EP0805686A1 - Collagen-based methods and formulations for the treatment of immune system-mediated diseases - Google Patents

Collagen-based methods and formulations for the treatment of immune system-mediated diseases

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Publication number
EP0805686A1
EP0805686A1 EP96902148A EP96902148A EP0805686A1 EP 0805686 A1 EP0805686 A1 EP 0805686A1 EP 96902148 A EP96902148 A EP 96902148A EP 96902148 A EP96902148 A EP 96902148A EP 0805686 A1 EP0805686 A1 EP 0805686A1
Authority
EP
European Patent Office
Prior art keywords
collagen
type
tolerance
immune system
derivatives
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96902148A
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German (de)
English (en)
French (fr)
Inventor
Thomas B. Neff
George R. Martin
Karl A. Piez
Taina A. Pihlajaniemi
Kari I. Kivirikko
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ACADEMY OF FINLAND
Fibrogen Inc
Original Assignee
ACADEMY OF FINLAND
Fibrogen Inc
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Application filed by ACADEMY OF FINLAND, Fibrogen Inc filed Critical ACADEMY OF FINLAND
Publication of EP0805686A1 publication Critical patent/EP0805686A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention is in the field of autoimmune disease and other immune system- mediated disease treatments employing collagen and/or derivatives thereof.
  • Immune system-mediated diseases may be either B cell-mediated (i.e. , antibody-mediated) or T cell-mediated. Additionally, immune system-mediated diseases may be caused by immune complexes formed between antibodies and antigens (either foreign or autologous). Many immune system-mediated diseases involve an undesirable inflammatory response, e.g.
  • 95/10301 describes the use of oral ingested cholera toxin conjugates to decrease a delayed hypersensitivity reaction and control experimental autoimmune encephalitis in mouse models.
  • Methods described herein employ one or more collagens and/or collagen derivatives so as to reduce or eliminate an immune response that is important for the pathogenesis of a given immune system- mediated disease.
  • the methods and formulations designed to induce tolerance to antigens involved in the disease process are particularly useful in view of the shortcomings of existing techniques for treating chronic immune system-mediated diseases.
  • the subject invention provides novel methods and compositions for the treatment of immune system-mediated diseases, including rheumatoid arthritis.
  • the subject compositions comprise one or more different types of collagen or collagen derivatives.
  • collagen and/or collagen derivatives may be used to treat specific immune system-mediated diseases.
  • the collagen(s) and/ or collagen(s) derivatives used in the subject compositions may be either obtained from natural sources or produced by recombinant genetic engineering techniques.
  • Another aspect of the invention is to provide methods for treating immune system-mediated diseases by administering an effective amount of the subject collagen- containing compositions.
  • the methods of the invention include the administration of the subject collagen(s) and/or collagen derivative(s) containing compositions into the intestine so as to induce immune tolerance, i.e. , oral administration.
  • Another aspect of the invention is to provide the subject collagen and/or collagen derivative containing compositions formulated for administration to a patient.
  • Preferred formulations of the invention are designed for the release of collagen(s) and/or collagen derivatives in the intestine so as to contact intestinal lymphoid tissue.
  • the subject compositions are formulated for oral admimstration.
  • Another aspect of the invention is to provide collagen derivatives that comprise at least one tolerance inducing epitope, and preferably a plurality of tolerance inducing epitopes.
  • Such collagen derivatives may comprise a plurality of different collagen- derived tolerance inducing epitopes and are referred to herein as tolerance epitope polypeptides.
  • Tolerance epitope polypeptides may also comprise amino acid resolve sequences derived from non-collagenous proteins.
  • the mucosa binding collagen conjugates of the invention comprise one or more collagen molecules linked to a mucosa binding molecule.
  • a variety of different collagen and/or collagen derivatives may be used as the collagen component of the mucosa binding collagen conjugates of the invention.
  • a variety of mucosa binding molecules may be used as the mucosa binding molecule component of the mucosa binding collagen conjugates of the invention
  • suitable mucosa binding components include mucosa binding structures derived from bacterial toxins, bacterial fimbriae, viral attachment proteins, and plant lectins.
  • a particularly preferred mucosa binding components are the ⁇ subunit of cholera toxin and the ⁇ subunit of E. coli heat labile enterotoxin.
  • Another aspect of the invention is to provide methods of treating immune system mediated diseases by administering an effective amount of a mucosa binding collagen conjugate.
  • Yet another aspect of the invention is to provide formulations for use in the treatment of immune system-mediated diseases, wherein the formulation comprise a mucosa binding collagen conjugate of the invention.
  • the formulations are adapted for oral administration.
  • the invention involves the use of one or more different collagens and/or collagen derivatives to induce oral tolerance in a specific population of immune cells so as to provide a method of treating immune system-mediated diseases.
  • Methods of using various collagens and/or collagen derivatives to treat immune system-mediated diseases and compositions for use in such methods are provided herein.
  • the methods of the invention involve the oral admimstration of a collagen or collagens found in a specific tissue so as to induce the suppression (immunological tolerance) of inflammation against the tissue (or tissues) from which the collagen is found to occur in nature.
  • the correspondence between a specific collagen type and its (e.g. tissue type) may be found in various art references, including Fukai, et al.
  • Collagen (and collagen derivative) containing compositions for use in such methods are also provided.
  • the invention also provides many different types of mucosa binding collagen conjugates and methods for their use in treating immune system- mediated diseases.
  • One aspect of the invention is to use minor collagens, i.e. , comparatively rare collagens found to be naturally associated with major collagens, to induce immune tolerance so as to reduce the severity of an undesirable immune response, thereby providing the basis of a treatment for arthritis and related immune system-mediated diseases; however, the invention also provides for the use of major collagens to induce immune tolerance.
  • Cartilage contains diverse species of collagen that provide the major extracellular scaffolding for the surrounding tissue. Data has been found to suggest that type II collagen, a major collagen, may have the capacity to suppress inflammation in cartilage.
  • the subject invention is based, in part, upon the realization that minor collagen(s) naturally found to occur in combination with major collagen(s) may be important in inducing immune tolerance useful in treating immune system-mediated diseases such as rheumatoid arthritis. Accordingly, one aspect of the invention is to use minor collagens, either alone or in combination with major collagens, to induce immune tolerance that results in amelioration of the disease process in several immune system- mediated diseases.
  • the subject invention provides a variety of collagen and collagen derivative containing compositions.
  • Numerous different types of collagen are known to the person of ordinary skill in the art. At present nineteen different types of collagen have been discovered. A detailed description of structure and biological functions of the various different types of naturally occurring collagens can be found, among other places, in Ayad et al. , The Extracellular Matrix Facts Book, Academic Press, San Diego, CA; Burgeson, R. E., and Nimmi, "Collagen types: Molecular Structure and Tissue Distribution, " Clin. Orthop.. 282:250-272 (1992); Kielty, C. M., et_al., "The Collagen
  • Type I collagen is the major fibrillar collagen of bone and skin.
  • Type I collagen is a heterotrimeric molecule comprising two ⁇ J(I) chains and one ⁇ 2(I) chain. Details on preparing purified type I collagen can be found, among other places, in Miller, E. J. , and Rhodes, R. K. , Methods In Enzvmology. 82:33-64 (1982), Academic Press.
  • Type II collagen is a homotrimeric collagen comprising three identical l(II) chains.
  • Purified Type II collagen may be prepared by, among other methods, the procedure described in Miller, E. J., and Rhodes, R.K. Methods In Enzvmology.
  • Type III collagen is a major fibrillar collagen found in skin and vascular tissues.
  • Type III collagen is a homotrimeric collagen comprising three identical 0.1(111) chains. Methods for producing type III collagen can be found in, among other places, Byers, et al., Biochemistry. 13:5243-5248 (1974) and Miller E. J. and Miller, R. K., Methods in
  • Type IN collagen is found in basement membranes in the form of a sheet rather than fibrils.
  • Type IN collagen molecules are composed of three different alpha chains derived from six different genes, i.e. , type IV collagen comprises three of ⁇ l(IN), Q-2(IN), ⁇ .3(IV), ⁇ 4(IV), ⁇ 5(TV), and ⁇ 6(IV) chains. The particulars are expressed in a tissue-specific manner.
  • Type IV collagen may be purified by, among other methods, the procedures described in Furuto et al., D. K., and Miller, E. J., Methods in Enzvmology. 144:41-61 (1987), Academic Press.
  • Type V collagen is a fibrillar collagen found in bones, tendon, cornea, skin, and blood vessels. Type V collagen exists in both homotrimeric and heterotrimeric forms.
  • One type of Type V collagen is a heterotrimer of two ⁇ l(V) chains and ⁇ 2(V).
  • Another type of type V collagen is a heterotrimer of l(V), 2(V), and ⁇ 3(V).
  • Yet another type of type V collagen is a homotrimer of ⁇ l(V).
  • Type VI collagen has a small triple helical region and a large non-collagenous remainder portion. Type VI collagen is found in many connective tissues. Type VI collagen is a heterotrimer comprising ⁇ l(VI), ⁇ 2(VI), and ⁇ .3(NI) chains. Descriptions of how to purify type IV collagen can be found, among other places, in Wu, et al., Biochem. J.. 248:373-381 (1987), and Kielty, et al., J. Cell Sci.. 99:797-807. Type VII collagen is a fibrillar collagen found in particular epithelial tissues.
  • Type VII is a homotrimeric molecule of three ⁇ l(VII) chains. Descriptions of how to purify type VII collagen can be found in, among other places, Lundstrom, et al., _ Biol. Chem.. 261:9042-9048 (1986), and Bentz, et al., Proc. ⁇ atl. Acad. Sci. U.S.A.. 80:3168-3172 (1983). Type VIII collagen can be found in Descemet's membrane in the cornea. Type
  • Type IX collagen is a heterotrimer comprising two ⁇ l(VIII) and one ⁇ .2(VIII) chain. Methods for the purification of type VIII collagen can be found, among other places, in Benya and Badilla, J. Biol. Chem.. 261:4160-4169 (1986), and Kapoor, et al., Biochemistry. 25:3930-3937 (1986).
  • Type IX collagen is a fibril associated collagen which can be found in cartilage and vitreous humor.
  • Type IX collagen is a heterotrimeric molecule comprising ⁇ l(IX), ⁇ 2(IX), and ⁇ 3 (IX) chains.
