WO1998057655A1 - A method of enhancing antigen-specific peripheral immune tolerance - Google Patents

Abstract

There is disclosed a method of initiating or enhancing antigen-specific immune tolerance by administering an effective amount of flt3-ligand prior to, concurrent with, or subsequent to the mucosal administration of an immunotolerizing amount of the immunogenic molecule. Tolerance can be initiated or enhanced to angitens associated with autoimmune diseases, organ or tissue transplantation, food allergies, and immunogenic therapeutic products.

Description

TITLE A Method of Enhancing Antigen-Specific Peripheral Immune Tolerance

FIELD OF THE INVENTION

This invention relates to a method of enhancing the specific immuno tolerance to an antigen, and more particularly, to a method of enhancing tolerance to mucosally- administered antigens.

BACKGROUND OF THE INVENTION

It is essential for normal life that the intestinal mucosa is protected from pathogens. It is also important that immune responses are not directed against harmless or potentially beneficial antigens such as foods and commensal bacteria. Although the intestine is continually exposed to both pathogens and essential nutrients the mucosal immune system is able to discriminate effectively between harmful and innocuous antigens. The mechanisms which maintain the state of systemic tolerance are the subject of controversy. All aspects of immune regulation have been implicated for being responsible for antigen-specific tolerance, including clonal deletion, see for example, Chen, et al., Nature, 376: 177 (1995) and Marth et al., J. Immunol., 157:2348 (1996); anergy of T cells, see for example, Whitacre, et al., J. Immunol, 147:2155 (1991) and Van Houghten et al., J. Immunol., 157: 1337 (1996). The generation of active regulatory mechanisms have been implicated: these may include preferential activation of CD8+ T cells, γ_ T cells, cross-regulation of inflammatory Thl CD4+ T cells by Th2 cells, or the preferential production of inhibitory cytokines such as IL-4, IL-10 or TGF-β. While different mechanisms appear to predominate, under different experimental conditions it would not be surprising if many are activated simultaneously to ensure tolerance is maintained.

The mucosal administration of soluble proteins is one of the most effective means of inducing specific systemic immunological unresponsiveness. The phenomenon was first known as oral tolerance. Induction of oral tolerance by administration of an antigen to the peripheral immune system via the gut has been found to inhibit several forms of experimental autoimmune disease, including experimental autoimmune encephalomyelitis (EAE), diabetes, collagen-induced arthritis, uveitis and thyroiditis. Clinical trials that take advantage of this tolerization phenomenon are currently underway for a number of disorders including rheumatoid arthritis, multiple sclerosis, diabetes and uveitis. SUMMARY OF THE INVENTION

The present invention pertains to a method of augmenting peripheral immune tolerance to an antigen. More particularly, the invention relates to a method of using the polypeptide, flt3-ligand (flt3-L) to initiate or enhance peripheral immune tolerance to a mucosally-administered antigen. The method of the invention comprises administering a tolerance-enhancing amount of flt3-L prior to, concurrent with, or subsequent to the mucosal administration of an immunotolerizing amount of the antigen. The method of the invention finds utility in ameliorating the effects of autoimmune disorders, food allergies, or organ or tissue rejection following transplantation. In addition, the method of the invention finds use in initiating or augmenting the tolerance to pharmaceutical products whose effectiveness is diminished due to the generation of an immune response directed at the drug itself. Administration of flt3-L prior to, concurrent with or subsequent to the mucosal administration of the drug would enhance tolerance of the drug and provide greater efficiency in systemic drug dosing.

DETAILED DESCRIPTION OF THE INVENTION

Flt3-ligand ("flt3-L") is known to affect hematopoietic stem and progenitor cells and dendritic cells. It was surprisingly found that flt3-L also enhances tolerance to mucosally-administered antigen and allows lower dosages of antigen to be used in vivo. The invention provides for the use of a tolerance-enhancing, or tolerance-augmenting amount of flt3-L prior to, concurrent with, or subsequent to the mucosal administration of an immunotolerizing amount of the antigen.

The invention also relates to an improved method for suppressing autoimmune diseases, or suppressing immune responses to transplanted organs or tissue, which method comprises administering flt3-L in an amount effective to initiate or enhance tolerization to a mucosally-administered antigen associated with the disease.

As used herein, the term "mucosal administration" means administration via oral, nasal, lung, vaginal or rectal mucous membranes.

