Triple polypeptide complexes Number:7,148,020 from the United States Patent and Trademark Office (PTO) owispatent

Title: Triple polypeptide complexes

Abstract: The invention provides methods and materials related to treating and diagnosing autoimmune conditions. Specifically, the invention provides polypeptide compositions, nucleic acids, substantially pure polypeptides, host cells, and methods for identifying a mammal with an autoimmune condition, treating a mammal with an autoimmune condition, and enhancing tolerance in a mammal with an autoimmune condition.

Patent Number: 7,148,020 Issued on 12/12/2006 to Holmdahl,   et al.

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Inventors:	Holmdahl; Rikard (Lund, SE), Engstrom; Jan Ake (B{hacek over (a)}linge, SE), Kihlberg; Jan (Savar, SE), Burkhardt; Harald (Erlangen, DE)
Assignee:	Arexis AB (Molndal, SE)
Appl. No.:	10/194,441
Filed:	July 11, 2002

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Current U.S. Class:	435/7.1
Current International Class:	G01N 33/53 (20060101)
Field of Search:	514/12 530/350

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References Cited [Referenced By]

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U.S. Patent Documents

	4683195		July 1987		Mullis et al.
	4873191		October 1989		Wagner et al.
	5580859		December 1996		Felgner et al.
	5589466		December 1996		Felgner et al.
	5726243		March 1998		Fields
	5849323		December 1998		Braswell et al.

Foreign Patent Documents

	0 718 308		Dec., 1999		EP
	WO 96/20950		Jul., 1996		WO
	WO 97/19106		May., 1997		WO
	WO 98/33811		Aug., 1998		WO
	WO 99/10381		Mar., 1999		WO
	WO 00/12538		Mar., 2000		WO

Other References

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Immunol., 1998, 28:1534-1543. cited by other . Backlund et al., "Reversal of tolerance induced by transplantation of skin expressing the immunodominant T cell epitope of rat type II collagen entitles development of collagen-induced arthritis but not graft rejection," Eur. J. Immunol., 2002, 32:1773-1783. cited by other . Backlund et al., "Predominant selection of T cells specific for the glycosylated collagen type II epitope (263-270) in humanized transgenic mice and in rheumatoid arthritis," Proc. Natl. Acad. Sci. USA, 2002, 99(15):9611-9613. cited by other . Bengtsson et al., "Convergent synthesis of neoglycopeptides by coupling of 2-bromoethyl glycosides to cysteine and homocysteine residues in T cell stimulating peptides," Glycoconjugate Journal, 1998, 15:223-231. cited by other . Borgia and Fields, "Chemical synthesis of proteins," Trends Biotechnol., 2000, 18(6):243-251. cited by other . 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Selective inductionof interleukin-10 or interferon-.gamma. synthesis in small resting CD4.sup.+ T cells," Eur. J. Immunol., 1993, 23:523-529. cited by other . Corthay et al., "Epitope glycosylation plays a critical role for T cell recognition of type II collagen in collagen-induced arthritis," Eur. J. Immunol., 1998, 28:2580-2590. cited by other . Feng et al., "Acetyl-Terminated and Template-Assembled Collagen-Based Polypeptides Composed of Gly-Pro-Hyp Sequences 2. Synthesis and Conformational Analysis by Circular Dichroism, Ultraviolet Absorbance, and Optical Rotation," J. Am. Chem. Soc., 1996, 118:10351-10358. cited by other . Flegel and Sheppard, "A Sensitive, General Method for Quantitative Monitoring of Continuous Flow Solid Phase Peptide Synthesis," J. Chem. Soc. Chem. Commun., 1990, pp. 536-538. cited by other . Goodman et al. "A Template-Induced Incipient Collagen-Like Triple-Helical Structure," J. Am. Chem. Soc., 1996, 118:5156-5157. cited by other . Gossler et al., "Transgenesis by means of blastocyst-derived embryonic stem cell lines," Proc. Natl. Acad. Sci. USA, 1986, 83:9065-9069. cited by other . Grab et al., "Promotion of Fibroblast Adhesion by Triple-helical Peptide Models of Type I Collagen-derived Sequences," J. Biol. Chem., 1996, 271(21):12234-12240. cited by other . Greiche and Heidemann, "Collagen Model Peptides with Antiparallel Structure," Biopolymers, 1979, 18(9):2359-61. cited by other . Hansson et al. "A new animal model for relapsing polychondritis, induced by cartilage matrix protein (matrilin-1)," J. Clin. Invest., 1999, 104(5):589-598. cited by other . Hauselmann et al. "Can Collagen Type II Sustain a Methotrexate-Induced Therapeutic Effect in Patients with Long-Standing Rheumatoid Arthritis? A Double-Blind, Randomized Trial," Br. J. Rheumatol., 1998, 37:1110-1117. cited by other . Henkel et al., "Synthesis and Folding of Native Collagen III Model Peptides," Biochemistry, 1999, 38:13610-13622. cited by other . Hojo et al., "Synthesis and Structural Characterization of Triple-Helical Peptides Which Mimic the Ligand Binding Site of the Human Macrophage Scavenger Receptor," Tetrahedron, 1997, 53(42):14263-14274. cited by othe- r . Holm et al., "An Improved Synthesis of a Galactosylated Hydroxylsine Building Block and its use in Solid-Phase Glycopeptide Synthesis," Tetrahedron, 2000, 56:1579-1586. cited by other . Kihlberg and Ahman, "Glycosylated Peptide Hormones: Pharmacological Properties and Conformational Studies of Analogues of [1-Desamino,8-D-arginine]vasopressin," J. Med. Chem., 1995, 38:161-169. cited by other . Lauer-Fields et al., "Hydrolysis of Triple-helical Collagen Peptide Models by Matrix Metalloproteinases," J. Biol. Chem., 2000, 275(18):13282-13290. cited by other . Lo, "Transformation by Iontophoretic Microinjection of DNA: Multiple Integrations Without Tandem Insertions," Mol. Cell. Biol., 1983, 3(10):1803-1814. cited by other . Malmstrom et al., "Systemic versus cartilage-specific expression of a type II collagen-specific T-cell epitope determine the level of tolerance and susceptibility to arthritis," Proc. Natl. Acad. Sci. USA, 1996, 93:4480-4485. cited by other . Mechling and Bachinger, "The Collagen-like Peptide (GER).sub.15GPCCG Forms pH-dependent Covalently Linked Triple Helical Trimers," J. Biol. Chem., 2000, 275(19):14532-14536. cited by other . Michaelsson et al., "T Cell Recognition of Carbohydrates on Type II Collagen," J. Exp. Med., 1994, 180:745-749. cited by other . Michet et al., "Relapsing Polychondritis," Annals of Internal Medicine, 1986, 104:74-78. cited by other . Muller et al., "Heterotrimeric Collagen Peptides as Fluorogenic Collagenase Substrates: Synthesis, Conformational Properties, and Enzymatic Digestion," Biochemistry, 2000, 39:5111-5116. cited by other . Ottl and Moroder, "A New Strategy for Regioselective Interstrand Disulfide Bridging of Multiple Cysteine Peptides," Tetrahedron Letters, 1999, 40:1487-1490. cited by other . Sambrook et al., Molecular Cloning--A Laboratory Manual, Second Edition, 1989, Cold Spring Harbor Laboratory, Plainview, NY, pp. 7.39-7.52. cited by other . Schellekens et al., "Citrulline is an Essential Constituent of Antigenic Determinants Recognized by Rheumatoid Arthritis-specific Autoantibodies," J. Clin. Invest., 1998, 101(1):273-281. cited by other . Schnieke et al., "Human Factor IX Transgenic Sheep Produced by Transfer of Nuclei from Transfected Fetal Fibroblasts," Science, 1997, 278:2130-2133. cited by other . Tanaka et al., "Synthesis and stabilization of amino and carboxy terminal constrained collagenous peptides," J. Peptide Res., '998, 51(6):413-419. cited by other . Thompson et al., "Germ Line Transmission and Expression of a Corrected HPRT Gene Produced by Gene Targeting in Embryonic Stem Cells," Cell, 1989, 56:313-321. cited by other . van der Putten et al., "Efficient insertion of genes into the mouse germ line via retroviral vectors," Proc. Natl. Acad. Sci. USA, 1985, 82:6148-6152. cited by other . Wellner et al., "Synthesis of a C-Glycoside Analogue of .beta.-D-Galactosyl Hydroxynorvaline and Its Use in Immunological Studies," Chembiochem., 2000, 1:272-280. cited by other . Yan et al., "Specificity and Tcell receptor .beta. chain usage of a human collagen type II-reactive T cell clone derived from a healthy individual," Eur. J. Immunol., 1992, 22:51-56. cited by other.

Primary Examiner: Carlson; Karen Cochrane
Assistant Examiner: Rooke; Agnes
Attorney, Agent or Firm: Fish & Richardson P.C.

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Parent Case Text

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CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. application Ser. No. 60/305,048, which was filed on Jul. 12, 2001.

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Claims

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What is claimed is:

1. A method for detecting an antibody in a sample from a mammal, wherein said antibody has specificity for a triple polypeptide complex, wherein said triple polypeptide complex comprises three polypeptides, wherein each of said three polypeptides comprises a triple helix formation sequence, wherein each of said three polypeptides comprises at least one interpolypeptide linkage such that each polypeptide is attached to at least one of the other two polypeptides of said three polypeptides, and wherein at least one polypeptide of said three polypeptides comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:30 or 45; said method comprising: (a) contacting said sample with said triple polypeptide complex, and (b) determining the presence or absence of said antibody bound to said triple polypeptide complex, wherein the presence of bound antibody indicates that said sample contains said antibody.

2. The method of claim 1, wherein said mammal is a human.

3. The method of claim 1, wherein said sample is serum.

4. The method of claim 1, wherein said antibody is an anti-collagen antibody.

5. The method of claim 1, wherein said antibody is bound to a B-cell.

6. The method of claim 1, wherein said antibody is a circulating antibody.

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Description

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BACKGROUND

1. Technical Field

The invention relates to methods and materials involved in assessing and treating autoimmune conditions such as rheumatoid arthritis.

2. Background Information

Rheumatoid arthritis (RA) is an autoimmune, inflammatory disease that affects peripheral joints. The main genetic association is to the major histocompatibility complex class II region (HLA-DR), suggesting that T cell mediated autoimmune recognition of joint specific antigens is involved in the disease. In addition, B cell mediated autoimmune responses have been observed in rheumatoid joints. Specifically, B cells have been detected secreting IgG antibodies specific for type II collagen (CII). Further, mice transgenic for a particular human DR4 molecule were found to develop arthritis after immunization with CII. The T cell response in these immunized mice was predominantly directed towards one dominant epitope corresponding to the amino acid sequence at positions 261 273 of CII.

The collagens are a family of highly fibrous proteins, including fibril-forming, fibril-associated, and network-forming collagen types. CII is a fibril-forming collagen that serves as a major component of bone, cartilage, invertebral disc, notochord, and vitreous humor. Additionally, CII plays an important role in the development of RA.

SUMMARY

The invention involves methods and materials for assessing and treating autoimmune conditions such as rheumatoid arthritis. Specifically, the invention provides polypeptide compositions, nucleic acids, substantially pure polypeptides, host cells, and methods for identifying mammals with autoimmune conditions, treating mammals with autoimmune conditions, and enhancing tolerance in mammals with autoimmune conditions.

In general, the invention features a composition containing three polypeptides, wherein each polypeptide contains a triple helix formation sequence, and wherein each polypeptide contains at least two interpolypeptide linkages such that each polypeptide is covalently attached to at least one of the other two polypeptides of the three polypeptides. The triple helix formation sequence of at least one of the three polypeptides can contain (Gly-Pro-Hyp). The triple helix formation sequence of at least one of the three polypeptides can contain (Gly-Pro-Flp). At least one of the interpolypeptide linkages can include an Ahx-Lys bond. At least one of the interpolypeptide linkages can include a Cys-Cys bond. At least one of the three polypeptides can contain a (Gly-Xaa-Yaa).sub.n sequence, the n being an integer from 1 to 100. At least one of the three polypeptides can contain a (Gly-Pro-Hyp).sub.x(Gly-Xaa-Yaa).sub.y(Gly-Pro-Hyp).sub.z sequence, wherein the x, y, and z are independently integers from 1 to 100. At least one of the interpolypeptide linkages for each polypeptide can be located in an N-terminal region. At least one of the interpolypeptide linkages for each polypeptide can be located in a C-terminal region. At least one of the interpolypeptide linkages for each polypeptide can be located in an N-terminal region, and at least one of the interpolypeptide linkages for each polypeptide can be located in a C-terminal region. At least one of the three polypeptides can be covalently attached to the other two polypeptides of the three polypeptides. Each polypeptide can be covalently attached to the other two polypeptides of the three polypeptides. At least one of the three polypeptides can contain a modified amino acid residue (e.g., a glycosylated amino acid residue). Each polypeptide can contain a modified amino acid residue. At least one of the three polypeptides can contain an amino acid sequence at least about 80 percent identical to the sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, or 56. Each polypeptide can contain an amino acid sequence at least about 80 percent identical to the sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, or 56.

In another embodiment, the invention features a composition containing three polypeptides, wherein each polypeptide contains a triple helix formation sequence, wherein each polypeptide contains at least one interpolypeptide linkage such that each polypeptide is attached to at least one of the other two polypeptides of the three polypeptides, and wherein at least one of the three polypeptides contains an amino acid sequence at least about 80 percent identical to the sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, or 56. The triple helix formation sequence of at least one of the three polypeptides can contain (Gly-Pro-Hyp). The triple helix formation sequence of at least one of the three polypeptides can contain (Gly-Pro-Flp). At least one of the interpolypeptide linkages can contain an Ahx-Lys bond. At least one of the interpolypeptide linkages can contain a Cys-Cys bond. At least one of the three polypeptides can contain a (Gly-Xaa-Yaa).sub.n sequence, the n being an integer from 1 to 100. At least one of the three polypeptides can contain a (Gly-Pro-Hyp).sub.x(Gly-Xaa-Yaa).sub.y(Gly-Pro-Hyp).sub.z sequence, wherein the x, y, and z are independently integers from 1 to 100. At least one of the interpolypeptide linkages for each polypeptide can be located in an N-terminal region. At least one of the interpolypeptide linkages for each polypeptide can be located in a C-terminal region. At least one of the interpolypeptide linkages for each polypeptide can be located in an N-terminal region, and at least one of the interpolypeptide linkages for each polypeptide can be located in a C-terminal region. At least one of the three polypeptides can be covalently attached to the other two polypeptides of the three polypeptides. Each polypeptide can be covalently attached to the other two polypeptides of the three polypeptides. At least one of the three polypeptides can contain a modified amino acid residue (e.g., a glycosylated amino acid residue). Each polypeptide can contain a modified amino acid residue. Each polypeptide can contain at least two interpolypeptide linkages.

Another embodiment of the invention features a composition containing three polypeptides, wherein each polypeptide contains a triple helix formation sequence, wherein each polypeptide contains at least one interpolypeptide linkage such that each polypeptide is covalently attached to at least one of the other two polypeptides of the three polypeptides, and wherein at least one of the three polypeptides contains a modified amino acid residue. The modified amino acid residue can be a glycosylated amino acid residue. The modified amino acid residue can be a modified lysine residue. The modified amino acid residue can be lysine-dinitrophenyl. At least one of the three polypeptides can contain an amino acid sequence at least about 80 percent identical to the sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, or 56. Each polypeptide can contain an amino acid sequence at least about 80 percent identical to the sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, or 56.

Another embodiment of the invention features a composition containing three polypeptides, wherein each polypeptide contains at least one interpolypeptide linkage such that each polypeptide is attached to at least one of the other two polypeptides of the three polypeptides, and wherein at least one polypeptide of the three polypeptides contains (Gly-Pro-Flp). Each polypeptide of the three polypeptides can contain (Gly-Pro-Flp). At least one of the three polypeptides can contain an amino acid sequence at least about 80 percent identical to the sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, or 56. Each polypeptide can contain an amino acid sequence at least about 80 percent identical to the sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, or 56.

In another aspect, the invention features a method for detecting an antibody in a sample from a mammal (e.g., human), wherein the antibody has specificity for a triple polypeptide complex, wherein the triple polypeptide complex contains three polypeptides, wherein each of the three polypeptides contains a triple helix formation sequence, wherein each of the three polypeptides contains at least one interpolypeptide linkage such that each polypeptide is attached to at least one of the other two polypeptides of the three polypeptides, and wherein (i) each polypeptide contains at least two interpolypeptide linkages; (ii) at least one polypeptide of the three polypeptides contains an amino acid sequence at least about 80 percent identical to the sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, or 56; (iii) at least one polypeptide of the three polypeptides contains a modified amino acid residue; or (iv) at least one polypeptide of the three polypeptides contains (Gly-Pro-Flp); the method including: (a) contacting the sample with the triple polypeptide complex, and (b) determining the presence or absence of the antibody bound to the triple polypeptide complex, wherein the presence of bound antibody indicates that the sample contains the antibody. The sample can be serum. The antibody can be an anti-collagen antibody. The antibody can be bound to a B-cell. The antibody can be a circulating antibody.

In another embodiment, the invention features a method for detecting a T-cell in a sample from a mammal (e.g., human), wherein the T-cell is reactive to a triple polypeptide complex, wherein the triple polypeptide complex contains three polypeptides, wherein each of the three polypeptides contains a triple helix formation sequence, wherein each of the three polypeptides contains at least one interpolypeptide linkage such that each polypeptide is attached to at least one of the other two polypeptides of the three polypeptides, and wherein: (i) each polypeptide contains at least two interpolypeptide linkages; (ii) at least one polypeptide of the three polypeptides contains an amino acid sequence at least about 80 percent identical to the sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, or 56; (iii) at least one polypeptide of the three polypeptides contains a modified amino acid residue; or (iv) at least one polypeptide of the three polypeptides contains (Gly-Pro-Flp); the method including: (a) contacting the sample with the triple polypeptide complex, and (b) determining the presence or absence of T-cell activation, wherein the presence of the T-cell activation indicates that the sample contains the T-cell. The sample can be a blood sample. The T-cell can be a CD4.sup.+ T-cell.

Another aspect of the invention features a method of enhancing, in a mammal, tolerance to an endogenous polypeptide, the method including administering a composition to the mammal under conditions effective to enhance the tolerance, the composition containing three polypeptides, wherein each of the three polypeptides contains a triple helix formation sequence and at least one interpolypeptide linkage such that each of the three polypeptides is covalently attached to at least one of the other two polypeptides of the three polypeptides. The endogenous polypeptide can be a triple helical polypeptide (e.g., type II collagen). Each of the three polypeptides can contain at least two interpolypeptide linkages. At least one of the three polypeptides can contain an amino acid sequence at least about 80 percent identical to the sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, or 56. At least one of the three polypeptides can contain a modified amino acid residue. At least one polypeptide of the three polypeptides can contain (Gly-Pro-Flp).

Another aspect of the invention features a method of forming, in a mammal, a triple helical polypeptide-antibody complex, the method including administering an antibody to the mammal under conditions effective to form the triple helical polypeptide-antibody complex with a triple helical polypeptide, the antibody having specificity for a triple polypeptide complex, wherein the triple polypeptide complex contains three polypeptides, wherein each polypeptide contains a triple helix formation sequence and at least one interpolypeptide linkage such that each polypeptide is attached to at least one of the other two polypeptides of the three polypeptides, and wherein at least one polypeptide of the three polypeptides contains an amino acid sequence at least about 80 percent identical to the sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, or 56. The triple helical polypeptide can be type II collagen.

In another embodiment, the invention features a method of enhancing, in a mammal, tolerance to an endogenous polypeptide, the method including administering an isolated nucleic acid molecule to a somatic cell of the mammal under conditions effective to enhance the tolerance, wherein the nucleic acid molecule contains a nucleic acid sequence that encodes a polypeptide containing a triple helix formation sequence. The mammal can have arthritis. The endogenous polypeptide can be a triple helical polypeptide (e.g., type II collagen). The somatic cell can be a fibroblast or fibrocyte. The polypeptide can contain an amino acid sequence at least about 80 percent identical to the sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, or 56.

Another embodiment of the invention features a method of enhancing, in a mammal, tolerance to an endogenous polypeptide, the method including administering cells to the mammal under conditions effective to enhance the tolerance, wherein the cells contain an isolated nucleic acid molecule encoding a polypeptide containing a triple helix formation sequence. The mammal can have arthritis. The endogenous polypeptide can be a triple helical polypeptide (e.g., type II collagen). The polypeptide can contain an amino acid sequence at least about 80 percent identical to the sequence set forth in SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, or 56.

Unless otherwise defined, 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 invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. 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 definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a listing of the amino acid sequence of mouse and human CII. The mouse CII amino acid sequence is on top (SEQ ID NO:48), and the human CII amino acid sequence is on the bottom (SEQ ID NO:1). The dots in the human sequence indicate that that amino acid residue is identical to the amino acid residue listed in the mouse CII amino acid sequence.