  • Type X collagen is a homotrimeric compound of XI(x) chain. Type X collagen has been isolated from, among other tissues, hypertrophic cartilage found in growth plates.
  • Type XI collagen can be found in cartilaginous tissues associated with type II and type IX collagens, as well as other locations in the body.
  • Type XI collagen is a heterotrimeric molecule comprising ⁇ l(XI), ⁇ 2(XI), and ⁇ 3(XI) chains. Methods for purifying type XI collagen can be found, among other places, in Grant et al., In The Control of Tissue Damage. Glauert, A. M., Ed., El Savier, Amsterdam, pp. 3-28 (1988).
  • Type XII collagen is a fibril associated collagen found primarily associated with
  • Type I collagen Type XII collagen is a homotrimeric molecule comprising three ⁇ l(XII) chains. Methods for purifying type XII collagen and variants thereof can be found, among other places, in Dublet et al. , J. Biol. Chem.. 264:13150-13156 (1989), Lundstrum et al. , J. Biol. Chem.. 267:20087-20092 (1992), Watt et al., J. Biol. Chem.. 267:20093-20099 (1992).
  • Type XIII is a non-fibrillar collagen found, among other places, in skin, intestine, bone, cartilage, and striated muscle. A detailed description of the type XIII collagen may be found, among other places, in Juvonen, et al. J. Biol. Chem.. 267:24700-24707 (1992).
  • Type XIV is a fibril associated collagen.
  • Type XIV collagen is a homotrimeric molecule comprising three ⁇ l(XIV) chains. Methods for isolating type XIV collagen can be found, among other places, in Aubert-Foucher, et al., J. Biol. Chem.. 266: 19759-19764 (1992) and Watt, et al., J. Biol.
  • Type XV collagen is homologous in structure to type XVIII collagen. Information about the structure and isolation of type XV collagen can be found, among other places, in Myers et al, Proc. Natl. Aca. Sci. USA. 89: 10144-10148 (1992), Huebner et al. , Genomics. 14: 220-224 (1992), Kivirikko et al, J. Biol. Chem.. 269:4773-4779 (1994), and Muragaki, J. Biol. Chem.. 264:4042-4046 (1994).
  • Type XVII collagen is a fibril associated collagen, found in skin, lung fibroblast, keratinocytes, and elsewhere. Information on the structure of type XVI collagen and the gene encoding type XVI can be found, among elsewhere, in Pan et al, Proc. Natl. Acad. Sci. U.S.A.. 1989:6565-6569 (1992), and Yamaguchi, et al , J. Biochem.. 112:856-863 (1992).
  • Type XVIII collagen is homologous in structure to type XV collagen and can be isolated from the liver. Descriptions of the structures and isolation of type XVIII collagen can be found, among other places, in Rehn et al, Proc. Natl. Acad. Sci USA.
  • the above-collagen types may also be obtained by recombinant expression techniques, as described generally in U.S. Patent No. 5,405,757, PCT-published patent applications WO 93/07889 and WO94/16570, and related patents and applications.
  • the subject mucosa binding collagen conjugates comprise a collagen component and a mucosa binding component.
  • the two components may be linked directly to one another or may be linked to one another through one or more intermediary linking molecules.
  • Mucosa binding collagen conjugates may be used to treat a variety of immune system-mediated diseases. The treatment is effected through the induction of immune tolerance to one or more epitopes on the collagen component of the mucosa binding collagen conjugates.
  • the collagen component of the subject mucosa binding collagen conjugates may comprise one or more collagen or collagen derivative molecules.
  • the collagen molecules of the collagen component of the mucosa binding collagen conjugates may be the same or different from one another.
  • the collagen/collagen derivative molecules of the collagen component may be linked to one another, either directly or through linker intermediates.
  • the collagen/collagen derivatives of the collagen component are not necessarily linked to each other, but may be linked directly to the mucosa binding component of the subject mucosa binding collagen conjugates.
  • the mucosa binding component comprises one or more molecules capable of specifically binding to the mucosa cells of a patient for treatment.
  • a variety of different molecules may serve as the mucosa binding component.
  • These mucosa binding molecules may be derived from the mucosa binding structures of bacterial toxins, bacterial fimbriae, viral attachment proteins and plant lectins.
  • the use of mucosa binding structures from bacterial toxins is preferred.
  • Particularly preferred are the ⁇ subunits of cholera toxin and the ⁇ subunits of the heat-labile enterotoxin of E. coli.
  • the ⁇ subunits of cholera toxin and the ⁇ subunits of the heat-labile enterotoxin of E. coli have the property of specifically binding to ganglioside GM, in mucosal cells.
  • the toxin is preferably modified so as to significantly remove of destroy the cytotoxic properties of the toxin. Inactivation of cytotoxic properties my be accomplished in a number of ways well known to the person of ordinary skill the art including, for example, denaturation, mutation, and the like.
  • Other molecules that specifically bind to ganglioside GM may be used as the mucosa binding component of the mucosa binding collagen conjugates of the invention.
  • the mucosa binding molecules may be covalently joined to one another, either directly or indirectly, by means of an intermediary linker molecule(s). Alternatively, the mucosa binding molecules that form the mucosa binding component may associate with another by means of intermolecular attractive forces.
