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WO1993007291A1 - Glycanes associes a l'immunoglobuline d et leurs utilisations - Google Patents

Glycanes associes a l'immunoglobuline d et leurs utilisations Download PDF

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Publication number
WO1993007291A1
WO1993007291A1 PCT/US1992/008724 US9208724W WO9307291A1 WO 1993007291 A1 WO1993007291 A1 WO 1993007291A1 US 9208724 W US9208724 W US 9208724W WO 9307291 A1 WO9307291 A1 WO 9307291A1
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Prior art keywords
igd
glycan
subject
linkage
immune response
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Gertrude Jeanette Thorbecke
Ashok R. Amin
Joel D. Oppenheim
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New York University NYU
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New York University NYU
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55583—Polysaccharides

Definitions

  • the invention in the fields of immunology, biochemistry and medicine relates to glycans associated with IgD and their use in the stimulation of IgD receptor upregulation, in the augmentation or inhibition of immune responses and in the treatment of immune-mediated diseases.
  • Immunoglobulin D exists in both membrane-associated (surface IgD or sIgD) form and in secreted form. While the biological function of serum IgD, present at minute concentrations, is unknown, the membrane-bound form appears to play a role in a number of immunoregulatory processes (discussed below).
  • the ⁇ heavy chain of human IgD has four domains: V ⁇ , C ⁇ 1, C ⁇ 2 and C ⁇ 3. An exceptionally long hinge region is located between C ⁇ l and C ⁇ 2.
  • human IgD is a glycoprotein and contains carbohydrate in its heavy chain (Spiegelberg, H. et al., Biochemistry 9:2115-2122 (1970)).
  • the human ⁇ chain contains asparagine-linked oligosaccharides at residue 354 in C ⁇ 1 and at 445 and 496 in C ⁇ 3. There are also multiple 0-linked oligosaccharides attached to serine and threonine residues in the hinge region (Takahashi, N. et al., Proc. Natl. Acad. Sci. USA 79:2850-2854 (1982)).
  • the structures of the oligosaccharides at the three asparagine-linked glycosyl- ation sites (Mellis, S.J. et al., J. Biol. Chem. 258:11546- 11556 (1983)) and the O-glycosylation sites (Mellis, S.J.
  • Murine IgD also has a high content of N-linked glycans (Vasilov, R.G. et al., Eur. J. Immunol. 12:804-818 (1982); Argon, Y. et al., J. Immunol. 133:1627-1633 (1984)).
  • N-linked glycans Vasilov, R.G. et al., Eur. J. Immunol. 12:804-818 (1982); Argon, Y. et al., J. Immunol. 133:1627-1633 (1984)
  • IgD is expressed on the majority of mature B lympho-cytes (Rowe, D.S. et al., J. Exp. Med. 138:965-972 (1973)), although its role in the humoral immune response is still unclear.
  • the present inventors' laboratory has recently shown the presence of receptors for IgD (IgD-R) on T cells from mice harboring IgD-secreting plasmacytomas (TEPC-1017 or TEPC-1033) or in mice injected with purified IgD produced by these plasmacytomas.
  • IgD-R receptors for IgD
  • mice harboring IgD-secreting plasmacytomas TEPC-1017 or TEPC-1033
  • mice exhibit significantly enhanced antibody responses of all antibody isotypes except IgD (Xue, B. et al., J. EXP. Med. 159:103-113 (1984), Swenson, CD.
  • Such an augmented antibody response can be transferred from IgD- treated mice to normal mice by transfer of CD4 + , Lyt-1 + , CD8-T cells (Coico, R.F. et al., J. Exp. Med. 162:1852-1861 (1985)).
  • CD4 + , Lyt-1 + , CD8-T cells Coico, R.F. et al., J. Exp. Med. 162:1852-1861 (1985)
  • T cells that also express IgD-R on their surface, measured by their capacity to form rosettes with IgD-coated sheep erythrocytes (IgD-SRBC).
  • IgD-SRBC IgD-SRBC
  • T ⁇ cells augment B cell responses. Soluble IgD-binding factors released by T ⁇ cells may contribute to their immunoaugmenting properties (Adachi, M. et al., Proc. Natl. Acad. Sci. (USA) 85:554-558 (1988)). T ⁇ cells appear to interact more efficiently with slgD-bearing B cells subsequent to antigen-induced cross-linking of slgD molecules. This notion is based on observations from the present inventors' laboratory that (1) both primary and secondary antibody responses are augmented by injection of IgD prior to the first injection of antigen (Xue, B. et al., J. EXP. Med.
  • B cells with cross-linked slgD induce upregulation of IgD-R on T cells in vivo and in vitro (see below).
  • Cross-linking of slgD with the C ⁇ 3 specific monoclonal antibody H ⁇ a /1 causes such IgD-R upregulation. Since this antibody could sterically hinder interaction between IgD-R and the C ⁇ 3 domain of IgD, a role for C ⁇ 1 in this IgD-R upregulation is likely.
  • Fc receptors for other immunoglobulin isocypes (IgG, IgM, IgA and IgE) are present on cells of the lymphohemopoietic system such as macrophages, granulocytes and lymphocytes. Some FcR's trigger cellular functions such as phagocytosis, antibody-dependent cytotoxicity, and the secretion of potent mediators (Metzger, H. et al., FASEB J. 2:3-8 (1988); Kinet, P.E. Cell 57:351-353 (1989)). The Fc -R principally recognizes the C 2-hinge region of IgG (Revetch, J.V. et al.. in Metzger. H.
  • FceRI recognizes residues 301-376 roughly centered in the interface between C ⁇ 2 and C ⁇ 3 domains of IgE
  • the Fc ⁇ R recognizes the C ⁇ 3 region of IgM (Mathur, A. et al., J. Immunol. 140:143-147 (1988), Mathur, A. et al., J. Immunol. 141:1855-1862 (1988)).
  • IgD-R of T ⁇ cells for immunoglobulin of the IgD class was established by showing that IgD, at concentrations >120 ⁇ g/ml (1.0 ⁇ M), competitively inhibited the rosetting of T ⁇ cells with IgD- SRBC, whereas IgM, IgG1, IgG2, IgG3, IgA, and IgE failed to inhibit this interaction (Coico, R.F. et al., Nature 316:744-746 (1985), Coico, R.F. et al., Immunol. Rev. 105:45-67 (1988)).