FIG. 2 is a listing of a nucleic acid sequence that encodes human CII (SEQ ID NO:2).

FIG. 3 is a line graph plotting radioactive counts per minute (CPM) versus number of dendritic cells per well for cells treated with 10 .mu.g of [(Gly-Pro-Hyp).sub.5-CII.sub.(259-274)-(Gly-Pro-Hyp).sub.2].sub.3L.sub.Ah- x (linked polypeptide), CII.sub.(259-270) (unlinked polypeptides), or native rat CII. The results are given as the means of duplicates.

FIG. 4 is a line graph plotting percent incidence (frequency of arthritis) versus days post immunization for neonatal transgenic mice vaccinated with PBS, CII.sub.(256-270), or CII.sub.(256-264GHyl-270).

FIG. 5 is a line graph plotting percent incidence (frequency of arthritis) versus days post immunization for neonatal non-transgenic mice vaccinated with PBS, CII.sub.(256-270), CII.sub.(256-264Hyl-270), or CII.sub.(256-264GHyl-270).

FIG. 6 is a line graph plotting percent incidence (frequency of arthritis) versus days post immunization for adult female mice vaccinated with PBS, CII.sub.(256-270), or [(Gly-Pro-Hyp).sub.5-CII.sub.(259-274)-(Gly-Pro-Hyp).sub.2].sub.3L.sub.Ah- x.

FIG. 7 is a graph plotting molar elipticity ([.theta.]) verses X for [(Gly-Pro-Hyp).sub.5-CII.sub.(358-366Hyp-369)-(Gly-Pro-Hyp).sub.2]3L.sub.- Ahx as assessed by circular dichroism spectroscopy.

FIG. 8 is a graph plotting the amount of M2.139 monoclonal antibody binding verses dilution of [(Gly-Pro-Hyp).sub.5-CII.sub.(551-552Hyp-564)-(Gly-Pro-Hyp).sub.2].sub.3L- .sub.Ahx and a graph plotting the amount of C1 monoclonal antibody binding verses dilution of [(Gly-Pro-Hyp).sub.5-CII.sub.(358-366Hyp-369)-(Gly-Pro-Hyp).sub.2].sub.3L- .sub.Ahx.

FIG. 9 is a three-dimensional graph plotting ELISA reactivity (absorbance at 405 nm) for diluted serum binding to [(Gly-Pro-Hyp).sub.5-CII.sub.(358-366Hyp-369)-(Gly-Pro-Hyp).sub.2].sub.3L- .sub.Ahx.

FIG. 10 is a bar graph plotting ELISA reactivity (absorbance at 405 nm) detected in the indicated patient cohorts using either [(Gly-Pro-Hyp).sub.5-CII.sub.(358-366Hyp-369)-(Gly-Pro-Hyp).sub.2].sub.3L- .sub.Ahx- or [(Gly-Pro-Hyp).sub.5-CII.sub.(551-552Hyp-564)-(Gly-Pro-Hyp).sub.2].sub.3L- .sub.Ahx-coated plates. 95.sup.th and 75.sup.th percentiles are shown, and the bold horizontal lines indicate the median values. Significant differences in absorbance at 405 nm are indicated. The p-values were calculated according to the Mann-Whitney u-test; (n.s.=not significant).

FIG. 11 is a bar graph plotting ELISA reactivity (absorbance at 405 nm) detected in the indicated patient cohorts using [(Gly-Pro-Hyp).sub.5-CII.sub.(494-504Hyp)-(Gly-Pro-Hyp).sub.2].sub.3L.sub- .Ahx-coated plates. 95.sup.th and 75.sup.th percentiles are shown, and the bold horizontal lines indicate the median values. Significant differences in absorbance at 405 nm are indicated. The p-values were calculated according to the Mann-Whitney u-test; (n.s.=not significant).

FIG. 12 is a series of plots of two-color flow cytometry of in vitro stimulated T cells from RA patients. The X-axis represents fluorescence intensities for binding of a FITC-labelled anti-CD3 antibody, whereas the y-axis represents signal intensities for binding of a PE-labelled anti-IL-2 antibody. The cells were gated on the population large lymphoblasts according to the forward side scatter. The upper right quadrant of the different panels (indicated by *) represents the percentage of double positive cells.

FIG. 13 is a bar graph plotting the percent IL-2-producing CD3.sup.+ T cells present within samples obtained from RA patients. Each sample was treated with either no polypeptide composition (control), tetanus toxoid (TT), or the indicated polypeptide composition.

FIG. 14 is a series of bar graphs demonstrating recall responses in vitro towards CII antigens in skin transplanted mice after CII immunization. The indicated number of mice were either grafted with skin from transgenic mice (TSC) or from control littermates (CQ) four weeks before immunization with CII in adjuvant. Ten days later, draining lymphnode cells were restimulated with different antigens (50 mg/mL) for four days, and the proliferation was measured. After in vitro restimulation, supernatants were collected for determination of IFN-.gamma. content. Some mice were also thymectomized (Tx) two weeks before transplantation (b). Cell proliferation is presented as .DELTA.CPM (proliferation with antigen-proliferation without antigen)* p.ltoreq.0.05, ** p.ltoreq.0.01.

FIG. 15 is a table demonstrating arthritis development in TSC-skin transplanted recipients. Normal (A C) or thymectomized (D) mice were grafted with skin from either transgenic (TSC) or control littermates (CQ) mice and immunized with CII and adjuvant four weeks later. Five weeks later, all mice were given a boost injection of CII. A second boost was also given 10 weeks after the first immunization (C D). Abbreviations: Inc, incidence; AUC, area under the curve (of mean arthritis index); MMS, mean maximum score (at the end of experiment); MDO, mean day of onset. Results are given as number of diseased animals and mean values StDev of AUC, MMS and MDO.

FIG. 16 is two graphs representing arthritis indexes of TSC or control grafted mice. Mice were grafted four weeks prior to immunization with CII and adjuvant. Five weeks later, mice were given a boost injection of CII (A D). A second boost was also given 10 weeks after the first immunization (C D). In one experiment, mice were also thymectomized 2 weeks prior to skin transplantation (D). *p.ltoreq.0.05, *p.ltoreq.0.01, **p<0.001.

FIG. 17 is a table presenting anti-CII antibody titers in skin transplanted mice. The indicated number of mice were either grafted with skin from transgenic (TSC) or negative littermates (CQ) mice. In three separate experiments (A C), non-thymectomized mice and, in one experiment (E), thymectomized mice were grafted and immunized with CII as described in FIG. 15. After 5, 10, and 19 (only C, E, and F) weeks, blood samples were collected and used to determine anti-CII antibody titers. Non-grafted, euthymic TSC transgenic mice were also immunized as controls (F). The ratio of isotype IgG antibodies (IgG2a/IgG1) are given to compare different experiments separated by time and animal facilities. * Pooled results from three experiments with non-thymectomized mice. Antibody titers measured 19 weeks post immunization only includes animals from experiment C (i.e., n (CQ)=7; n (TSC)=10).

FIG. 18 is two graphs reporting the detection of transgenic CII from skin grafts. Hybridomas HCQ.4 and HCQ.10, specific for non-glycosylated and glycosylated CII.sub.(256-270)respectively, were tested for recognition of transgenic CII from skin grafts removed from non-thymectomized recipient mice 19 weeks after immunization by a CTLL assay. Glycosylated (CII.sub.(256-264GHyl-270)) or non-glycosylated (CII.sub.(256-270)) polypeptides were used as positive and negative controls. Both hybridomas respond to whole CII protein. The same results were also found from preparations made from grafts from thymectomized animals.

FIG. 19 is a listing of the amino acid sequence of mouse and human CII as presented in FIG. 1 with selected CII epitopes being identified via underline.

FIG. 20 is a listing of the nucleic acid sequence that encodes human CII as presented in FIG. 2 with selected CII epitopes being identified via underline. In addition, the amino acid sequence of each selected CII epitope is provided under the corresponding nucleic acid sequence.

FIG. 21 is a scheme depicting the synthesis of a polypeptide complex having interpolypeptide linkages in the C-terminal and N-terminal regions.

FIG. 22 is a scheme depicting the synthesis of a polypeptide complex having interpolypeptide linkages in the C-terminal region.

FIG. 23 is a graph plotting mean residue elipticity at 225 nm as a function of temperature for THP 2 and THP 4.

FIG. 24 is a graph plotting optical density against dilutions for [(Gly-Pro-Hyp).sub.5-CII.sub.(259-273T)-(Gly-Pro-Hyp).sub.2].sub.3-L.sub.- Ahx, [(Gly-Pro-Hyp).sub.5-CII.sub.(259-273T)-(Gly-Pro-Hyp).sub.1-CII.sub.(- 358-366Hyp)-(Gly-Pro-Hyp).sub.2].sub.3-L.sub.Ahx, and [(Gly-Pro-Hyp).sub.5-CII.sub.(358-366Hyp)-(Gly-Pro-Hyp).sub.2].sub.3-L.su- b.Ahxused as antigen in an ELISA assay. Standard deviations between OD values in the individual wells are depicted as bars through the points. In the legend, T-cell epitope refers to [(Gly-Pro-Hyp).sub.5-CII.sub.(259-273T)-(Gly-Pro-Hyp).sub.2].sub.3-L.sub.- Ahx; B-cell epitope refers to [(Gly-Pro-Hyp).sub.5-CII.sub.(358-366Hyp)-(Gly-Pro-Hyp).sub.2].sub.3-L.su- b.Ahx; and T+B-cell epitope refers to [(Gly-Pro-Hyp).sub.5-CII.sub.(259-273T)-(Gly-Pro-Hyp).sub.1-CII.sub.(358-- 366Hyp)-(Gly-Pro-Hyp).sub.2].sub.3-L.sub.Ahx.

FIG. 25 is a graph plotting IL-2 production as measured by ELISA as a function of polypeptide concentration. A.sup.q-restricted HRQ.2 hybridoma cells were incubated with CII.sub.(256-270), [(Pro-Hyp-Gly).sub.5-CII.sub.(257-258Hyp-273T-274)].sub.3-L.sub.Ahx, and KTA-[Gly-(Gly-Pro-Hyp).sub.5-Gly-CII.sub.(257-258Hyp-273T-274)].sub.3-L(F- ).sub.Ahx. In the legend, CII 256 270 refers to CII.sub.(256-270); single bound THP refers to [(Pro-Hyp-Gly).sub.5-CII.sub.(257-258Hyp-273T-274)].sub.3-L.sub.Ahx; double bound THP refers to KTA-[Gly-(Gly-Pro-Hyp).sub.5-Gly-CII.sub.(257-258Hyp-273T-274)].sub.3-L(F- ).sub.Ahx; and blank refers to media without polypeptide complexes.

FIG. 26 is a graph plotting IL-2 production as measured by ELISA as a function of polypeptide concentration. A.sup.q-restricted HRC.2 hybridoma cells were incubated with CII.sub.(256-270), [(Pro-Hyp-Gly).sub.5-CII.sub.(257-258Hyp-273T-274)].sub.3-L.sub.Ahx, and KTA-[Gly-(Gly-Pro-Hyp).sub.5-Gly-CII.sub.(257-258Hyp-273T-274)].sub.3-L(F- ).sub.Ahx. In the legend, CII 256 270 refers to CII.sub.(256-270); single bound THP refers to [(Pro-Hyp-Gly)5-CII.sub.(257-258Hyp-273T-274)].sub.3-L.sub.Ahx; double bound THP refers to KTA-[Gly-(Gly-Pro-Hyp).sub.5-Gly-CII.sub.(257-258Hyp-273T-274)].sub.3-L(F- ).sub.Ahx; and blank refers to media without polypeptide complexes.

FIG. 27 is a graph plotting IL-2 production as measured by ELISA as a function of polypeptide concentration. A.sup.q-restricted HRC.2 hybridoma cells were incubated with CII.sub.(259-273Hyp-274) and CII.sub.(259-267E-273Hyp-274) polypeptides. In the legend, Q(267)-peptide refers to CII.sub.(259-273Hyp-274); E(267)-peptide refers to CII.sub.(259-267E-273Hyp-274); and blank refers to media without polypeptide complexes.

FIG. 28 is a graph plotting IL-2 production as measured by ELISA as a function of polypeptide concentration. A.sup.q-restricted HDQ.9 hybridoma cells were incubated with CII.sub.(259-273Hyp-274) and CII.sub.(259-267E-273Hyp-274) polypeptides. In the legend, Q(267)-peptide refers to CII.sub.(259-273Hyp-274); E(267)-peptide refers to CII.sub.(259-267E-273Hyp-274); and blank refers to media without polypeptide complexes.

FIG. 29 is a graph plotting IL-2 production as measured by ELISA as a function of polypeptide concentration. A.sup.q-restricted HCQ.4 hybridoma cells were incubated with CII.sub.(259-273Hyp-274) and CII.sub.(259-267E-273Hyp-274) polypeptides. In the legend, Q(267)-peptide refers to CII.sub.(259-273Hyp-274); E(267)-peptide refers to CII.sub.(259-267E-273Hyp-274); and blank refers to media without polypeptide complexes.

FIG. 30 is a graph plotting IL-2 production as measured by ELISA as a function of polypeptide concentration. DR4-restricted 1259 hybridoma cells were incubated with CII.sub.(259-273Hyp-274) and CII.sub.(259-267E-273Hyp-274) polypeptides. In the legend, Q(267)-peptide refers to CII.sub.(259-273Hyp-274); E(267)-peptide refers to CII.sub.(259-267E-273Hyp-274); and blank refers to media without polypeptide complexes.

DETAILED DESCRIPTION

The invention provides methods and materials related to assessing and treating autoimmune conditions such as rheumatoid arthritis. Specifically, the invention provides polypeptide compositions, nucleic acids, substantially pure polypeptides, host cells, and methods for identifying mammals with autoimmune conditions, treating mammals with autoimmune conditions, and enhancing tolerance in mammals with autoimmune conditions. For the purpose of this invention, the term "autoimmune condition" refers to any condition resulting from a mammal's body tissue being attacked by that mammal's own immune system. For example, a patient with an autoimmune condition can have antibodies in their blood that target their own body tissues. Examples of autoimmune conditions include, without limitation, rheumatoid arthritis, relapsing polychondritis, systemic lupus erythematosus, psoriasis arthritis, anylosing spondylitis, chronic stages of asthma, Sjorgren's syndrome, and multiple sclerosis.

Polypeptide Compositions

The invention provides polypeptide compositions. A polypeptide composition can contain three polypeptides arranged in a triple helical conformation. Additionally, a polypeptide composition can contain three polypeptides with each polypeptide having at least one interpolypeptide linkage such that each polypeptide of the three polypeptides is covalently attached to at least one of the other two polypeptides. The term "interpolypeptide linkage" as used herein refers to any bond or series of bonds that covalently connects two polypeptides. An interpolypeptide linkage can be a bond or series of bonds formed between the side group of a unit from one polypeptide and the side group of a unit from the other polypeptide. Any linkage can be used to link polypeptides within a polypeptide composition. For example, adding .epsilon.-aminohexanoic acid (Ahx) to the three available amino groups of Lys-Lys-Tyr-Gly-resin allows three distinct polypeptides of a polypeptide composition to be synthesized in parallel. In this case, each polypeptide contains at least one interpolypeptide linkage with at least one of the other two polypeptides of the polypeptide composition, and each interpolypeptide linkage is located at or near the C-terminal ends of the three polypeptides.

In addition, interpolypeptide linkages can be added at or near the last stages of polypeptide synthesis to allow the polypeptides to be linked at or near their N-terminal ends. For example, each N-terminal .alpha.-amino group of three distinct polypeptides can be linked by an amide bond to a tricarboxylic acid such as Kemp triacid (KTA, cis,cis-1,3,5-trimethylcyclohexane-1,3,5-tricarboxylic acid; Goodman et al., J. Am. Chem. Soc., 118:5156 5157 (1996) and Feng et al., J. Am. Chem. Soc., 118:10351 10358 (1996)) or 1,2,3-propanetricarboxylic acid (Greiche and Heidemann, Biopolymers, 18:2359 2361 (1979)). Alternatively, a glutamic acid dipeptide, in which the two side-chain carboxylic acid groups as well as the .alpha.-carboxylic acid group are individually coupled to one of three distinct polypeptides, can be used to form a polypeptide composition where each of the three polypeptides contains at least one interpolypeptide linkage with at least one of the other two polypeptides of the polypeptide composition, and where each interpolypeptide linkage is located at or near the N-terminal ends of the three polypeptides (Hojo et al., Tetrahedron, 53:14263 14274 (1997)). Other examples of interpolypeptide linkages include, without limitation, disulfide knots formed between cysteine residues (Ottl and Moroder, Tetrahedron Lett., 40:1487 1490 (1999)) or other thiol-containing units located, for example, at or near the N- or C-terminus of the connected polypeptides. In such disulfide knots, a single cysteine residue or thiol-containing unit can be incorporated into two distinct polypeptides, while two cysteine residues or thiol-containing units are incorporated into a third polypeptide. Oxidation of the cysteine residues or thiol-containing units can then covalently link the three polypeptide strands to each other such that each of the three polypeptides contains at least one interpolypeptide linkage with at least one of the other two polypeptides of the polypeptide composition. Cysteine residues or thiol-containing units such as 3-mercaptopropionic acid can be located at or near the N-terminus of each of polypeptides to be linked. In addition, cysteine residues or thiol-containing units can be alkylated with an alkyl tribromide or triiodide. Examples of such alkyltrihalogenides include, without limitation, 1,2,3-tribromo- or triiodomethylpropane as well as compounds obtained by coupling each of the three carboxylic acid groups of Kemp triacid to one of the amino groups of a diamine such as 1,2-diaminoethane followed by attachment of .alpha.-bromo or .alpha.-iodo acetic acid to the other amino group of the diamine. In addition, lysine residues can be included at or near the N-terminus of each of the polypeptides to be connected such that the amino groups of these lysine residues can be linked by treatment with glutaraldehyde. Treatment with glutaraldehyde leads to the formation of imines with the amino groups of the lysine residues.

Any unit can be incorporated into the polypeptides of a polypeptide composition. The term "unit" as used herein with reference to the sequence of a polypeptide refers to any of the twenty conventional amino acid residues as well as any other chemical structure that can be incorporated into a sequence including, without limitation, ornithine (Orn), citrulline (Cit), .epsilon.-aminohexanoic acid (Ahx). Hydroxylated amino acids such as 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp or simply Hyp), (5R)-5-hydroxy-L-lysine (Hyl), allo-hydroxylysine (aHyl), and 5-hydroxy-L-norvaline (Hnv) can be incorporated into a sequence. Glycosylated amino acids such as amino acids containing monosaccharides (e.g., D-glucose, D-galactose, D-mannose, D-glucosamine, and D-galactosamine) or combinations of monosaccharides also can be incorporated into a polypeptide sequence. Other examples of modified chemical structures that can be incorporated into a sequence include, without limitation, 2-aminoadipic acid (Aad), 3-aminoadipic acid (bAad), beta-alanine or beta-aminopropionic acid (bAla), 2-aminobutyric acid (Abu), 4-aminobutyric acid or piperidinic acid (4Abu), 6-aminocaproic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (bAib), 2-aminopimelic acid (Apm), 2, 4-diaminobutyric acid (Dbu), desmosine (Des), 2,2-diaminopimelic acid (Dpm), 2,3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), isodesmosin (Ide), allo-isoleucine (alle), N-methylglycine or sarcosine (MeGly), N-methylisoleucine (Melle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), and norleucine (Nle). Specific modifications can include, without limitation, ornithine modifications of arginine (OrnR) or citrulline modifications of arginine (CitR). Further examples of chemical structures that can be incorporated into a sequence include, without limitation, .beta.-D-galactopyranosyl-5-hydroxy-L-lysine with single or multiple deoxygenations and 2-O-.alpha.-D-glucopyranosly-.beta.-D-galactopyranosyl-5-hydroxy-L-lysine with single or multiple deoxygenations. In addition, one or more hydroxyl groups of a unit can be replaced with fluorine. For example, the hydroxy group of 3-hydroxyproline (3Hyp) can be replaced with fluorine to create 3-fluoroproline (3Flp), or the hydroxy group of 4-hydroxyproline (4Hyp) can be replaced with fluorine to create 4-fluoroproline (4Flp). Further, units having C- or S-glycosidic linkages can replace the O-glycosidic linkages. It will be appreciated that a single polypeptide can contain any combination of units. For example, a single polypeptide can contain twelve conventional amino acids, eight hydroxylated amino acids, two glycosylated amino acids, and one ornithine in any order.

Units also can be placed together to form a triple helix formation sequence. The term "triple helix formation sequence" as used herein refers to any sequence of units of a polypeptide that can form a stable triple helical conformation through non-covalent interactions with any two other polypeptides under optimal conditions. Examples of triple helix formation sequences include, without limitation, (Gly-Xaa-Yaa).sub.n, where Xaa and Yaa can be any unit and n can be any integer greater than three (e.g., any integer greater than 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or 1000). Thus, a polypeptide containing (Gly-Pro-Arg).sub.8 can be a polypeptide having a triple helix formation sequence.

A polypeptide composition within the scope of the invention can contain polypeptides having a sequence of units connected by amide bonds (--CONH--) or any other bond including, without limitation, modified amide bonds such as those modified by N-methylation (--CONMe--), N-alkylation (--CONR--), or reduction (--CH.sub.2NH--) as well as isosteres bonds such as methylene ether bonds (--CH.sub.2O--), methylene thioether bonds (--CH.sub.2S--), vinyl group bonds(--CH.dbd.CH--), ethylene group bonds (--CH.sub.2CH.sub.2--), ketomethylene group bonds (--COCH.sub.2--), thioamide bonds (--CSNH--), and sulfone bonds (--CH.sub.2SO--). It will be appreciated that a single polypeptide can contain a sequence of units connected by any combination of bonds. For example, a single polypeptide can contain a sequence of units connected exclusively by amide bonds or by a combination of amide bonds, methylene ether bonds, and sulfone bonds.

A polypeptide composition can contain any sequence. Typically, a polypeptide composition contains an amino acid sequence corresponding to at least a portion of the amino acid sequence (e.g., at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, or more amino acid residues) of CII (e.g., human, rat, or mouse CII). The amino acid sequence of human CII is set forth in SEQ ID NO:1 as well as FIG. 1. The nucleic acid sequence that encodes human CII is set forth in SEQ ID NO:2 as well as FIG. 2.

A polypeptide composition can contain a polypeptide having an epitope. The term "epitope" as used herein refers to a sequence of units (e.g., an amino acid sequence) that is recognized by a lymphocyte (e.g., a B cell or a T cell). The term "B cell epitope" as used herein refers to a sequence of units that is recognized by a B cell. For example, an amino acid sequence corresponding to a sequence from CII recognized by a B cell can be a B cell epitope. Examples of CII B cell epitopes include, without limitation, CII.sub.(256-270)(Gly-Glu-Pro-Gly-Ile-Ala-Gly-Phe-Lys-Gly-Glu-Gln-Gly-Pro- -Lys; SEQ ID NO:3), CII.sub.(358-366) (Gly-Ala-Arg-Gly-Leu-Thr-Gly-Arg-Pro; SEQ ID NO:4), CII.sub.(358-369) (Gly-Ala-Arg-Gly-Leu-Thr-Gly-Arg-Pro-Gly-Asp-Ala; SEQ ID NO:5), CII.sub.(259-274) (Gly-Ile-Ala-Gly-Phe-Lys-Gly-Glu-Gln-Gly-Pro-Lys-Gly-Glu-Pro-Gly; SEQ ID NO:6), CII.sub.(494-504) (Leu-Val-Gly-Pro-Arg-Gly-Glu-Arg-Phe-Pro; SEQ ID NO:7), CII.sub.(551-564) (Met-Pro-Gly-Glu-Arg-Gly-Ala-Ala-Gly-Ile-Ala-Gly-Pro-Lys; SEQ ID NO:8), CII.sub.(932-936) (His-Arg-Gly-Phe-Thr; SEQ ID NO:9), CII.sub.(687-698) (Arg-Gly-Ala-Gln-Gly-Pro-Pro-Gly-Ala-Thr-Gly-Phe; SEQ ID NO:10), CII.sub.(777-783) (Ala-Gly-Gln-Arg-Gly-Ile-Val; SEQ ID NO:11), CII.sub.(124-142) (Gly-Pro-Arg-Gly-Leu-Pro-Gly-Glu-Arg-Gly-Arg-Thr-Gly-Pro-Ala-Gly-Ala-Ala-- Gly; SEQ ID NO:12), CII.sub.(208-220) (Gly-Asn-Pro-Gly-Thr-Asp-Gly-Ile-Pro-Gly-Ala-Lys-Gly; SEQ ID NO:13), CII.sub.(182-193) (Ala-Arg-Gly-Pro-Glu-Gly-Ala-Gln-Gly-Pro-Arg; SEQ ID NO:14), and CII.sub.(368-381) (Asp-Ala-Gly-Pro-Gln-Gly-Lys-Val-Gly-Pro-Ser-Gly-Ala-Pro; SEQ ID NO:15).

The term "T cell epitope" as used herein refers to a sequence of units that is recognized by a T cell. For example, an amino acid sequence corresponding to a sequence from CII recognized by a T cell can be a T cell epitope. Examples of CII T cell epitopes include, without limitation, CII.sub.(259-274) (Gly-Ile-Ala-Gly-Phe-Lys-Gly-Glu-Gln-Gly-Pro-Lys-Gly-Glu-Pro-Gly; SEQ ID NO:6), CII.sub.(335-349) (Pro-Ser-Gly-Leu-Ala-Gly-Pro-Lys-Gly-Ala-Asn-Gly-Asp-Pro-Gly; SEQ ID NO:16), CII.sub.(374-388) (Lys-Val-Gly-Pro-Ser-Gly-Ala-Pro-Gly-Glu-Asp-Gly-Arg-Pro-Gly; SEQ ID NO:17), CII.sub.(404-418) (Phe-Pro-Gly-Pro-Lys-Gly-Ala-Asn-Gly-Glu-Pro-Gly-Lys-Ala-Gly; SEQ ID NO:18), CII.sub.(593-607) (Pro-Pro-Gly-Pro-Ala-Gly-Ala-Asn-Gly-Glu-Lys-Gly-Glu-Val-Gly; SEQ ID NO:19), CII.sub.(707-721) (Pro-Pro-Gly-Ala-Asn-Gly-Asn-Pro-Gly-Pro-Ala-Gly-Pro-Pro-Gly; SEQ ID NO:20), CII.sub.(224-238) (Ala-Pro-Gly-Ile-Ala-Gly-Ala-Pro-Gly-Phe-Pro-Gly-Pro-Arg-Gly; SEQ ID NO:21), CII.sub.(88-95) (Gly-His-Arg-Gly-Tyr-Pro-Gly-Leu; SEQ ID NO:22), CII.sub.(791-798) (Gly-Glu-Arg-Gly-Phe-Pro-Gly-Leu; SEQ ID NO:23), and CII.sub.(931-938) (Gly-His-Arg-Gly-Phe-Thr-Gly-Leu; SEQ ID NO:24).

An epitope can be used in a native form. For example, an epitope can have an amino acid sequence identical to a sequence from CII (e.g., amino acid residues 259 274). In addition, an epitope can be a mutated version of a native sequence. For example, the fifth unit of an epitope can be replaced with a different unit. Mutated epitopes can contain any number of additions, deletions, substitutions, or combinations thereof. For example, in one embodiment a mutated epitope can be a CII T cell epitope with amino acid residues 260 270, where the glutamine residue at position 267 is substituted with a glutamic acid residue (e.g., CII.sub.(260-267E-270). An epitope also can be used in a modified form. For example, in one embodiment a modified epitope can be a CII B cell epitope with amino acid residues 259 274, where the proline at residue 273 is hydroxylated (e.g., CII.sub.(259-273Hyp-274)). Any type of modification can be used. For example, a modification can include, without limitation, hydroxylation or glycosylation. Examples of modified epitopes include, without limitation, CII.sub.(358-366Hyp) (Gly-Ala-Arg-Gly-Leu-Thr-Gly-Arg-Hyp; SEQ ID NO:25), CII.sub.(358-366Hyp-369) (Gly-Ala-Arg-Gly-Leu-Thr-Gly-Arg-Hyp-Gly-Asp-Ala; SEQ ID NO:26), CII.sub.(259-273Hyp-274) (Gly-Ile-Ala-Gly-Phe-Lys-Gly-Glu-Gln-Gly-Pro-Lys-Gly-Glu-Hyp-Gly; SEQ ID NO:27), CII.sub.(494-504Hyp) (Leu-Val-Gly-Pro-Arg-Gly-Glu-Arg-Gly-Phe-Hyp; SEQ ID NO:28), CII.sub.(551-552Hyp-564) (Met-Hyp-Gly-Glu-Arg-Gly-Ala-Ala-Gly-Ile-Ala-Gly-Pro-Lys; SEQ ID NO:29), CII.sub.(358-360CtR-365CtR-366Hyp-369) (Gly-Ala-CtR-Gly-Leu-Thr-Gly-CtR-Hyp-Gly-Asp-Ala; SEQ ID NO:30), CII.sub.(358-360OnR-365OnR-366Hyp-369) (Gly-Ala-OnR-Gly-Leu-Thr-Gly-OnR-Hyp-Gly-Asp-Ala; SEQ ID NO:31), CII.sub.(124-129Hyp-142) (Gly-Pro-Arg-Gly-Leu-Hyp-Gly-Glu-Arg-Gly-Arg-Thr-Gly-Pro-Ala-Gly-Ala-Ala-- Gly; SEQ ID NO:32), CII.sub.(208-210Hyp-216Hyp-220) (Gly-Asn-Hyp-Gly-Thr-Asp-Gly-Ile-Hyp-Gly-Ala-Lys-Gly; SEQ ID NO:33), and CII.sub.(368-381Hyp) (Asp-Ala-Gly-Pro-Gln-Gly-Lys-Val-Gly-Pro-Ser-Gly-Ala-Hyp; SEQ ID NO:34). Additionally, any combination of modifications can be used. For example, an epitope can have two hydroxylated units and one glycosylated unit. An epitope also can contain a unit that has more than one modification. For example, the lysine at residue 264 of the CII epitope CII.sub.(256-270) can be both hydroxylated and glycosylated (e.g., CII.sub.(256-264Ghyl-270)).

A polypeptide can contain one epitope or more than one (e.g., 2, 3, 4, 5, or more) epitope. For example, a polypeptide can contain four contiguous B cell epitopes. Alternatively, a polypeptide can contain four B cell epitopes each separated by any number, type, and combination of units or linkages. A polypeptide also can contain different combinations of B cell and T cell epitopes. For example, both a B cell epitope and a T cell epitope can be incorporated into a polypeptide.

Each distinct polypeptide of a polypeptide composition can contain any sequence. For example, a single polypeptide can contain Gly-Pro-Thr-Ser-Ser-Leu (SEQ ID NO:35), the CII B cell epitope CII.sub.(259-274) (SEQ ID NO:6), and Met-Glu-Met-Gly-Gly-Leu-Arg-Hyp (SEQ ID NO:36). Such a polypeptide can be represented as Gly-Pro-Thr-Ser-Ser-Leu-CII.sub.(259-274)-Met-Glu-Met-Gly-Gly-Leu-Arg-Hyp (SEQ ID NO:37). In some cases, a polypeptide of a polypeptide composition can contain repeating units. For example, a polypeptide can contain two methionines followed by Gly-Pro-Arg-Gly-Pro-Arg-Gly-Pro-Arg (SEQ ID NO:38) followed by Glu-Ser-Phe-Leu-Glu-Ser-Phe-Leu-Glu-Ser-Phe-Leu-Glu-Ser-Phe-Leu-Glu-Ser-P- he-Leu (SEQ ID NO:39). Such a polypeptide can be represented as Met-Met-Gly-Pro-Arg-Gly-Pro-Arg-Gly-Pro-Arg-Glu-Ser-Phe-Leu-Glu-Ser-Phe-L- eu-Glu-Ser-Phe-Leu-Glu-Ser-Phe-Leu-Glu-Ser-Phe-Leu (SEQ ID NO:40) or (Met).sub.2(Gly-Pro-Pro).sub.3(Glu-Ser-Phe-Leu).sub.5 (SEQ ID NO:40). A polypeptide of a polypeptide composition can contain any number of interpolypeptide linkages. Any polypeptide of a polypeptide composition can be linked to any other polypeptide of the polypeptide composition.

In one embodiment, the three polypeptides of a polypeptide composition can each contain a sequence that extends in the N-terminal direction from an Axh unit that is attached to one of the three available amino groups on the two Lys residues of a Lys-Lys-Tyr-Gly-resin. In this case, the portion containing the three Ahx residues attached to the Lys-Lys-Tyr-Gly sequence can be represented as L.sub.Ahx. Thus, a polypeptide composition containing three (Gly-Pro-Hyp).sub.6-CII.sub.(259-274)-(Gly-Pro-Hyp).sub.2 polypeptides each extending from one of the Ahx units of L.sub.Ahx can be represented as [(Gly-Pro-Hyp).sub.6-CII.sub.(259-274)-(Gly-Pro-Hyp).sub.2].sub.3L.sub- .Ahx.

In another embodiment, the three polypeptides of a polypeptide composition can each contain a sequence that extends in the N-terminal direction from an Axh unit that is attached to one of the three available amino groups on the two Lys residues of a Lys-Lys-Phe(F)-Tyr-Gly-resin. In this case, the portion containing the three Ahx residues attached to the Lys-Lys-Phe(F)-Tyr-Gly sequence can be represented as L(F).sub.Ahx. Thus, a polypeptide composition containing three (Gly-Pro-Hyp).sub.6-CII.sub.(259-274)-(Gly-Pro-Hyp).sub.2 polypeptides each extending from one of the Ahx units of L(F).sub.Ahx can be represented as [(Gly-Pro-Hyp).sub.6-CII.sub.(259-274)-(Gly-Pro-Hyp).sub.2].sub.3L(F).sub- .Ahx.

In some embodiments, each polypeptide of a polypeptide composition can contain a sequence that extends in the C-terminal direction from a Gly unit that is attached to KTA. Thus, a polypeptide composition containing three (Gly-Pro-Hyp).sub.6-CII.sub.(259-274)-(Gly-Pro-Hyp).sub.2 polypeptides each extending from one of the Ahx units of L.sub.Ahx to a Gly unit attached to KTA can be represented as KTA-[Gly-(Gly-Pro-Hyp).sub.6-CII.sub.(259-274)-(Gly-Pro-Hyp).sub.2].sub.3- L.sub.Ahx. Such complexes have interpolypeptide linkages in both the N-terminal and C-terminal regions. An example of such a polypeptide complex is provided in FIG. 21.

It is noted that a polypeptide composition containing three distinct polypeptides can contain no interpolypeptide linkages. Such a polypeptide composition can be a triple helix polypeptide composition where three distinct polypept