  • the ⁇ subunits of cholera toxin form a 5 molecule "ring" structure with allowed to associate.
  • suitable toxin molecules include subunits S2, S3, S4 and/or S5 of Bordatella pertussis toxin, diphtheria toxin, diphtheria toxin ⁇ fragment, shiga toxin, shiga-like toxins, shiga toxin ⁇ subunit, shiga-like toxin ⁇ subunit, cholera toxin, E. coli heat-labile toxin.
  • suitable bacterial fimbriae include E. coli K88, K99, 987P, F41, CFA/I,
  • CFA/II (CS1, CS2 and/or CS3), CFA/IV (CS4, CS5 and/or CS6), P fimbriae, Vibrio cholera toxin co-regulated pili (TCP), mucus sensitive hemagglatin (MSHA), fucose- sensitive hemagglatinin (FSITH), B. pertussis filamentous hemagglutinin and the like.
  • suitable viral attachment proteins include Influenza hemagglutinin, Sendai Virus hemagglatin.
  • Suitable lectin include both plant lectins and animal lectins, soluble lactose-binding lectins, selecting, collecting, helix pomatin hemagglutinin, concanavalin A, Wheat-germ agglutinin, Phytohemagglutinin, aurin ricin.
  • Immunoglobins that are specific for mucosal cell antigens may also be used as mucosal binding components.
  • the separate collagen compound and mucosa binding component of the mucosa binding components may be linked to one another by a variety of means.
  • the two components may be coupled together by means of chemical cross-linking agents such as N-succinimidyl (3-(2-pyridyl-dithio) propionate, dimethyl-3,3'-dithiobispropionimidate, 2-iminothiolane, N-succinimidyl-(4 azidophenyl)-l, 3-dithioprionate, ethyl-4- azidophenyl-1, 4-dithiobutyrimidate, diethyl malonimidate, 2-iminothiolane, N,N'-p- phenylenedimaleimide, and the like.
  • chemical cross-linking agents such as N-succinimidyl (3-(2-pyridyl-dithio) propionate, dimethyl-3,3'-dithiobispropionimidate, 2-iminothiolane, N-succinimidyl-(4 azidophenyl)-l, 3-di
  • the mucosa binding collagen conjugates of the invention may be single polypeptides that are fusion protein formed between the collagen component and the mucosa binding molecule component. These fusion proteins may be produced by conventional in vitro genetic engineering techniques. Another aspect of the invention is to provide pol nucleotide sequences encoding mucosa binding collagen conjugate fusion proteins. Yet another aspect of the invention to provide host cells for the recombinant production of mucosa binding collagen conjugate fusion proteins.
  • the mucosa binding molecule components may be obtained by purifying from their naturally occurring source organism. Alternatively, the mucosa binding molecule components may be produced by recombinant DNA techniques. Methods of purifying mucosa binding molecules, or alternatively, recombinantly producing such molecules suitable for use in the invention are well known to the person of ordinary skill in the art. For example, the recombinant production of cholera toxin ⁇ subunit has been described in Sanchez and Holmgren, Proc. Natl. Acad. Sci. (USA). 86:481-485 (1989) and production of the ⁇ subunit of e,. coli heat labile enterotoxin has been described in
  • compositions of the invention comprise one or more purified collagens and/or collagen derivatives.
  • the subject compositions comprise specific combinations of collagens and/or collagen(s) derivatives are provided.
  • Table 1 provides a list of collagen types (indicated by Arabic numeral rather than Roman numerals) that may be used to treat specific diseases. Each of the collagen types listed as being useful for treating a specific disease, either alone or in a composition combining one or more collagen molecules that are suitable for treating the disease of interest.
  • rheumatoid arthritis may be treated by composi- tions comprising type I collagen, type II collagen, type III collagen, type V collagen, type VI collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIV collagen, or any combination thereof comprising two or more of the collagens indicated as being suitable for the treatment of a specific disease condition.
  • compositions for the treatment of rheumatoid arthritis comprising at least one compound selected from the group consisting of type I collagen, type II collagen, type III collagen, type V collagen, type VI collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen and type IV collagen.
  • Collagen derivatives corresponding to a given collagen type may also be used in addition to or in place of a specific collagen type indicated in the compositions of Table 1.
  • a composition for the treatment of rheumatoid arthritis as indicated in Table 1, may comprise type II collagen, a type II collagen derivative, multiple different type II collagen derivatives, or a mixture of type II collagen and type II collagen derivatives.
  • the subject compositions comprise type IX collagen and/or type IX collagen derivatives thereof, either with or without type II collagen and/or derivatives of type II collagen.
  • the invention provides compositions that comprise type XI collagen and/or derivatives thereof, either with or without type II collagen and/or derivatives thereof.
  • the invention consists of compositions comprising type IX collagen and/or derivatives thereof and type XI collagen, either with or without type II collagen and/or derivatives thereof.
  • the subject compositions comprise type II collagen derivatives.
  • the mucosa binding collagen conjugates of the invention may be adapted for the treatment of specific immune system-mediated diseases.
  • the mucosa binding collagen conjugates may be adapted by selecting the collagen component of the mucosa binding collagen conjugate in accordance with the information in Table 1.
  • Purified collagens for use in the methods and compositions of the invention subject inventions may be isolated from animal or human tissues; however, the use of human collagens in the subject compositions and methods is preferred when the subject to be treated is a human.
  • collagens for use in the methods and compositions of the invention may be produced by recombinant DNA technology. A description of how to produce type II by recombinant DNA technology technique can be found, among other places, in PCT-published patent applications WO 93/07889 and WO94/ 16570, which are herein incorporated by reference.
  • recombinant production techniques described in these PCT publications may readily be adapted so as to produce many different types of collagen, human or otherwise, by recombinant DNA techniques.
  • human collagens produced by recombinant DNA technology are used in compositions of the invention.
  • Recombinant DNA technology avoids ethical and economic problems associated with obtaining adequate quantities of human tissue.
  • the methods and compositions of the invention may comprise many different types of collagen derivatives.
  • Collagen derivatives may vary from naturally occurring collagens in several respects. Collagen derivatives may be non-glycosylated or glycosylated differently than naturally-occurring collagens. Desired glycosylation patterns may be produced by a variety of methods, including direct chemical modification and enzymatically catalyzed glycosylation and deglycosylation reactions. Desired glycosylation patterns may also be produced by inhibiting or deleting enzymes necessary for producing the naturally-occurring glycosylation patterns found on collagens.
  • Collagen derivatives have a glycosylation pattern that differs from a corresponding naturally-occurring collagen, but are otherwise essentially the same in structure (except for the amount of proline and/or lysine residues that have been hydroxylated) are referred to herein as "variably glycosylated collagens. " Collagen derivatives also include various fragments of naturally-occurring collagens. Such collagen derivatives also include various fragments of naturally-occurring collagens. Such collagen fragments may be produced by, among other methods, chemically or enzymatically cleaving one or more peptide bands. Collagen derivatives may also contain one or more amino acid residue differences as compared with corresponding amino acid residue positions is a naturally-occurring collagen.
  • Collagen derivatives containing such amino acid residue substitutions may be produced by a variety of methods including genetic engineering techniques and by in vitro peptide synthesis. Additional collagen derivatives may be produced by varying the amount of hydroxylysines and/or hydroxyprolines present in a given molecule, by the varied expression of lysine hydroxylases, and/or proline hydroxylases, wherein the hydroxylase genes (recombinant or otherwise) are also expressed in a host cell for the expression of recombinant collagen, or derivatives thereof.
  • Collagen derivatives for use in the compositions and methods of the invention have the capacity to reduce the undesired immune reaction in the immune system- mediated disease of interest when administered to a patent either alone or as part of a composition of the invention, including compositions comprising a plurality of collagen and/or collagen derivatives.
  • Collagen derivatives suitable for use in those methods and compositions of the invention for the treatment of rheumatoid arthritis may be determined by employing in vitro tests of T cell from rheumatoid arthritis patients, wherein the tests could determine if a given collagen derivative can stimulate suppressor
  • T cells induce clonal anergy, or induce other forms of immune tolerance.
  • Methods of measuring antigen tolerance induction are well known to persons of ordinary skill in the art and can be found, for example, in Paul, W. E., editor, Fundamental Immunology. 2nd Ed., Raven Press: New York (1994).
  • collagen derivatives for use in the treatment of other immune system-mediated disorders may be determined to be suitable for use in treating a given immune system-mediated disease by in vitro tests on immune system cell isolated from patients suffering from the disease of interest.
  • a collagen molecule, collagen derivative or mucosa binding collagen conjugate suitable for use in the methods of the invention comprises at least one tolerance inducing epitope.
  • the term "tolerance inducing epitope” as used herein refers to epitopes on collagen that have the property of inducing immune tolerance with respect to a pathogenic immune response in an immune system-mediated disease, independent of other portions of the collagen molecule from which the "tolerance-inducing epitope" was derived.
  • Individual collagen molecules may comprise one or more tolerance inducing epitopes. Additionally, the tolerance-inducing epitope or epitopes on a given collagen molecule may vary in accordance with the specific immune system-mediated disease to be treated by the collagen.
  • the invention also provides for collagen derivatives consisting of amino acid residue sequences that comprise one or more collagen tolerance- inducing epitopes; such novel polypeptides are referred to herein as "tolerance epitope polypeptides.
  • Tolerance epitope polypeptides may comprise a plurality of identical tolerance-inducing epitopes.
  • Tolerance epitope polypeptides of the invention may also comprise one or more non- identical tolerance-inducing epitopes derived from the same or different collagen molecules.
  • Embodiments of the subject tolerance epitope polypeptides include polypeptides in which the different epitopes are arranged in a regular repeating pattern and polypeptides in which the various tolerance-inducing epitopes are arranged randomly.
  • Tolerance epitope polypeptides comprise a plurality of tolerance-inducing epitopes; while tolerance epitope polypeptides may be of virtually any size, the person of ordinary skill in the art will appreciate that certain technical problems arise in synthesizing and using very large polypeptides, e.g., solubility, stability, etc. Accordingly, the tolerance epitope polypeptides of the invention preferably comprise the
  • tolerance inducing epitopes more preferably in the range of to 250 tolerance- inducing epitopes and still more preferably in the range of 50 to 100 tolerance-inducing epitopes.
  • Tolerance-inducing epitopes may be glycosylated or non-glycosylated, depending upon whether the carbohydrate matrix are considered to be part of a tolerance-inducing epitope.
  • Tolerance epitope polypeptides may be produced by conventional genetic engineering techniques such as those described, for example, in
  • tolerance epitope polypeptides may comprise one or more non-collagen derived amino acid residue sequences, whereby the collagen derived tolerance-inducing epitope region on the tolerance epitope polypeptide serves as a carrier for the non-collagen derived amino acid sequence so that immune tolerance can also be induced against the non-collagen derived amino acid residue sequence.
  • some embodiments of the subject tolerance epitope polypeptides are polypeptides that act as "carriers" for inducing immune tolerance against non-collagen proteins that may be involved in the pathogenesis of immune system mediated diseases.
  • the subject invention provides tolerance epitope polypeptides that comprise regions of myelin basic protein that have been shown to be important in the development of the pathogenic immune response in multiple sclerosis and multiple tolerance inducing epitopes.
  • Polynucleotide sequence encoding tolerance epitope polypeptides may be produced by, among other methods, in vitro polynucleotide synthesis and the manipulation of previously-isolated collagen-encoding polynucleotides.
  • Tolerance epitope polypeptides may be designed for the treatment of specific immune system-mediated diseases in accordance with the information provided in Table 1. Tolerance epitope polypeptides may comprise tolerance-inducing epitopes derived from collagen or collagens found in a specific tissue so as to induce the suppression
  • a tolerance epitope polypeptides comprising tolerance-inducing epitopes of type ⁇ , type IX, and type XI collagen may be used in place of (or in addition to) a mixture of type II, type IX, and type XI collagens.
  • Tolerance-inducing epitopes for a given collagen and a given immune system- mediated disease may be readily determined by a person of ordinary skill in the art of immunology. For example, certain subpopulations of lymphocytes in a person wiring from (or likely to develop) a specific immune system-mediated disease have receptors capable of binding tolerance inducing options. Wherein a putative tolerance-inducing epitopes may be obtained by making systematic deletions in a collagen molecule of interest.
  • Another aspect of the invention is to provide methods of treating immune system- mediated diseases.
  • treatment or “treating” as used herein with reference to a disease refer both to prophylaxis and to the amelioration of symptoms already present in an individual. It will be appreciated by the person of ordinary skill in the art that a treatment need not be completely effective in preventing the onset of a disease or in reducing the symptoms associated with the disease. Any reduction of the severity of symptoms, delay in the onset of symptoms, or delay in the progression of severity of symptoms is desirable to a patient. Persons at risk of developing a given immune system-mediated disease may be treated prophylactically based on any of a variety of factors suggesting the possible onset of an immune system-mediated disease, e.g. , family history, genetic markers, early symptoms, and the like.
  • Immune system-mediated diseases that may be treated by the subject methods include, but are not limited to, the diseases listed in Table 1.
  • the subject methods of the invention comprise the step of administering an effective amount of a composition of the invention, e.g., collagens, collagen derivatives, or mucosa binding collagen conjugates.
  • a composition of the invention e.g., collagens, collagen derivatives, or mucosa binding collagen conjugates.
  • Preferred compositions for use in treating specific immune system-mediated diseases are provided in Table 1, as described in the preceding sections.
  • the compositions administered to the subject comprise variably glycosylated collagens or mucosa binding collagen conjugates, wherein the collagen component comprises variably glycosylated collagen molecules.
  • the compositions administered in the subject methods are admimstered so that the active components, i.e.
  • collagens and/or collagen derivatives contact the lymphoid tissue of the gut, e.g. , Peyer's patches or other similar sites, so that immune tolerance is induced.
  • Such administration may be effected, by among many possible methods, through the use of formulations comprising the subjected compositions that are designed for oral administration, i.e. , the active components are not destroyed or inactivated in the mouth, stomach, or other portions of the digestive system prior to contacting the appropriate gut lymphoid tissue.
  • the treatment methods of the invention may also comprise the steps of administering additional pharmaceutical compounds for the treatment of immune system-mediated diseases, such as anti-inflammatory agents and the like.
  • the dosage at which the subject compositions are administered may vary within a wide range and will depend on various factors such as for example the severity of the inflammation, the age of the patient, etc., and may have to be individually adjusted.
  • the amount of collagen(s) and/or collagen(s) derivatives which may be administered per day may be in the range of from about 0.001 mg to about 200 mg.
  • the amount of collagen and/or collagen derivatives administered is low, thereby favoring the induction of immune tolerance by suppression rather than clonal anergy.
  • the pharmaceutical compositions containing the collagen(s) and/or collagen(s) derivatives may suitably be formulated so that they provide doses within these ranges either as single dosage units or as multiple dosage units.
  • the optimal dosage of tolerance inducing compositions for use in the methods of the invention will vary in accordance with a number of factors.
  • the terms "dosage” and “dose” as used herein, unless indicated otherwise, may refer not only to a single administration of a composition but may be used to refer to the total amount of a given pharmaceutical composition administered over a selected period of time and involving multiple individual administrations.
  • Factors affecting the optimal dosage include the choice of collagen molecule or molecules (and/or collagen derivatives) administered to the patient, the specific mucosa binding molecules selected, the age of the patient, the severity of the disease, other diseases that may be present in the patient, inert components in the formulation, adjuvants, and the like.
  • Oral tolerance may be mediated by active cellular suppression in which regulatory T cells that suppress the activation and proliferation of lymphocytes specific for tolerized antigen. Another mechanism of oral tolerance induction is clonal anergy in which T lymphocytes having a suitable receptor are rendered unresponsive.
  • compositions may be formulated as pharmaceutical compositions so as to be adapted for certain types of admimstration to mucosal surfaces, e.g. , oral, topical, and inhalation.
  • the preferred form of formulation for oral administration in a form where the collagen and/or collagen derivatives in the composition come into contact with intestinal lymphoid tissue, e.g. , Peyer's patches.
  • compositions of the invention may be administered topically, orally, intranasally, by injection or by inhalation in the form of a pharmaceutical compositions comprising a collagen(s) and/or collagen(s) derivatives in the form of the original compound or optionally in the form of a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier which may be a solid, semi-solid or liquid diluent or an ingestible capsule, and such preparations comprise a further aspect of the invention.
  • a pharmaceutically acceptable carrier which may be a solid, semi-solid or liquid diluent or an ingestible capsule, and such preparations comprise a further aspect of the invention.
  • the collagen(s) and/or collagen(s) derivatives and mucosa binding collagen conjugates may also be used with carrier material.
  • the collagen(s) and/or collagen(s) derivatives will comprise between 0.05 and 99%, or between 0J and 99% by weight of the preparation, for example between 0.5 and 20% for preparations intended for injection and between 0J and 50% for preparations intended for oral administration.
  • the active ingredient may be mixed with a solid, pulverulent carrier, for example lactose, saccharose, sorbitol, mannitol, a starch such as potato starch, corn starch, amylopectin, laminaria powder or citrus pulp powder, a cellulose derivative or gelatine and also may include lubricants such as magnesium or calcium stearate or a Carbowax ® or other polyethylene glycol waxes and are compressed to form tablets or cores for dragees.
  • a solid, pulverulent carrier for example lactose, saccharose, sorbitol, mannitol, a starch such as potato starch, corn starch, amylopectin, laminaria powder or citrus pulp powder, a cellulose derivative or gelatine and also may include lubricants such as magnesium or calcium stearate or a Carbowax ® or other polyethylene glycol waxes and are compressed to form tablets or cores for dragees.
  • the cores may be coated, for example, with concentrated sugar solutions which may contain gum arabic, talc and/or titanium dioxide, or alternatively with a film forming agent dissolved in easily volatile organic solvents or mixtures of organic solvents.
  • Dyestuffs can be added to these coatings, for example, to distinguish between different contents of active substance.
  • the active substance may be admixed with a Carbowax ® or a suitable oil as e.g. sesame oil, olive oil, or arachis oil.
  • Hard gelatine capsules may contain granulates of the active substance with solid, pulverulent carriers such as lactose, saccharose, sorbitol, mannitol, starches (for example) potato starch, corn starch or amylopectin), cellulose derivatives or gelatine, and may also include magnesium stearate or stearic acid as lubricants.
  • solid, pulverulent carriers such as lactose, saccharose, sorbitol, mannitol, starches (for example) potato starch, corn starch or amylopectin), cellulose derivatives or gelatine, and may also include magnesium stearate or stearic acid as lubricants.
  • compositions of the invention may also be formulated so as to provide a sustained release.
  • sustained release tablets By using several layers of the active drug, separated by slowly dissolving coatings sustained release tablets may be obtained.
  • Another way of preparing sustained release tablets is to divide the dose of the active drug into granules with coatings of different thicknesses and compress the granules into tablets together with the carrier substance.
  • the collagen(s) and/or collagen(s) derivatives and mucosa binding collagen conjugates may also be incorporated in slowly dissolving tablets made, for instance, of fat and wax substances or evenly distributed in a tablet of an insoluble substance such as a physiologically inert plastic substance.
  • the tablets, dragees etc. may be enteric-coated, that is provided with a layer of gastric juice-resistant enteric film or coating having such properties that it is not dissolved at the acidic pH in the gastric juice.
  • enteric coatings may be mentioned cellulose acetate phthalate, hydroxypropyl-methylcellulose phthalates such as those sold under the trade names HP 55 and HP 50, and Edragit ® L and Eudragit ® S.
  • Liquid preparations for oral application may be in the form of elixirs, syrups or suspensions, for example solutions containing from about 0.1 % to 20% by weight of active substance, sugar and a mixture or ethanol, water glycerol, propylene glycol and optionally aroma, saccharine and/or carboxymethylcellulose as a dispersing agent.
  • purified as used herein in reference to collagens denotes that the indicated molecules are present in the substantial absence of other biological macromole- cules, e.g. , polynucleotides, proteins, and the like.
  • purified as used herein preferably means at least 95 % by weight, more preferably at least 99.8% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons can be present).
  • isolated refers to a protein molecule separated not only from other proteins that are present in the natural source of the protein, but also from other proteins, and preferably refers to a protein found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same.
  • isolated and purified do not encompass proteins present in their natural source.
  • Groups of 10 DBA/lmice were immunized with either human type II collagen (CII), mammalian cell derived recombinant type II collagen (rCII-f ), or insect cell derived recombinant type II collagen (rCII-).
  • CII human type II collagen
  • rCII-f mammalian cell derived recombinant type II collagen
  • rCII-f insect cell derived recombinant type II collagen
  • the collagen was dissolved in 0.01M acetic acid and then emulsized in a 1 to 1 ration with Complete Freud's Adjuvent prior to immunization.
  • Each mouse received 100 ⁇ g of protein subcutaneously in the tail.
  • Two groups received two doses of 100 ⁇ g rCII+ or rCII-, thereby receiving in total 200 ⁇ g of rCII + or rCII-.
  • the first 100 ⁇ g dose of protein was administered as described above.
  • the second 100 ⁇ g dose of protein was emulsified in incomplete Freud's adjuvent and administered three weeks after admimstration of the first dose.
  • antibodies represent the mean levels/group (expressed in units) against native human type II collagen sera collected four weeks after immunization.
  • Ovalalbumin, human type II collagen (CII), mammalian cell derived recombinant type II collagen (rCII+), or insect cell derived recombinant type II collagen (rCII-) were admimstered intravenously to groups of 10 DBA/1 mice.
  • the collagens (CII, rCII+ and rCII-) were dissolved in 0.01M acetic acid and dialyzed against PBS prior to administration. Either 33 ⁇ g or 33 ⁇ g of protein was administered daily for three days such that a total of either 100 ⁇ g or 1000 ⁇ g of protein was administered to each test mouse.
  • Four days after administration of the last dose the mice were then immunized with CII.
  • the incidence of arthritis at six (6) weeks after immunization is set forth at Table 3. TABLE 3
  • OVA (1000 ⁇ g) 9/10 100.8 ⁇ 40 rCII + (1000 ⁇ g) 0/10 120 ⁇ 5.3 p ⁇ 0.0005 rCII + (100 ⁇ g) 0/10 14.3 ⁇ 6 p ⁇ 0.0005 rC ⁇ - (1000 ⁇ g) 0/10 14.9 ⁇ 7 p ⁇ 0.0005 rC ⁇ - (100 ⁇ g) 0/10 8.5 ⁇ 4 p ⁇ 0.0005 c ⁇ (looo ⁇ g) 0/10 5.5 ⁇ 1 p ⁇ 0.0005
  • Antibodies represent the mean levels/group (expressed in units) against native human type II collagen using sera collected four (4) weeks after immunization. The statistics are reported using Student's T test.
  • mice Groups of ten to twelve DBA/1 mice were orally administered either
  • Ovalbumin human type II collagen (CII), mammalian cell derived recombinant type II collagen (rCII+), or insect cell derived recombinant type II collagen (rCII-).
  • the collagens were dissolved in 0.01M acetic acid and administered four (4) times per week for two weeks for a total of eight doses. Either 10 ⁇ g or 100 ⁇ g was administered daily so that mice received a total of either 80 ⁇ g or 800 ⁇ g of protein. Three (3) days after receipt of the last dose, the mice were then immunized with CII. Table 4 provides the incidence of arthritis at five weeks after immunization.
  • Antibodies represent the mean levels per group (expressed in units) against native human type II collagen using sera collected four weeks after immunization and statistics are reported using Student's T test.
  • Type III recombinant collagen and human types I and III collagen were tested in blood samples from four donors and tested, using routine platelet aggregation assays. All types of collagen demonstrated platelet aggregation activity. More specifically, the percentage of aggregation of 10 ⁇ g of recombinant type III collagen is equivalent to the percent aggregation of either 0.3 ⁇ g of type III or 0.6 ⁇ g of type I collagen.

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US5856446A (en) * 1995-07-07 1999-01-05 Autoimmune Inc. Method of treating rheumatoid arthritis with low dose type II collagen
WO1998021951A1 (en) * 1996-11-18 1998-05-28 Emisphere Technologies, Inc. Methods and compositions for inducing oral tolerance in mammals
US7097845B2 (en) 1997-04-23 2006-08-29 Jacob Sten Petersen Combinations of antigen and mucosal binding component for inducing specific immunological tolerance
DE19804210A1 (de) * 1998-02-03 1999-08-12 Biosyn Arzneimittel Gmbh Rekombinante Mistellektine
MXPA01001510A (es) * 1998-08-10 2003-08-20 Fibrogen Inc Composiciones hemostaticas de colageno tipo i y tipo iii para usarse como sellador vascular y vendaje para heridas.
KR102577697B1 (ko) * 2019-11-13 2023-09-14 주식회사 나이벡 염증 및 상처 치료용 펩타이드
BR112022011557A2 (pt) * 2019-12-12 2022-08-30 Ineb Inst De Engenharia Biomedica Material fetal descelularizado do núcleo pulposo e métodos para a obtenção de composições farmacêuticas a serem utilizadas no tratamento da degeneração do disco intervertebral e dor nas costas
DE102021214899A1 (de) * 2021-12-22 2023-06-22 Gelita Ag Typ I-Kollagenpeptid zur therapeutischen Verwendung
JP7357189B1 (ja) * 2022-07-14 2023-10-06 株式会社リナイス 魚類軟骨由来のコラーゲン含有組成物

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