As used herein, the term "flt3-ligand" or "flt3-L" refers to a genus of polypeptides that are described in United States Patent No. 5,554,512, EP 0627487 A2, WO 94/28391, and Lyman et al., Cell, 75:1157 (1993) and Lyman et al., Blood, 83, No. 10:2795 (1994) all incorporated herein by reference. A human flt3-L cDNA was deposited with the American Type Culture Collection, Rockville, Maryland, USA (ATCC) on August 6, 1993 and assigned accession number ATCC 69382. The deposit was made under the terms of the Budapest Treaty. Flt3-L can be made according to the information described in the documents cited above using methods per se well-known in the art. A cDNA encoding murine flt3-L is disclosed in SEQ ID NO: l. The amino acid sequence for murine flt3-L is shown in SEQ ID NO:2. A cDNA encoding human flt3-L is disclosed in SEQ ID NO:3. The amino acid sequence for human flt3-L is shown in SEQ ID NO:4. The definition of flt3-L includes soluble or truncated forms of flt3-L, which are described in the documents cited above, and described infra. Specifically, flt3-L means a polypeptide which binds flt3 and which comprises (a) the sequence of amino acids 28-x of SEQ ID NO:2, wherein x is an amino acid selected from the group consisting of amino acids 163 to 231; (b) amino acids 28-y of SEQ ID NO:4, wherein y is an amino acid selected from the group consisting of amino acids 160 to 235; or (c) an amino acid sequence that is at least 90% identical or homologous to the amino acid sequences of (a) or (b).

As described supra., an aspect of the invention is the use of soluble flt3-L polypeptides. Soluble flt3-L polypeptides comprise all or part of the extracellular domain of a native flt3-L but generally lack the transmembrane region that would cause retention of the polypeptide on a cell membrane. Soluble flt3-L polypeptides advantageously comprise the native (or a heterologous) signal peptide when initially synthesized to promote secretion, but the signal peptide is cleaved upon secretion of flt3-L from the cell. Soluble flt3-L polypeptides encompassed by the invention retain the ability to bind the flt3 receptor. Indeed, soluble flt3-L may also include part of the transmembrane region or part of the cytoplasmic domain, provided that the soluble flt3-L protein can be secreted.

Soluble flt3-L may be identified (and distinguished from its non-soluble membrane-bound counterparts) by separating intact cells which express the desired protein from the culture medium, e.g., by centrifugation, and assaying the medium (supernatant) for the presence of the desired protein. The presence of flt3-L in the medium indicates that the protein was secreted from the cells and thus is a soluble form of the desired protein.

Soluble forms of flt3-L possess many advantages over the native bound flt3-L protein. Purification of the proteins from recombinant host cells is feasible, since the soluble proteins are secreted from the cells. Further, soluble proteins are generally more suitable for intravenous administration. Examples of soluble flt3-L polypeptides include those comprising a substantial portion of the extracellular domain of a native flt3-L protein. Such soluble mammalian flt3-L proteins comprise amino acids 28 through 188 of SEQ ID NO:2 or amino acids 28 through 182 of SEQ ID NO:4. In addition, truncated soluble flt3-L proteins comprising less than the entire extracellular domain are included in the invention. Such truncated soluble proteins are represented by the sequence of amino acids 28-163 of SEQ ID NO:2, and amino acids 28-160 of SEQ ID NO:4. When initially expressed within a host cell, soluble flt3-L may additionally comprise one of the heterologous signal peptides described below that is functional within the host cells employed. Alternatively, the protein may comprise the native signal peptide, such that the mammalian flt3-L comprises amino acids 1 through 188 of SEQ ID NO:2 or amino acids 1 through 182 of SEQ ID NO:4. Truncated flt3-L, including soluble polypeptides, may be prepared by any of a number of conventional techniques, in particular those described in United States Patent No. 5,554,512, EP 0627487 A2, WO 94/28391, which have been incorporated herein by reference.

The term "biologically active" as it refers to flt3-L, means that the flt3-L is capable of binding to flt3.

"Isolated" means that flt3-L is free of association with other proteins or polypeptides, for example, as a purification product of recombinant host cell culture or as a purified extract.

A flt3-L variant is a polypeptide substantially homologous to native flt3-L, but which has an amino acid sequence different from that of native flt3-L (human, murine or other mammalian species) because of one or more deletions, insertions or substitutions. The variant amino acid sequence preferably is at least 80% identical to a native flt3-L amino acid sequence, most preferably at least 90% identical. The percent identity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981). The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Variants may comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as He, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known. Naturally occurring flt3-L variants are also encompassed by the invention. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the flt3-L protein, wherein the flt3-L binding property is retained. Alternate splicing of mRNA may yield a truncated but biologically active flt3-L protein, such as a naturally occurring soluble form of the protein, for example. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the flt3-L protein (generally from 1-5 terminal amino acids).

Antigens to which tolerization can be enhanced as a result of this invention include those associated with diseases such as arthritis, multiple sclerosis, diabetes, uveitis, and thyroiditis. For example, type II collagen and human gρ-39 are autoantigens associated with rheumatoid arthritis. Autoantigens associated with multiple sclerosis are myelin antigens, such as myelin basic protein or proteolipid protein. Antigens associated with food allergies can also be subject to enhanced tolerization. An autoantigen associated with uveitis is the S-antigen. An autoantigen associated with diabetes can be for example, insulin, glutamic acid decarboxylase, carboxypeptidase H, heat shock proteins, or insulin secretory granule proteins. In addition, tissue or organ antigens from donors can administered according to the invention to provide tolerance thereof. Any antigen or autoantigen known to be associated with a disease or condition may be suitable for oral or mucosal administration. Furthermore, it is preferred, but not critical, that the entire antigen be mucosally-administered to induce tolerance. However, tolerogenic peptide fragments of the antigen or autoantigen can be administered mucosally to induce tolerance.

The invention also finds use in situations where a pharmaceutical product is immunogenic. Many marketed and development stage drugs are of limited use for chronic administration due to development of antibodies against the recombinant protein. The development of such antibodies can result from differences between recombinant and native proteins, for example, missing or incorrect post translational modifications, changes in glycosylation patterns, or because the amino acid backbone has been modified. For example beta-interferon is produced in E. coli and lacks any glycosylation and recent reports indicate that some patients have developed antibodies against the drug. The effectiveness of such a drug can be diminished due to the neutralizing antibodies generated against such drug. Where a drug is known or suspected to be immunogenic, flt3-L may be useful when used according to the invention to initiate or enhance immune tolerance to such immunogenic drug. In addition, many drug delivery vehicles are immunogenic. For example, in gene therapy adenovirus, adeno-associated virus, herpes simplex virus, lend virus and retroviruses are known to be immunogenic. Further, patients that have not yet undergone treatment with a known or suspected immunogenic drug or delivery vehicle could benefit from a regimen of flt3-L administered prior to, concurrent with or subsequent to the mucosal administration of the immunogenic product. It may be beneficial in some instances to practice the claimed invention by mucosally tolerizing a patient with an immunogenic protein and flt3-L prior to beginning the treatment regimen with the same drug. This protocol would allow for specific immune suppression against the protein prior to beginning full treatment.

By way of example only, patients who have generated an immune response against beta-interferon could benefit from flt3-L administration prior to, concurrent with or subsequent to the oral administration of beta-interferon. Oral administration of the beta-interferon would induce immunologic tolerance to the drug which would be enhanced by administration of flt3-L. This allows for more effective systemic dosing of the beta-interferon product.

Flt3-L polypeptides of the invention can be formulated according to known methods used to prepare pharmaceutically useful compositions. Flt3-L can be combined in admixture, either as the sole active material or with other known active materials, with pharmaceutically suitable diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Co. In addition, such compositions can contain flt3-L complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of flt3-L.

Flt3-L can be administered topically, parenterally, or by inhalation. The term "parenteral" includes subcutaneous injections, intravenous, intramuscular, intracisternal injection, or infusion techniques. These compositions will typically contain an effective amount of the flt3-L, alone or in combination with an effective amount of any other active material. Such dosages and desired drug concentrations contained in the compositions may vary depending upon many factors, including the intended use, patient's body weight and age, and route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration can be performed according to art-accepted practices.

In addition to the above, the following examples are provided to illustrate particular embodiments and not to limit the scope of the invention.

EXAMPLE Enhancement of Oral Tolerance in Mice

(A) Administration offlt3-L in vivo. A well-established model of oral tolerance is described in Mowat A.M., Immunol. Today, 8:93 (1987) and in Garside et al., J. Immunol., 154:5649 (1995), each of which is incorporated herein by reference. Following the described procedures for the model, female C57BL/6 mice (6-8 weeks of age) were obtained from Taconic Laboratories (Germantown, NY) and were maintained in accordance with approved ethical guidelines. Mice were injected intraperitoneally (ip) once daily with purified CHO-derived human flt3-L (lOμg) for 12 days. CHO-derived flt3-L was obtained following the procedures described in Brasel et al., Blood, 88:2004 (1996). Control mice were injected i.p. with either purified human IgGl (lOμg; Sigma) or PBS (lOOμl) for the same period. Control and flt3-L mice were injected with PBS or flt3- L, respectively, for ten days prior to and for two days after oral administration of antigen.

(B) Administration of Antigen (Ovalbumin). The mice were fed a single dose of ovalbumin (OVA, Sigma Chemical Co.) at concentrations of either 25mg, 0.5mg or 0.0 lmg in 0.2ml saline by gavage needle. Ten days after OVA feeding, all mice were immunized subcutaneously with lOOμg OVA in 50μl adjuvant (RIBI adjuvant; RIBI Immunochemicals).

(C) Measurement of PLN Cell Proliferation. Two weeks after immunization, half of the mice were sacrificed and the draining popliteal lymph nodes (PLN) removed for assessment of antigen specific proliferative capability. Cells isolated from PLN two weeks after immunization were cultured at a density of 2 x 10⁵ cells/well in 96-well flat- bottomed plates in a total volume of 200ul with OVA (lmg/ml) in complete medium (DMEM/10%FCS). Proliferation was assessed at 72 hours by the addition of lμCi/well ³H thy mi dine (Amersham) 18 hours before harvesting. The amount of radioactivity incorporated into the DNA was measured using a Matrix-96 cell harvester (Inotech) and direct beta counter (Packard). The results are shown in Figure 1(b) and are reported as mean cpm + 1SD of quadruplicate wells from PLN pooled from five mice, and are representative of two additional experiments. Lymph node cells isolated from control mice that were fed 25mg OVA exhibited significantly decreased proliferative responses to OVA compared with saline-fed control mice (p<0.01, ##). However, other doses of OVA had little effect. All groups of mice that were both treated with flt3-L and fed OVA displayed significantly decreased proliferative responses compared with saline-fed flt3-L- treated mice (p<0.01, ##) and were significantly more tolerant than equivalently fed control mice (p<0.05,*; p<0.01,**). These results illustrate the dose-dependent tolerogenic effects of feeding OVA on T cell-dependent immune responses.

(D) Measurement of Delayed-Type Hypersensitivity (DTH) response. Twenty-one days after immunization, the remaining half of the mice were assayed for systemic delayed-type hypersensitivity (DTH) responses by measuring the increase in footpad thickness 24 hours after challenge with lOOμg heat- aggregated OVA in 50ul saline as described by Garside, et al., Id. The results are shown in Figure 1(a). The data are reported in as the mean increase in footpad response ± 1SEM from groups of five mice, and are representative of three experiments. The control mice that were fed 25mg OVA had decreased antigen-specific DTH responses compared with saline-fed control mice (p<0.05, #), whereas no decrease was observed in mice fed other doses of OVA. DTH responses were significantly decreased in flt3-L-treated mice that were fed all doses of OVA compared with saline-fed flt3-L-treated mice (p<0.05,#; p<0.01,##). DTH responses in these mice were also significantly decreased compared with equivalent control mice (p<0.05,*; p<0.01**). (E) Measurement of Humoral Immunity by Serum ELISA. Mice were exsanguinated and serum analyzed for antigen-specific antibody titres. Plates (Nunc Maxisorp) were coated with lμg/well OVA, quenched with PBS/5% FCS and washed with PBS/0.1% Tween-20. Samples were diluted in PBS/FCS, titrated and antigen- specific serum antibodies detected with alkaline phophatase-conjugated anti-IgG (Sigma), anti-IgGl (Pharmingen) or anti-IgG2a (Pharmingen). Enzyme activity was detected with p-nitrophenyl phosphate disodium (Sigma). The amount of reaction product was assessed on an ELISA plate reader at O.D.405nm using the Deltasoft program. The results are shown in Figure 2 and are presented as mean OD + SEM from groups of five individual mice and are representative of four experiments. It is apparent that control mice that were fed 25mg OVA had significantly decreased IgG, IgGl and IgG2a serum antibody titres (p<0.01); however, other doses of fed OVA had no effect. All flt3-L-treated mice that were fed any dose of OVA displayed significantly diminished antigen-specific IgG, IgGl and IgG2a production (p<0.01).

Unexpectedly, mice treated with flt3-L prior to feeding OVA had more profound tolerance than that seen in equivalent OVA fed control mice, as determined by both in vivo and in vitro parameters. The enhanced unresponsiveness induced by flt3-L treatment was most dramatic in mice that were fed low doses of antigen. Mice pretreated with flt3- L before feeding 0.5mg or 0.0 lmg OVA displayed significantly decreased T cell responses, unlike control animals fed the same doses of antigen. The magnitude of tolerance induced by feeding low doses of OVA to flt3-L- treated mice was comparable to that seen in control mice fed the highest dose of antigen. Thus, flt3-L treatment prior to feeding antigen promotes the induction of peripheral tolerance even using doses of protein that are normally nontolerogenic.

These data demonstrate that treating mice with flt3-L can initiate or enhance the induction of antigen-specific unresponsiveness by mucosal administration of antigen.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: IMMUNEX CORPORATION

(ii) TITLE OF INVENTION: A Method of Enhancing Antigen-Specific Peripheral Immune Tolerance

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Stephen L. Malaska, Immunex Corporation

(B) STREET: 51 University Street

(C) CITY: Seattle

(D) STATE: Washington

(E) COUNTRY: US

(F) ZIP: 98101

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM/ PC Compatible

(C) OPERATING SYSTEM: MS-DOS/Windows 95

(D) SOFTWARE: Word for Windows 95, Version 7.0a

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: -to be assigned-

(B) FILING DATE: 12 June 1998

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Malaska, Stephen L.

(B) REGISTRATION NUMBER: 32,655

(C) REFERENCE/DOCKET NUMBER: 2855-WO

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (206) 587-0430

(B) TELEFAX: (206) 233-0644

(C) TELEX: 756822

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 879 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..25

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 855..879

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 57..752

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

GTCGACTGGA ACGAGACGAC CTGCTCTGTC ACAGGCATGA GGGGTCCCCG GCAGAG 56

ATG ACA GTG CTG GCG CCA GCC TGG AGC CCA AAT TCC TCC CTG TTG CTG 104 Met Thr Val Leu Ala Pro Ala Trp Ser Pro Asn Ser Ser Leu Leu Leu 1 5 10 15

CTG TTG CTG CTG CTG AGT CCT TGC CTG CGG GGG ACA CCT GAC TGT TAC 152

Leu Leu Leu Leu Leu Ser Pro Cys Leu Arg Gly Thr Pro Asp Cys Tyr

20 25 30

TTC AGC CAC AGT CCC ATC TCC TCC AAC TTC AAA GTG AAG TTT AGA GAG 200

Phe Ser His Ser Pro lie Ser Ser Asn Phe Lys Val Lys Phe Arg Glu

35 40 45

TTG ACT GAC CAC CTG CTT AAA GAT TAC CCA GTC ACT GTG GCC GTC AAT 248 Leu Thr Asp His Leu Leu Lys Asp Tyr Pro Val Thr Val Ala Val Asn 50 55 60

CTT CAG GAC GAG AAG CAC TGC AAG GCC TTG TGG AGC CTC TTC CTA GCC 296 Leu Gin Asp Glu Lys His Cys Lys Ala Leu Trp Ser Leu Phe Leu Ala 65 70 75 80

CAG CGC TGG ATA GAG CAA CTG AAG ACT GTG GCA GGG TCT AAG ATG CAA 344 Gin Arg Trp lie Glu Gin Leu Lys Thr Val Ala Gly Ser Lys Met Gin 85 90 95

ACG CTT CTG GAG GAC GTC AAC ACC GAG ATA CAT TTT GTC ACC TCA TGT 392 Thr Leu Leu Glu Asp Val Asn Thr Glu lie His Phe Val Thr Ser Cys 100 105 110

ACC TTC CAG CCC CTA CCA GAA TGT CTG CGA TTC GTC CAG ACC AAC ATC 440 Thr Phe Gin Pro Leu Pro Glu Cys Leu Arg Phe Val Gin Thr Asn lie 115 120 125

TCC CAC CTC CTG AAG GAC ACC TGC ACA CAG CTG CTT GCT CTG AAG CCC 488 Ser His Leu Leu Lys Asp Thr Cys Thr Gin Leu Leu Ala Leu Lys Pro 130 135 140

TGT ATC GGG AAG GCC TGC CAG AAT TTC TCT CGG TGC CTG GAG GTG CAG 536 Cys lie Gly Lys Ala Cys Gin Asn Phe Ser Arg Cys Leu Glu Val Gin 145 150 155 160

TGC CAG CCG GAC TCC TCC ACC CTG CTG CCC CCA AGG AGT CCC ATA GCC 584 Cys Gin Pro Asp Ser Ser Thr Leu Leu Pro Pro Arg Ser Pro lie Ala 165 170 175

CTA GAA GCC ACG GAG CTC CCA GAG CCT CGG CCC AGG CAG CTG TTG CTC 632 Leu Glu Ala Thr Glu Leu Pro Glu Pro Arg Pro Arg Gin Leu Leu Leu 180 185 190

CTG CTG CTG CTG CTG CCT CTC ACA CTG GTG CTG CTG GCA GCC GCC TGG 680 Leu Leu Leu Leu Leu Pro Leu Thr Leu Val Leu Leu Ala Ala Ala Trp 195 200 205

GGC CTT CGC TGG CAA AGG GCA AGA AGG AGG GGG GAG CTC CAC CCT GGG 728 Gly Leu Arg Trp Gin Arg Ala Arg Arg Arg Gly Glu Leu His Pro Gly 210 215 220

GTG CCC CTC CCC TCC CAT CCC TAGGATTCGA GCCTTGTGCA TCGTTGACTC 779 Val Pro Leu Pro Ser His Pro 225 230

AGCCAGGGTC TTATCTCGGT TACACCTGTA ATCTCAGCCC TTGGGAGCCC AGAGCAGGAT 839 TGCTGAATGG TCTGGAGCAG GTCGTCTCGT TCCAGTCGAC 879

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 231 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Met Thr Val Leu Ala Pro Ala Trp Ser Pro Asn Ser Ser Leu Leu Leu 1 5 10 15

Leu Leu Leu Leu Leu Ser Pro Cys Leu Arg Gly Thr Pro Asp Cys Tyr 20 25 30

Phe Ser His Ser Pro lie Ser Ser Asn Phe Lys Val Lys Phe Arg Glu 35 40 45

Leu Thr Asp His Leu Leu Lys Asp Tyr Pro Val Thr Val Ala Val Asn 50 55 60

Leu Gin Asp Glu Lys His Cys Lys Ala Leu Trp Ser Leu Phe Leu Ala 65 70 75 80

Gin Arg Trp lie Glu Gin Leu Lys Thr Val Ala Gly Ser Lys Met Gin 85 90 95

Thr Leu Leu Glu Asp Val Asn Thr Glu lie His Phe Val Thr Ser Cys 100 105 110

Thr Phe Gin Pro Leu Pro Glu Cys Leu Arg Phe Val Gin Thr Asn lie 115 120 125

Ser His Leu Leu Lys Asp Thr Cys Thr Gin Leu Leu Ala Leu Lys Pro 130 135 140

Cys lie Gly Lys Ala Cys Gin Asn Phe Ser Arg Cys Leu Glu Val Gin 145 150 155 160

Cys Gin Pro Asp Ser Ser Thr Leu Leu Pro Pro Arg Ser Pro lie Ala 165 170 175

Leu Glu Ala Thr Glu Leu Pro Glu Pro Arg Pro Arg Gin Leu Leu Leu 180 185 190

Leu Leu Leu Leu Leu Pro Leu Thr Leu Val Leu Leu Ala Ala Ala Trp 195 200 205

Gly Leu Arg Trp Gin Arg Ala Arg Arg Arg Gly Glu Leu His Pro Gly 210 215 220

Val Pro Leu Pro Ser His Pro 225 230

( 2 ) INFORMATION FOR SEQ ID NO : 3 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 988 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 30..734

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

CGGCCGGAAT TCCGGGGCCC CCGGCCGAA ATG ACA GTG CTG GCG CCA GCC TGG 53

Met Thr Val Leu Ala Pro Ala Trp 1 5

AGC CCA ACA ACC TAT CTC CTC CTG CTG CTG CTG CTG AGC TCG GGA CTC 101 Ser Pro Thr Thr Tyr Leu Leu Leu Leu Leu Leu Leu Ser Ser Gly Leu 10 15 20

AGT GGG ACC CAG GAC TGC TCC TTC CAA CAC AGC CCC ATC TCC TCC GAC 149 Ser Gly Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser Ser Asp 25 30 35 40

TTC GCT GTC AAA ATC CGT GAG CTG TCT GAC TAC CTG CTT CAA GAT TAC 197 Phe Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr 45 50 55

CCA GTC ACC GTG GCC TCC AAC CTG CAG GAC GAG GAG CTC TGC GGG GGC 245 Pro Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly 60 65 70

CTC TGG CGG CTG GTC CTG GCA CAG CGC TGG ATG GAG CGG CTC AAG ACT 293 Leu Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr 75 80 85

GTC GCT GGG TCC AAG ATG CAA GGC TTG CTG GAG CGC GTG AAC ACG GAG 341 Val Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu 90 95 100

ATA CAC TTT GTC ACC AAA TGT GCC TTT CAG CCC CCC CCC AGC TGT CTT 389 lie His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu 105 110 115 120

CGC TTC GTC CAG ACC AAC ATC TCC CGC CTC CTG CAG GAG ACC TCC GAG 437 Arg Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr Ser Glu 125 130 135

CAG CTG GTG GCG CTG AAG CCC TGG ATC ACT CGC CAG AAC TTC TCC CGG 485 Gin Leu Val Ala Leu Lys Pro Trp lie Thr Arg Gin Asn Phe Ser Arg 140 145 150

TGC CTG GAG CTG CAG TGT CAG CCC GAC TCC TCA ACC CTG CCA CCC CCA 533 Cys Leu Glu Leu Gin Cys Gin Pro Asp Ser Ser Thr Leu Pro Pro Pro 155 160 165 TGG AGT CCC CGG CCC CTG GAG GCC ACA GCC CCG ACA GCC CCG CAG CCC 581 Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gin Pro 170 175 180

CCT CTG CTC CTC CTA CTG CTG CTG CCC GTG GGC CTC CTG CTG CTG GCC 629 Pro Leu Leu Leu Leu Leu Leu Leu Pro Val Gly Leu Leu Leu Leu Ala 185 190 195 200

GCT GCC TGG TGC CTG CAC TGG CAG AGG ACG CGG CGG AGG ACA CCC CGC 677 Ala Ala Trp Cys Leu His Trp Gin Arg Thr Arg Arg Arg Thr Pro Arg 205 210 215

CCT GGG GAG CAG GTG CCC CCC GTC CCC AGT CCC CAG GAC CTG CTG CTT 725 Pro Gly Glu Gin Val Pro Pro Val Pro Ser Pro Gin Asp Leu Leu Leu 220 225 230

GTG GAG CAC TGACCTGGCC AAGGCCTCAT CCTGCGGAGC CTTAAACAAC 774

Val Glu His 235

GCAGTGAGAC AGACATCTAT CATCCCATTT TACAGGGGAG GATACTGAGG CACACAGAGG 834

GGAGTCACCA GCCAGAGGAT GTATAGCCTG GACACAGAGG AAGTTGGCTA GAGGCCGGTC 894

CCTTCCTTGG GCCCCTCTCA TTCCCTCCCC AGAATGGAGG CAACGCCAGA ATCCAGCACC 954

GGCCCCATTT ACCCAACTCT GAACAAAGCC CCCG 988

(2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 235 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu 1 5 10 15

Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr Gin Asp Cys Ser Phe 20 25 30

Gin His Ser Pro lie Ser Ser Asp Phe Ala Val Lys lie Arg Glu Leu 35 40 45

Ser Asp Tyr Leu Leu Gin Asp Tyr Pro Val Thr Val Ala Ser Asn Leu 50 55 60

Gin Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gin 65 70 75 80

Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gin Gly 85 90 95

Leu Leu Glu Arg Val Asn Thr Glu lie His Phe Val Thr Lys Cys Ala 100 105 110

Phe Gin Pro Pro Pro Ser Cys Leu Arg Phe Val Gin Thr Asn lie Ser 115 120 125

Arg Leu Leu Gin Glu Thr Ser Glu Gin Leu Val Ala Leu Lys Pro Trp 130 135 140 lie Thr Arg Gin Asn Phe Ser Arg Cys Leu Glu Leu Gin Cys Gin Pro 145 150 155 160

Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala 165 170 175

Thr Ala Pro Thr Ala Pro Gin Pro Pro Leu Leu Leu Leu Leu Leu Leu 180 185 190

Pro Val Gly Leu Leu Leu Leu Ala Ala Ala Trp Cys Leu His Trp Gin 195 200 205

Arg Thr Arg Arg Arg Thr Pro Arg Pro Gly Glu Gin Val Pro Pro Val 210 215 220

Pro Ser Pro Gin Asp Leu Leu Leu Val Glu His 225 230 235

Claims

What is claimed is:

1. A method of initiating or enhancing antigen-specific immune tolerance comprising administering an effective amount of a polypeptide prior to, concurrent with, or subsequent to the mucosal administration of a immunotolerizing amount of the antigen, wherein the polypeptide is selected from the group consisting of:

(a) a polypeptide comprising amino acids 28-x of SEQ ID NO:2, wherein x is an amino acid selected from the group consisting of 163 to 231;

(b) a polypeptide comprising amino acids 28-y of SEQ ID NO:4, wherein y is an amino acid selected from the group consisting of 160 to 235; and

(c) a polypeptide that has at least 90% identity to the polypeptides of either (a) or (b) above, wherein the polypeptide is capable of binding flt3 receptor.

2. A method according to claim 1, wherein the antigen is associated with arthritis.

3. A method according to claim 2, wherein the antigen is selected from the group consisting of type II collagen and human glycoprotein-39.

4. A method according to claim 1, wherein the antigen is associated with diabetes.

5. A method according to claim 4, wherein the antigen is selected from the group consisting of insulin, glutamic acid decarboxylase, carboxypeptidase H, heat shock proteins, and insulin secretory granule proteins.

6. A method according to claim 1 , wherein the antigen is associated with uveitis.

7. A method according to claim 6, wherein the antigen is the S-antigen.

8. A method according to claim 1, wherein the antigen is associated with multiple sclerosis.

9. A method according to claim 8, wherein the antigen is selected from the group consisting of myelin basic protein and proteolipid protein.

10. A method of initiating or enhancing tolerance to an antigen selected from the group consisting of type II collagen and human glycoprotein-39 comprising administering an effective amount of a polypeptide prior to, concurrent with, or subsequent to the mucosal administration of a immunotolerizing amount of type II collagen, wherein the polypeptide is selected from the group consisting of:

(a) a polypeptide comprising amino acids 28-x of SEQ ID NO:2, wherein x is an amino acid selected from the group consisting of 163 to 231 ;

(b) a polypeptide comprising amino acids 28-y of SEQ ID NO:4, wherein y is an amino acid selected from the group consisting of 160 to 235; and

(c) a polypeptide that has at least 90% identity to the polypeptides of either (a) or (b) above, wherein the polypeptide is capable of binding flt3 receptor.

11. A method of initiating or enhancing tolerance to an antigen selected from the group consisting of insulin, glutamic acid decarboxylase, carboxypeptidase H, heat shock proteins, and insulin secretory granule proteins, comprising administering an effective amount of a polypeptide prior to, concurrent with, or subsequent to the mucosal administration of a immunotolerizing amount of the antigen, wherein the polypeptide is selected from the group consisting of:

(a) a polypeptide comprising amino acids 28-x of SEQ ID NO:2, wherein x is an amino acid selected from the group consisting of 163 to 231 ;

(b) a polypeptide comprising amino acids 28-y of SEQ ID NO:4, wherein y is an amino acid selected from the group consisting of 160 to 235; and

(c) a polypeptide that has at least 90% identity to the polypeptides of either (a) or (b) above, wherein the polypeptide is capable of binding flt3 receptor.

12. A method of initiating or enhancing tolerance to an antigen selected from the group consisting of myelin basic protein and proteolipid protein, comprising administering an effective amount of a polypeptide prior to, concurrent with, or subsequent to the mucosal administration of a immunotolerizing amount of the antigen, wherein the polypeptide is selected from the group consisting of: (a) a polypeptide comprising amino acids 28-x of SEQ ID NO:2, wherein x is an amino acid selected from the group consisting of 163 to 231;

(b) a polypeptide comprising amino acids 28-y of SEQ ID NO:4, wherein y is an amino acid selected from the group consisting of 160 to 235; and

(c) a polypeptide that has at least 90% identity to the polypeptides of either (a) or (b) above, wherein the polypeptide is capable of binding flt3 receptor.

13. A method of initiating or enhancing tolerance to the S-antigen comprising administering an effective amount of a polypeptide prior to, concurrent with, or subsequent to the mucosal administration of a immunotolerizing amount of the S-antigen, wherein the polypeptide is selected from the group consisting of:

(a) a polypeptide comprising amino acids 28-x of SEQ ID NO:2, wherein x is an amino acid selected from the group consisting of 163 to 231;

(b) a polypeptide comprising amino acids 28-y of SEQ ID NO:4, wherein y is an amino acid selected from the group consisting of 160 to 235; and

(c) a polypeptide that has at least 90% identity to the polypeptides of either (a) or (b) above, wherein the polypeptide is capable of binding flt3 receptor.

14. A method of initiating or enhancing immune tolerance of a therapeutic immunogenic molecule comprising administering an effective amount of a polypeptide prior to, concurrent with, or subsequent to the mucosal administration of a immunotolerizing amount of the therapeutic molecule, wherein the polypeptide is selected from the group consisting of:

(a) a polypeptide comprising amino acids 28-x of SEQ ID NO:2, wherein x is an amino acid selected from the group consisting of 163 to 231;

(b) a polypeptide comprising amino acids 28-y of SEQ ID NO:4, wherein y is an amino acid selected from the group consisting of 160 to 235; and

(c) a polypeptide that has at least 90% identity to the polypeptides of either (a) or (b) above, wherein the polypeptide is capable of binding flt3 receptor.