  • T cells Exposure of T cells either in vitro or in vivo to either (a) oligomeric secreted IgD molecules (TEPC-1017 or TEPC-1033), (b) antigen-crosslinked monomeric secreted IgD (such as B1-8. ⁇ 1, a monoclonal antibody of IgD isotype) or (c) B-cell surface IgD which is crosslinked (by either anti-IgD or anti-Ig), results in upregulation of their IgD-R (Coico, R.F. et al., Proc. Natl. Acad. Sci. (USA) 11:559-563 (1988)). B cells with cross-linked sIgM do not induce IgD-R upregulation.
  • oligomeric secreted IgD molecules such as B1-8. ⁇ 1, a monoclonal antibody of IgD isotype
  • B-cell surface IgD which is crosslinked (by either anti-IgD or anti-Ig), results in upregulation of their I
  • the cytokines IL-2, IL-4 and IFN- known to participate in the immune response and in host defenses, also induce IgD-R upregulation on CD4 + T lymphocytes (including cloned T cells) (Coico, R.F. et al., Immunol. Rev. 105:45-67 (1988), Coico, R.F. et al., J. Immunol. 138:4-6 (1987), Amin, A.R. et al., Proc. Natl . Acad. Sci. (USA) 85:9179-9183 (1988)).
  • IgD-R on T-helper cells are not exclusively Fc receptors, but also bind equally well to the Fd piece of IgD, which consists of the C ⁇ 1 and the C ⁇ hinge domains. This domain specificity could not be explained by the amino acid sequence, as these domains have only 26% homology (Tucker, P.W. et al., Ann. NY Acad. Sci. 399:26-40 (1982)).
  • mutant IgD molecules containing either only the C ⁇ 1 or only the C ⁇ 3 domain, each bind to the lectin
  • Griffonia simplicifolia-1 This lectin is known to bind specifically to N-linked glycans from murine IgD
  • the present inventors discovered that the binding of IgD to the IgD-R is blocked by several preparations, including:
  • the present invention provides a method for inhibiting the binding of IgD with an IgD receptor comprising providing to a cell having an IgD receptor an effective amount of an IgD-associated glycan.
  • the invention provides a method for inhibiting an immune response involving the interaction of T and B lymphocytes in a subject, comprising administering to the subject an effective amount of an IgD-associated glycan, thereby inhibiting the response.
  • Also provided is a method for treating a subject with an immune-mediated disease comprising administering to the subject an effective amount of an IgD-associated glycan, thereby treating the disease.
  • the diseases for which this method is useful include autoimmune diseases in which antibody formation is enhanced such as systemic lupus erythematosus, rheumatoid arthritis, thyroiditis and the like.
  • the present invention is further directed to a method for increasing the number of IgD receptors on the surface of a T lymphocyte having such receptors, comprising contacting the cell with an effective amount of an IgD-associated glycan, thereby increasing the number of the receptors.
  • the present invention includes a method for inhibiting an immune response in a subject, comprising:
  • lymphocytes including CD8+ T lymphocytes from the subject obtained from the subject;
  • the above method is used for enhancing an immune response in a subject, the method comprising:
  • lymphocytes including CD4+ T lymphocytes from the subject obtained from the subject;
  • the invention provides a method for enhancing an immune response in a subject being immunized with an antigen or vaccine, comprising administering to the subject, in combination with the. antigen or vaccine, an effective amount of an IgD-associated glycan.
  • the glycan is preferably in non-polymerized or non-aggregated form.
  • the glycan is preferably in polymerized or aggregated form, or is a neoglycan.
  • a neoglycan comprising two or more of the same or different IgD-associated glycan monomers linked to a polymer.
  • a neoglycan may be a neoglycoprotein, wherein the polymer is a protein.
  • the protein is serum albumin.
  • the glycan is preferably one selected from the group consisting of:
  • R 1 is NeuAc in an ⁇ 2 ⁇ 3 linkage or H
  • R 2 is NeuAc in an ⁇ 2 ⁇ 6 linkage or H
  • R 3 is GlcNAc in a ⁇ 1 ⁇ 4 linkage or H
  • R 4 is Fuc in an ⁇ 1 ⁇ 6 linkage or H and R 5 is Glc in an a1 ⁇ 3 linkage or H.
  • the invention is further directed to pharmaceutical compositions comprising the glycans and neoglycans described above and to the use of the pharmaceutical compositions in the methods described above.
  • Figure 1 is a schematic representation of the constant region of the ⁇ chain of human IgD (WAH) (Mellis et al., supra). Essential structural features of the heavy chain constant region are depicted with emphasis on oligosaccharide structure and localization (after F.W. Putnam et al., Ann. N.Y. Acad. Sci. 399:41-68 (1982)).
  • the GalNAc-rich segment of the hinge is shown in an expanded view. Symbols (+) and (-) represent the highly charged segment of the hinge which is extremely sensitive to proteolytic degradation. Microheterogeneity of N- and O-glycosidically linked oligosaccharides is reflected by the presence or absence ( ⁇ ) of terminal sugar residues.
  • Asn 354, 445 and 496 refer to locations of the N-glycosidically linked oligosaccharides. These residue numbers were calculated beginning at the N-terminus of the ⁇ chain (SEQ ID NO:4). Their positions with respect to the first residue of the constant region is also indicated schematically below.
  • TP refers to the tail piece of the secreted form of IgD. Relative sizes of oligosaccharide units and protein domains are not drawn to scale.
  • Figure 2 shows the structure of the triantennary high mannose oligosaccharides found linked to Asn354 of human IgD (WAH) (Mellis et al., supra), and indicates the abundance of various species at this site.
  • Figure 3 shows the structure of the dibranched complex and bisected dibranched complex oligosaccharides found linked to Asn445 of human IgD (WAH) (Mellis et al., supra), and indicates the abundance of various species at this site.
  • Figure 4 shows the structure of the dibranched complex and bisected dibranched complex oligosaccharides found linked to Asn496 of human IgD (WAH) (Mellis et al., supra), and indicates the abundance of various species at this site.
  • Figure 5 is a graph showing cross-inhibition of rosette formation by Gen.24 and KWD6 IgD.
  • Indicator SRBC were coupled to mutant myeloma protein Gen.24 and mutant hybridoma IgD, KWD6.
  • Figure 6 shows gel patterns of SDS-PAGE analysis of affinity-purified, naturally-degraded TEPC-1017 IgD fragments. Partially degraded IgD fragments were subjected to SDS-PAGE (7%) under non-reducing conditions (lanes A-C). Lane A: naturally degraded TEPC-1017 IgD molecules showing fragment sizes of 130, 100, 70 and 66 kDa.
  • Lane B 4M MgCl 2 eluate of anti-Fc ⁇ (H ⁇ a /1)-adherent TEPC-1017 IgD fragments showing fragment sizes of 130 and 100 kDa.
  • Lane C H ⁇ a /1non-adherent Fab ⁇ fragments showing molecular weights of 70 and 66 kDa.
  • Lane D intact TEPC-1017 IgD molecules, reducing conditions, showing heavy (54 kDa) and light (25 kDa) chain molecules.
  • Lane E same as C, but under reducing conditions.
  • Lanes F and G 9% SDS-PAGE of Fab ⁇ fragments, Western blotted and probed with anti-Fab (Lane F) and anti-IgD (Lane G). Positions of molecular weight markers given in kDa.
  • Figure 7 is a graph showing the induction of murine T cell IgD-R by aggregated mutant IgD molecules or IgD fragments.
  • Figure 8 is a graph (A) and dot blot (B) showing the inability of deglycosylated IgD to inhibit IgD-rosetting by induced splenic T cells.
  • DG-IgD IgD lacking N-linked glycans
  • IgD per assay respectively.
  • One ⁇ g of IgD, DG-IgD or IgG was dot blotted onto nitrocellulose paper in sets of three (Panel B).
  • Set 1 was stained with Coomassie blue R-250.
  • Set 2 was reacted with phosphatase-conjugated sheep anti-mouse IgD (Pel-Freeze).
  • Set 3 was reacted with peroxidase
  • Figure 9 is a graph showing the carbohydrate specificity of IgD-R.
  • BALB/c splenic T cells induced to express IgD-R with IL-4 (10 U/ml) were treated with various carbohydrates as blocking agents at the doses indicated.
  • the % Inhibition was calculated as described in Example I.
  • IL-4- induced T ⁇ cells rosetted with IgD-coated RBC in the absence of any blocking agent had a mean IgD-RFC value of 25 ⁇ 2% Results are the mean ⁇ standard error of three determinations.
  • Figure 10 is a graph showing competitive inhibition of human IgD-rosetting by low molecular weight IgD fragments.
  • Human PBL were isolated from blood, and incubated with plastic dishes coated with cross-linked human IgD to upregulate human IgD-R. These cells were then rosetted with IgD-coated Ox-RBC in the presence or absence of various concentrations of potential inhibitors consisting of proteinase K (PK) digested IgD or IgG.
  • PK proteinase K
  • Figure 11 is a gel pattern from a 4-20 % SDS PAGE indicating the total digestion of human IgG and IgD by Proteinase K.
  • Five mg samples of human IgD (160 kD) or IgG (150 kD) purified from myeloma serum were each digested with 2 mg of proteinase K for 12 hrs prior to analysis. Note that the immunoglobulins have been completely digested into low molecular weight ( ⁇ 14 kDa) fractions.
  • Figure 12 is a graph showing that heat-denatured IgD inhibits binding to IgD-R.
  • Human PBL were first incubated with cross-linked human IgD to upregulate IgD-R, and then incubated with 50 or 100 ⁇ g control or heat-denatured IgD
  • the present invention is based on the present inven ⁇ tors' unexpected discovery that, among cellular receptors for immunoglobulin, most of which are classical Fc receptors (because they bind the Fc portion of the appropriate immunoglobulin chain), the receptor for IgD (IgD-R) is unique. This uniqueness is characterized by the absolute requirement for the presence of carbohydrate on the IgD molecule for binding to the IgD-R.
  • a ligand likely the true physiological ligand, for the IgD-R is a glycan structure.
  • the present inventors have conceived of methods for using glycans to modulate immune responses.
  • glycocan means an unsubsti ⁇ tuted, although possibly branched, carbohydrate, usually a polysaccharide, which represents the carbohydrate portion of a glycoside after it is separated (e.g., by hydrolysis) from the non-carbohydrate portion of the molecule.
  • IgD-associated glycan any glycan structure found linked to an IgD molecule, as well as any structural variant or chemical derivative thereof capable of binding to an IgD-R and disrupting the binding of the IgD-R to IgD (or an appropriate IgD-like ligand).
  • IgD-associated glycan any glycan structure found linked to an IgD molecule, as well as any structural variant or chemical derivative thereof capable of binding to an IgD-R and disrupting the binding of the IgD-R to IgD (or an appropriate IgD-like ligand).
  • a “neoglycan” is a non-natural glycan-containing structure, such as, for example, a protein, polyamino acid, or other polymer to which one or more glycan monomers are linked.
  • a preferred neoglycan is a "neoglycoprotein," which comprises a protein to which is linked one or more glycan monomers not naturally linked to the protein.
  • the neoglycan may comprise a non-protein polymer backbone.
  • liposomes (described below) into which the glycans or neoglycoproteins of interest are incorporated.
  • a preferred neoglycoprotein is a serum albumin to which are attached two or more IgD-associated glycan monomers.
  • Glycoproteins of which IgD is an example, are polymers which have one or more carbohydrate chains attached to a polypeptide. Glycoproteins may contain from 4% (by weight) to more than 60% carbohydrate.
  • the carbohydrate chains may be "oligosaccharides” having 2-10 carbohydrate residues or short “polysaccharides,” usually having 10-25 residues, although some are as large as 150 residues. However, it is common in the art to term chains of 10-25 carbohydrate residues on glycoproteins as “oligosaccharides” rather than "polysaccharides” (Schachter, H. Clin. Biochem. 17, 3-14 (1984)). If the polypeptide chain of a glycoprotein is degraded to an oligopeptide, for example, by proteolytic digestion or other means, the residual molecule with carbohydrate still attached is called a "glycopeptide.”
  • the linkage between the oligosaccharide and the protein is a glycosidic linkage, the result of a condensation (dehydration) reaction between an amino acid side chain on the protein and the anomeric carbon on the first residue of the oligosaccharide.
  • carbohydrate linkages to protein are either N-glycosides (carbohydrate linked to the amido nitrogen of asparagine) or O-glycosides (carbohydrate linked to the hydroxyl oxygen of serine, threonine or, rarely, hydroxy- lysine).
  • N-glycosides are more commonly found in mammalian glycoproteins than O-glycosides, but a single glycoprotein may have multiple chains, some of which are O-glycosides and some of which are N-glycosides.
  • the carbohydrates in O-glycosides are usually short
  • N-glycosides are generally somewhat longer (7-25 residues) than in O-glycosides.
  • the neutral sugar monomers (monosaccharides) in a polymer will usually be represented by a three letter abbreviation, e.g., Glc, glucose; Gal, galactose, etc. (see list below). For clarity, the common configuration and ring size of the sugar is implied in the abbreviation. Thus, Glu is D-glucopyranose (6-membered ring), Fuc is L-fucopyranose, etc.
  • Acidic or basic groups on a monosaccharide will be represented by additional letters appended to the three-letter abbreviation above, e.g., GlcNAc, N-acetylglucosamine, or 5-N-acetylneuraminic acid, abbreviated NeuAc.
  • the bond between two monosaccharides will be represented by a 4-character group which includes the anomeric configuration of the sugar on the left (either a or ⁇ ), the carbon number of the anomeric carbon in the bond (sugar on the left), an arrow pointed toward the hydroxyl carbon in the bond, and the carbon number of the hydroxyl carbon in the bond (sugar on the right).
  • Man ⁇ 1 ⁇ 4GlcNAc represents a bond between C-1 of ⁇ -D-mannose and C-4 of N-acetylglucosamine.
  • References to specific linkages may be written in the same manner, e.g., a ( ⁇ 1 ⁇ 3) -linkage, or simplified ( ⁇ 1,3- specific).
  • Man ⁇ 1 ⁇ 3 (Man ⁇ 1 ⁇ 6)Man ⁇ 1 ⁇ 4GlcNAc ⁇ 1 ⁇ 4GlcNAc-Asn which can also be represented as:
  • oligosaccharides occur in the non-reducing end of the molecule (left end as written above), i.e., in the sugars attached to the ⁇ 1,3-and ⁇ 1,6-linked mannose residues of the core.
  • Three major types of asparagine-linked oligosaccharides are found: high-mannose, complex and hybrid oligosaccharides. I n high-mannose oligosaccharides, all the residues attached to the core are mannose residues, with the total number being 5-9 (including the three in the core). These additional mannoses can have branched structures relative to the core mannoses, resulting in an "antenna-like" structure with 2 to 4 branches.
  • the oligosaccharide is a complex oligosaccharide.
  • each of these GN-initiated branches from the core is termed an antenna.
  • antienna is usually applied only to the branches of a hybrid or complex structure, as opposed to high-mannose structures, even if they are branched.
  • each core mannose can have several N-acetylglucosamine residues attached to it, there can be more than 2 antennae on complex oligosaccharides and the resulting structures are termed biantennary (2 GN-antennae), triantennary (3 GN-antennae), tetraantennary (4 GN-antennae), etc.
  • N-linked saccharides described for human IgD have biantennary (Asn496 and Asn 445) or triantennary (Asn354) structures (see Figures 1-4) (Table III of Mellis et al., J. Biol. Chem. 258:11546-11556 (1983)).
  • the third major type of asparagine-linked oligosac-charide is a hybrid type composed of elements of the other two types.
  • the ⁇ 1, 6-linked core mannose has only mannose residues attached to it (i.e., like a high-mannose structure), while the ⁇ 1,3-linked core mannose has one or more GN-initiated antennae attached to it (like a complex structure).
  • the mannose-containing arm of the hybrid structure is termed a single antenna, even if it has branch points within it.
  • N-acetylglucosamine residue is linked to the ⁇ 1 ,4-linked core mannose (the innermost mannose). Structures which contain this residue are said to be “bisected", since the residue comes between the branches formed by the remainder of the structure.
  • the bisecting N-acetylglucosamine residue contains no substituents and, in fact, limits the addition of further residues to the structure during oligosaccharide synthesis. Examples of a bisected complex structure, which is found on Asn496 of human IgD, is:
  • asparagine residues within a single IgD chain may contain an oligosaccharide chain and all chains on a single IgD are not necessarily of the same type or sequence, as is shown in Mellis et al. (supra).
  • IgD-associated N-linked glycans useful according to the present invention are shown in Figures 1-4 as those structures associated with Asn354, Asn445 and Asn496 of the human IgD as defined in the WAH myeloma protein (Mellis et al., supra).
  • the IgD-associated O-linked glycans useful according to the present invention are shown in Figure 1, associated with Ser109 of the C ⁇ 1 region, and Thr126, Thr127, Thr131 and Thr132 of the hinge region.
  • the O-linked glycan may include sialic acid (NeuAc) linked in an ⁇ 2,3 linkage to the Gal or in an ⁇ .2,6 linkage to the GalNAc.
  • the glycans of the present invention may be in either monomeric form, wherein they are non-polymerized and non-aggregated. Alternatively, the glycans of the present invention may be polymerized or aggregated.
  • a polymerized glycan preferably comprises one or more monomeric units attached to a carrier molecule or material. More than one glycan monomer linked to a carrier molecule is referred to as a "neoglycan" (described above).
  • carriers include proteins, such as serum albumin, or synthetic molecules such as an appropriate polyamino acid, or another polymer which will permit the glycans to maintain their structure in a manner that will enable them to bind to the IgD-R in accordance to the present invention.
  • the carrier when used for in vivo administration, is one that is itself non-immunogenic in the species to which the glycan is being administered.
  • a preferred carrier protein is human serum albumin.
  • the glycan may be attached to the protein using any of a number of means, using well-known chemical reactions (Kieda, C. et al., FEBS Lett. 99:29-332 (1979); Monsigny, M. et al., Biol. Cell. 51:187-196 (1984)).
  • a preferred glycan according to the present invention is an IgD-associated glycan, described for human or murine IgD in Mellis et al., supra). and depicted in Figures 1-4.
  • the most preferred glycans according to the present invention are the biantennary complex structures shown m Figure 1, 3 and 4.
  • the glycan or glycan polymer is incubated with T cells having IgD-R and with appropriate indicator particles, preferably erythrocytes, coated with the IgD of the appropriate species.
  • appropriate indicator particles preferably erythrocytes coated with human IgD
  • ox erythrocytes coated with human IgD are preferred.
  • sheep erythrocytes coated with murine IgD are preferred.
  • Inhibition of rosette formation by the glycan being tested is an indication that the glycan has the requisite properties for use according to the present invention.
  • one of ordinary skill in the art can determine without undue experimentation whether a particular glycan or glycan polymer has the capacity to interact with the IgD-R.
  • the murine IgD-R as well as the lectin GS-1, recognize only IgD among all murine immunoglobulins, the N-linked glycans associated with the IgD molecule appear to be totally specific for IgD. This specificity is apparently reflected in the functional properties of IgD since it, and no other immunoglobulin, augments antibody production (Coico et al., 1988, supra). There appears to be a species specificity in the binding of IgD to the T cell IgD-R. For example, human IgD does not block rosette formation of mouse T cells with mouse IgD. Similarly, mouse IgD does not block rosette formation of human T cells with human IgD.
  • a chimeric mouse-human IgD with known antigen specificity to the dansyl hapten
  • mouse myeloma cells does not bind to human IgD-R.
  • the IgD-associated glycan should either be derived from the species being treated, or alternatively should have a structure that is efficacious in that species.
  • One of ordinary skill in the art will be able to test a given glycan structure for its utility according to the present invention by using the methods described herein.
  • the IgD-R molecule appears to be a member of a family of cell-surface lectin-like molecules which are becoming recognized in the art. These include the IgE binding receptor known as Fc ⁇ RII or CD23, the ELAM-1 molecule (also termed LECAM-2) and the MEL-14 molecule (also termed LECAM-1) (Ikuta, K. et al., Proc. Natl. Acad. Sci. USA 84:819-823 (1987); Lasky, L.A. et al., Cell 56:1045-1055 (1989); Siegelman, M. et al., Science 241:1165 (1989); Bevilacqua, M.P.
  • the FceRII has a lectin-like domain important for the interaction with IgE, but it differs from the IgD-R as described herein in that it does not predominantly recognize carbohydrate moieties of IgE (Bettler et al., Proc. Natl. Acad. Sci. USA 86:7118-7122 (1989); Vercelli et al., Nature 338:649-651 (1989)).
  • LEO adhesion molecules Some of the carbohydrate ligands of the leukocyte-endothelial cell (LEO adhesion molecules have been identified (Brandlev et al., Cell 63:861-863 (1990); Picker et al.. Cell 66:921-933 (1991)). Transfection of a specific ⁇ (1,3) fucosyl-transferase cDNA into nonmyeloid cell lines results in the de novo expression of functional ligands for LECAM-2 and cell adhesion to this molecule (Lowe, J.B. et al., Cell 63:475-484 (1990)).
  • the pentapeptide GRGDS (SEQ ID NO:1), which is the sequence of fibronectin recognized by integrins (Hynes, R., Cell 48:549-554 (1987)), does not inhibit IgD rosetting.
  • the IgD-R is distinguishable from the GRGDS binding molecule.
  • Gal ⁇ 1 ⁇ 6Glc melibiose
  • Gal ⁇ 1 ⁇ 4Glc lactose
  • the lectin specificity of murine IgD-R resembles that of GS-1. Therefore, according to the present invention, the lectin GS-1 is useful as a reagent in the isolation of murine IgD-associated glycans that inhibit IgD-rosetting and are useful in various embodiments of the present invention.
  • an affinity column comprising GS-1 bound to a solid support can be used to isolate and purify from a complex mixture a glycan, glycoprotein or glycopeptide which binds to the IgD-R and can be used in accordance with the present invention.
  • surface IgD on B cells acts not only as a receptor for antigen, but also serves as a ligand for IgD-R on T helper cells. Because of its lectin-like properties, described herein, the IgD-R on T ⁇ cells is concluded to be an adhesion molecule. This is further supported by the ability of the IgD-R to recognize and bind the C ⁇ 1 region of B-cell surface IgD (Richards, M.L. et al., J. Immunol. 144:2638-2646 (1990); Amin, A.R. et al., Research Immunol. 141:94-99 (1990)).
  • T-B cell interaction central to the generation of an immune response. This interaction provides a mechanism for intracellular signalling in addition to the various secondary signals that may be generated in the T-helper cells via engagement of the IgD-R or in the B cells via engagement of their slgD.
  • IgD-R upregulation involves triggering of the cell in that inhibitors of protein kinase C inhibit the upregulation.
  • Stimulation of T cells by IgD or the glycans of the present invention stimulates the translocation of protein kinase C without a concomitant Ca ++ flux.
  • B cell slgD and the T helper cell IgD-R is the enhanced production of all antibodies of all isotypes except IgD itself (Amin, A. et al., supra). This results in an augmented primary and secondary immune responses.
  • the glycans described herein can be used to disrupt these T cell-B cell interactions, and are thus useful in the prevention or treatment of immune-mediated diseases, such as autoimmune diseases, that involve T cell-B cell interactions in the generation of pathological or otherwise undesired antibodies.
  • An effective amount of an IgD-associated glycan for inhibiting the binding of IgD with an IgD receptor according to the present invention is between about 0.01 and 5 mg/ml. More preferably, the amount is between about 0.1 and 1 mg/ml.
  • the glycan is preferably administered in an monomeric or soluble form, and is preferably given between about one week and about one day prior to immunization with an antigen.
  • An effective amount of an IgD-associated glycan for inhibiting an immune response involving the interaction of T and B lymphocytes in a subject according to the present invention is between about 0.01 and 1000 mg/kg body weight. More preferably, the dose is between about 0.1 and 100 mg/kg.
  • a subject suffering from such a disease is administered an effective amount of an IgD-associated glycan according to the present invention.
  • a dose of between about 0.01 and 100 mg/kg body weight is administered. More preferably, the dose is between about 0.1 and 100 mg/kg.
  • Administration may be over a prolonged period, ranging from several days to several months or years, depending on the nature and severity of the disease.
  • the glycan is preferably administered to such subjects in non-polymerized or non- aggregated form.
  • autoimmune diseases which involve undesired antibody production as an underlying cause or as a consequence of the pathophysiology.
  • diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, multiple sclerosis, autoimmune thyroiditis (Hashimoto's thyroiditis), Graves' disease, inflammatory bowel disease, autoimmune uveoretinitis, polymyositis and certain types of diabetes (see, Theofilopoulos, A., In: D.P. Stites, et al., eds., Basic and Clinical Immunology. Lange Medical Publica- tions, Los Altos, CA, 1988)).
  • human T cells also express receptors for IgD (Coico, R.F. et al., J. Immunol. 145:3556-3561 (1990)).
  • human IgD-R also exhibit lectin-like properties and are specific for glycans associated with human IgD.
  • Human IgD-R on CD4- and CD8-bearing human peripheral blood lymphocytes are upregulated by incubation with IgD, as well as with antibodies specific for CD3, CD4 or CD8.
  • IgD itself, an IgD-R-binding fragment, or most preferably, an IgD-associated glycan, or any other ligand for the IgD-R, may act as a substitute for a CD3, CD4 or CD8 ligand in engaging the T cell and triggering or enhancing a response.
  • An effective amount of an IgD-associated glycan for increasing the number of IgD receptors on the surface of a T lymphocyte having such receptors, according to the present invention is between about 0.001 and 1 mg/ml. More preferably, the amount is between about 0.01 and 0.1 mg/ml.
  • the glycans of the present invention are useful not only in modulating T-B interactions involving T helper cells but are also useful in modulating the action of other types of T cells on B cell responses.
  • the glycans may be used to upregulate the IgD-R on CD8+ cytotoxic T cells and stimulate cytotoxic activity for IgD-bearing target cells.
  • the glycans may be used to disrupt interactions between T helper cell and T effector cell subsets and B cells.
  • T suppressor cells are generally of the CD8+ subclass
  • the glycans of the present invention are also useful in modulating the activity of these cells. For example, stimulation of T suppressor cells by binding to and upregulating the IgD-R may enhance suppressor cell activity and thereby diminish unwanted immune responses, such as those associated with immune-mediated disease.
  • lymphocytes preferably peripheral blood lymphocytes
  • lymphocytes may be removed from a subject, incubated in vitro with an effective amount of an IgD-associated glycan, as described herein, to upregulate the IgD-R.
  • Such cells may then be reintroduced into the subject in order to stimulate the immune system generally, or to enhance an immune response to a particular antigen, such as a vaccine.
  • Unfractionated populations of cells may be used, or, alternatively, isolated populations of T cells or T cell subsets (e.g., CD4+ cells cells) may be prepared, using conventional methods, before or after treatment with the glycan.
  • Conditions for incubating the cells and concentrations of the glycans or neoglycans are those described above and in the Examples for inducing the IgD-R.
  • Upregulation of the immune response is useful in any condition resulting in a weakened immune system, in particular in immunodeficiency disease such as AIDS or congenital immunodeficiency diseases.
  • CD8+ cells commonly have suppressor cell activity and act to inhibit an immune response.
  • an IgD-associated glycan to upregulate the IgD-R, as described above, and administering these cells to a subject, it is possible to inhibit an immune response and to treat an immune-mediated disease such as an autoimmune disease.
  • unfractionated cells or whole T cells may be treated to upregulate the IgD-R, followed by fractionation and administration of only the CD8+ T cell subset.
  • the methods of the present invention may be used to enhance the immune response which becomes deficient in a proportion of aged individuals. For example, about half of aged humans do not upregulate IgD-R as well as younger adults (see Examples below). This measure correlates with total anti-influenza antibody titers in subjects immunized with influenza vaccine. Thus, individuals that did not upregulate their IgD-R had lower antibody titers, and individuals that were low responders to influenza were predictably less able or unable to upregulate their IgD-R in response to an appropriate stimulus. Therefore, according to the present invention, IgD-associated glycans may be used to upregulate the IgD-R and to increase antibody responses. In this manner, they may serve as immunological "adjuvants.”
  • the present invention provides a completely novel approach to modulating the immune response toward either greater or lesser responsiveness. Greater immune responsiveness is desirable in situations in which a subject suffers from an immunodeficiency either congenital or acquired, for example, in AIDS. In contrast, in a number of disease states, the immune system is hyperactive or generates undesired immune responses. This occurs primarily in the case of autoimmune disease, but is also observed in allergy, graft rejection and in graft-versus-host disease.
  • the boosting of an immune response is highly desirable when immunizing or vaccinating an individual with an antigen or vaccine, which will protect that individual from a disease, such as an infectious disease.
  • the glycans of the present invention may therefore be used in combination with an immunizing antigen preparation, such as a vaccine preparation, to provide such an adjuvant effect.
  • an immunizing antigen preparation such as a vaccine preparation
  • the glycan and vaccine may be combined into a single formulation.
  • the vaccine and glycan may be given separately, either on a concurrent or sequential basis.
  • an effective amount of an IgD-associated glycan for enhancing an immune response in a subject being immunized with an antigen or vaccine according to the present invention is between about 0.01 and 1000 mg/kg body weight. More preferably, the amount is between about 0.1 and 100 mg/kg.
  • the glycan is preferably in an aggregated or polymerized form.
  • CD4+ T cells treated with an IgD-associated glycan may be administered to a subject to enhance the immune response.
  • the preferred animal subject of the present invention is a mammal.
  • mammal an individual belonging to the class Mammalia.
  • the invention is particularly useful in the treatment of human subjects.
  • treating is intended the administering to subjects of the IgD-associated glycans of the present invention for purposes which may include prevention, amelioration, or cure of the disease.
  • the present invention is also directed to a pharmaceutical composition
  • a pharmaceutical composition comprising a glycan or neoglycan of the present invention.
  • the pharmaceutical composition may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically (see below).
  • the preparations particularly those preparations which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active compound(s), together with the excipient.
  • the pharmaceutical composition may be administered by any means that achieve their intended purpose. Amounts and regimens for the administration of the glycan can be determined readily by those with ordinary skill in the clinical art of treating the particular disease.
  • administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes.
  • administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulation may be used simultaneously to achieve systemic administration of the glycan.
  • Suitable formulations for oral administration include hard or soft gelatin capsules, dragees, pills tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
  • Solid dosage forms in addition to those formulated for oral administration include rectal suppositories.
  • Suitable injectable solutions include intravenous subcutaneous and intramuscular injectable solutions.
  • Suitable excipients are, in particular, fillers such as cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone.
  • disintegrating agents may be added.
  • Auxiliaries are, above all, flow-regulating agents and lubricants.
  • Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices. Dye stuffs or pigments may be added to the tablets or dragee coatings.
  • Possible pharmaceutical preparations which can be used rectally include, for example, suppositories which consist of a combination of the glycan with a suppository base, for example, natural or synthetic triglycerides, or paraffin hydrocarbons.
  • suppositories which consist of a combination of the glycan with a suppository base, for example, natural or synthetic triglycerides, or paraffin hydrocarbons.
  • gelatin rectal capsules it is also possible to use gelatin rectal capsules.
  • Suitable formulations for parenteral administration include aqueous solutions of the glycan in water-soluble form.
  • suspensions of the glycan compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension Optionally, the suspension may also contain stabilizers.
  • compositions according to the present invention are liposomes
  • the glycan is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers.
  • the glycan may be present both in the aqueous layer and in the lipidic layer, inside or outside, or, in any event, in the non-homogeneous system generally known as a liposomic suspension.
  • the glycan may be chemically linked to one of the liposome constituents.
  • the hydrophobic layer, or lipidic layer generally, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surface active substances such as dicetylphosphate, stearylamine or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • mice 6-8 week old, were obtained from Charles River Breeding Labs.
  • Two IgD secreting plasmacytomas TEPC-1017 and 1033 (Finkelman, F.D. et al., J. Immunol. 126:680-687 (1981)) were maintained i.p. in pristane primed BALB/c mice.
  • the hybridoma B1-8. ⁇ 1 secreting IgD specific for the hapten 4-hydroxy-3-nitrophenyl-acetyl was a kind gift from Dr. K. Rajewsky
  • (2H10) of helper phenotype and specific for cytochrome c was maintained in Click's/RPMI 1640. It was kindly provided by Dr. R.H. Schwartz (NIAID, NIH, Bethesda, MD).
  • H ⁇ a /1 and AMS-15 are monoclonal antibodies specific for Fc ⁇ and Fd ⁇ respectively (Zitron, I.M. et al., J. Exp.
  • F(ab') 2 fragments of IgG were received from Dr. W.O. Weigle
  • rIL-4 Recombinant IL-4 (rIL-4) produced by myeloma transfectants
  • Gal, GalNAc, GlcNAc, and mannose were from Sigma (St. Louis, MO.), melibiose (Mel) from Pfanstiehl Lab. (Waukegan, IL.), and lactose (Lac) from Fluka (Ronkonkoma, NY).
  • the purified polysaccharides from Pneumococcus and Klebsiella Heidelberger, M, et al.. Immunochem. 13:67-80 (1989)
  • Spleen T cells were prepared by sequential depletion of adherent cells at 37°C in Petri dishes (1400-1, Nunclon, Rockilde, Denmark) and of B cells by negative selection (Wysocki, L.I. et al., Proc. Natl. Acad. Sci. (USA) 75:2844-2848 (1978)) at 4°C on Petri dishes coated with affinity-purified anti-mouse Ig (remaining Ig + cells ⁇ 1%). Rosette-forming cell (RFC) assay
  • Mouse splenic T cells or human peripheral blood lymphocytes (PBL) (2.5 ⁇ 10 6 ) were incubated at 37°C for 18 hrs. in 1 ml of RPMI-1640 containing 2% fetal calf serum (FCS) with rIL-4 (10 U/ml) or crosslinked IgD (100 ⁇ g/ml) overnight. These resulting T ⁇ cells were tested for IgD-R in RFC assays. (Coico, R.F. et al., Nature 316:744-746 (1985)). SRBC were coated with either purified IgD, Gen.24, KWD1, KWD6 or bovine serum albumin (BSA) by the CrCl 3 -coupling method (Poston, P.W.
  • Rosette inhibition assays were carried out using IgD-R + T cells which had been induced with rIL-4 or IgD.
  • IgD-R + T cells were incubated with various purified test proteins, (e.g., IgD, Gen.24, KWD1, Fab ⁇ , Fc ⁇ , or KWD6), on ice for 30 min. and then allowed to interact with IgD-SRBC in a total volume of 300 ⁇ l. Percentages of IgD rosette inhibition were calculated with the formula:
  • IgD was also purified by affinity chromatography on an IgD specific GS-1-Sepharose column and eluted with galactose (Oppenheim, J.D. et al.,
  • Mutant IgD molecules Gen.24, KWD1 and KWD6 were affinity purified over a goat anti-mouse IgD-Sepharose column, as monitored by double diffusion in agarose gel, SDS-PAGE and ELISA.
  • Papain was purchased from Pierce Chemicals, Inc., (Rockford, IL), pronase was from Calbiochem (San Diego,
  • Purified TEPC-1017 IgD was digested at 37°C using immobilized papain as prescribed by the manufacturer. Fc ⁇ fragments were subsequently isolated by affinity chromatography with the Fc ⁇ -specific, H ⁇ a /1-Sepharose column. Fab ⁇ fragments of naturally degraded purified IgD were also isolated by passage through this column (See Figure 6). SDS-PAGE was performed under reducing and non- reducing conditions. Molecular weight markers are given in kDa.
  • Low Mr IgD fragments were prepared by pronase (F1) or proteinase K (F2) digestion of purified TEPC-1033 IgD.
  • the lowest Mr components ( ⁇ 5000 Da) of these digests were obtained by gel filtration on Sephadex G-25; the position of the retained small molecules was determined with tryptophan as standard. The retained fractions from these columns were pooled and lyophilized.
  • the low Mr fraction from IgG2a was similarly prepared by pronase digestion of UPC 10 myeloma protein (F3). Unfractionated fragments, obtained by complete digestion of TEPC-1017 IgD, 3A6 hybridoma IgA (F4) and TEPC-187 IgM (F5) with proteinase K, were found to be have Mr of ⁇ 5 kDa on analysis by 4-20% SDS-PAGE.
  • Neoglycoproteins comprising monosaccharide- substituted bovine serum albumin (BSA) were prepared as previously described (Kieda et al., supra: Monsigny et al., supra); p-nitrophenyl glycosides (Sigma, St. Louis) were reduced to p-aminophenyl glycosides, converted into glycosidophenyl isothiocyanates and coupled to BSA up to approximately 20 sugar residues/mole.
  • BSA monosaccharide- substituted bovine serum albumin
  • IgD was treated with N-glycanase (PNGase F) as previously described (Oppenheim et al., supra).
  • Ten mg purified TEPC-1017 IgD was treated with N-glycanase according to the manufacturer's specifications (Genzyme, Cambridge, MA).
  • a positive control, transferrin was treated similarly. All samples were then tested for the presence of glycans by dot-blot analysis using staining with digoxigenin succinyl- ⁇ -amidocaproic acid hydrazide (Boehringer Mannheim Biochemica Glycan Detection Kit) to monitor the reaction.
  • the partially deglycosylated IgD (DG-IgD) and the released asparagine-linked (N-linked) oligosaccharides were purified as follows: The reaction mixture was passed through a PD-10 desalting column according to the manufacturer's specifications (Pharmacia). The protein and
  • the released glycans (in the salt fraction) were purified by chromatography on GS-1 Sepharose column.
  • the bound glycans were eluted with glycine-HCL, pH 3.0.
  • the sample was neutralized and lyophilized. After dissolving in 2 ml of distilled water, the carbohydrate content was estimated by the Anthrone method.
  • the DG-IgD was analyzed for its reactivity with peroxidase conjugated GS-1 and anti-IgD antibody in agarose gel immunodiffusion and in dot blot assays.
  • the released glycans were shown to inhibit immunoprecipitin reactions between IgD and GS-1 in immunodiffusion gels.
  • Mouse IgD unlike human IgD, lacks A C ⁇ 2 heavy chain domain (Tucker, P.W. et al., Science 208:1353-1360 (1980)) and consists of the C ⁇ 1, C ⁇ -hinge and C ⁇ 3 domains.
  • binding the ability of mutant IgD molecules lacking one or more heavy chain domains to CD4 + T ⁇ cells was tested in rosetting assays.
  • the ability of the mutant IgDs to inhibit rosetting of CD4 + T hybridoma cells or splenic T ⁇ cells with IgD-coated SRBC was examined.
  • KWD1 lacks the C ⁇ 1 domain (Mountz, J.D. et al., J. Immunol. 145:1583-1591 (1990)), and KWD6 lacks both C ⁇ l and C ⁇ -hinge (Table 1). The deletions of these domains were confirmed by Northern blots and by ELISA with monoclonal anti- ⁇ antibodies (Table 1).
  • Gen.24 produced by a spontaneous variant of the IgD-producing plasmacytoma, TEPC-1017, includes C ⁇ l and part of C ⁇ -hinge but lacks C ⁇ 3 (Thiele, C.J. et al., J. Immunol.
  • TEPC-1017 is a 260 kDa dimer
  • its effectiveness is comparable to that of Gen.24 (100 kDa) and KWDl (90 kDa).
  • all of the mutant proteins when coating SRBC, resulted in rosette formation by either CD4 + splenic T ⁇ cells or 2H10 (CD4 + , IgD-R + ) T hybridoma cells, though KWD1-coated RBC were somewhat less effective (Table 1).
  • KWD6 and Gen.24 despite their lack of C ⁇ 1 + C ⁇ -hinge and C ⁇ 3 domains, respectively, each contain some structure or determinant recognized by the IgD-R.
  • the identity of this determinant was further examined (See Figure 5) by RFC cross-blocking experiments.
  • KWD6 and Gen.24 were equally effective in blocking rosetting with Gen.24-coated SRBC, while KWD6 was quantitatively more effective than Gen.24 in blocking rosetting with KWD6-coated SRBC.
  • the tailpiece of the secreted murine IgD is considerably longer (21 residues) than that of human IgD
  • Fab ⁇ was less effective an inhibitor of IgD-rosetting than was the Gen.24 molecule. This discrepancy could be explained by the lower avidity of a single ⁇ chain compared to a double ⁇ chain.
  • the effectiveness of the Fab ⁇ in rosette inhibition supports the conclusion that the C-terminal amino acid residues of secreted IgD are not necessary for binding to IgD-R.
  • the receptor on T cells for IgD is not limited to the Fc region, and it therefore should be referred to as IgD-R rather than a Fc ⁇ receptor.
  • TEPC-1033 IgD 50 ⁇ g/assay.
  • Expt. 3 boiled fragments were from 75 ⁇ g TEPC-1017 IgD, IgM or IgA. Since all fragments had Mr ⁇ 5 kDa, fragments were not further fractionated before assay. In Expts. 1 and 2, low Mr fractions were adsorbed by passage over lectin GS-1- coupled Sepharose 4B.
  • Lectin GS-1 Binds to and Absorbs IgD-RFC-Inhibiting Moietv
  • GS-1 Griffonia simplicifolia
  • IgD which was completely digested by protease retained its ability to form a precipitate with GS-1 upon double diffusion in agar. Neither intact nor digested IgM or IgA showed any such precipitation reaction.
  • N-linked sugars were removed from IgD by treatment with the enzyme N-glycanase.
  • This "deglycosylated" IgD (DG-IgD) no longer bound to GS-1 and failed to cause significant inhibition of IgD-rosetting ( Figure 8A).
  • DG- IgD caused 12 ⁇ 13% inhibition as compared to 74 ⁇ 2% inhibition by monomeric B1-8. ⁇ IgD and 80 ⁇ 4% inhibition by dimeric TEPC-1017 IgD.
  • the DG-IgD retained its ability to bind to anti-IgD antibodies ( Figure 8B).
  • the carbohydrates released from IgD during hydrolysis by N-glycanase were purified using affinity chromatography with GS-1-Sepharose and tested for their capacity to block rosetting (Table 3).
  • a dose-dependent inhibition of IgD-rosetting was obtained when the indicator erythrocytes were coated with intact IgD and also when they were coated with the mutant IgD molecules Gen.24 or KWD6 (Table 3).
  • the concentration which resulted in 50% inhibition (IC 50 ) was approximately 10-15 ⁇ g of N-glycans per assay.
  • both the mutant IgD proteins contain N-linked glycans.
  • TEPC-1017 IgD IgD (50) 4 ⁇ 1 (86) 3 ⁇ 0.4 (88) TEPC-1017 IgD Glycan (5) 9 ⁇ 0.3 (68) 8 ⁇ 0.5 (69) TEPC-1017 IgD Glycan (2.5) ND 19 ⁇ 2 (27)
  • Splenic T cells were induced to express IgD-R by overnight incubation with IL-4 (10 U/ml).
  • Blocking agents were all derived from TEPC-1017 IgD. Low Mr fractions containing N-glycans from IgD were passed over GS-1- Sepharose. Adherent glycans were eluted with gly conflict-HCl, pH
  • GS-1 lectin binds N-acetylgalactosamine (GalNAc) as well as galactose (Gal) (Murphy, L.A. et al..
  • IgD-RFC mannose
  • Glc glucose
  • Lac lactose
  • Mel melibiose
  • Neoglycoproteins based on bovine serum albumin such as ⁇ -D-GalNAc-BSA, ⁇ -D-GlcNAc-BSA and ⁇ -D-Gal-BSA, when added at a concentration of 3 ⁇ M protein to T ⁇ cells, blocked IgD-rosetting by 76.1%, 43% and 39.8% respectively.
  • BSA bovine serum albumin
  • Dimeric TEPC 1017 IgD causes 50% inhibition at 0.15 ⁇ M.
  • ⁇ -D-Lac-BSA, ⁇ -D-Man-BSA, ⁇ -D-Man-6- Phosphate-BSA, ⁇ -L-Fuc-BSA and ