKL dialyzed against 0. IX PBS 5.0% Mouse plasma (used as a source of mouse albumin) 28.33%

Acrylate polymer 28.33%

Cellulose polymer 28.33%

Trehalose 5.0%

Tween-20 0.01% Chitosan 5.0%

The oral formulation was administered to mice, and intestinal M cells were induced. The successful induction of intestinal M cells using this oral formulation of GST- RANKL documents that oral formulations can be produced for the delivery of RANKL. A variety of such enteric release oral formulations such as standard soft gel gelatin capsules that can be filled with liquid ingredients during the manufacturing process, also induce the production of M cells. It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described invention. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

1. A method of increasing a mucosal immune response, comprising selecting a subject in need of an increased mucosal immune response; administering to the subject a therapeutically effective amount of RANKL or an agonist of RANK, wherein the RANKL or the agonist of RANK administration results in the differentiation of microfold (M) cells in the intestine; and administering to the subject a therapeutically effective amount of an antigen of interest, wherein the therapeutically effective amount of RANKL or the agonist of RANK is administered sufficiently prior to the administration of antigen of interest to allow M cells to differentiate, thereby increasing the mucosal immune response to the antigen.

2. The method of claim 1, wherein the antigen is administered orally.

3. The method of claim 1, wherein the antigen is from a pathogen and is administered as a vaccine.

4. The method of claim 1, wherein the agonist of RANK is an antibody that specifically binds RANK.

5. The method of claim 4, wherein RANKL or the agonist of RANK is administered one day to one week prior to administering the antigen

6. The method of claim 1, wherein RANKL or the agonist of RANK is administered intra-nasally or orally.

7. The method of claim 1, wherein the antigen is targeted to the M cells.

8. The method of claim 7, wherein the antigen is targeted using an M cell-specific antibody, a lectin or an adhesin molecule specific for M cells.

9. The method of claim 1, wherein the antigen is administered with a mucosal adjuvant.

10. The method of claim 9, wherein the mucosal adjuvant is cholera toxin.

11. The method of claim 1, wherein the antigen is administered as a controlled release formulation.

12. The method of claim 1, further comprising measuring the number of M cells in a sample from the subject.

13. A method for increasing drug delivery to a mucosa, comprising administering to a subject a therapeutically effective amount of RANKL or an agonist of RANK; and administering to the subject a therapeutically effective amount of an agent, wherein the therapeutically effective amount of RANKL or the agonist of

RANK is administered sufficiently prior to the agent to allow M cells to differentiate and increase agent uptake; thereby increasing the delivery of the agent to the mucosa.

14. The method of claim 13, wherein the RANKL, the agonist of RANK, the agent, or any combination thereof are administered in a microp article.

15. The method of claim 14, wherein the particle is a liposome or a Lactobacillus.

16. The method of claim 14, wherein RANKL is administered in the Lactobacillus and wherein the drug is administered in a microparticle.

17. The method of claim 14, wherein the microparticle has a diameter of 100 nanometers to 10 microns.

18. The method of claim 13, wherein RANKL is administered intranasally and wherein the mucosa is nasal mucosa.

19. The method of claim 13, wherein the agent is administered orally, and wherein the mucosa is the intestinal mucosa.

20. The method of claim 13, wherein the therapeutically effective amount of RANKL is administered about one day to about one week prior to the administration of the agent.

21. The method of claim 13, wherein the agent is conjugated to an microfold (M) cell ligand.

22. The method of claim 21, wherein the M cell ligand is the reovirus sigma 1 attachment protein.

23. The method of claim 21, wherein the M cell ligand is an antibody that specifically binds M cells.

24. The method of claim 13, wherein the agent is a therapeutic protein.

25. The method of claim 24, wherein the therapeutic protein is administered in Lactococcus lacti.

26. The method of claim 13, wherein the agent is an antigen, a vaccine, a chemical compound, or an antibody.

27. The method of claim 13, wherein the mucosa is the mucosa of the small intestine, the colon or the nasal mucosa.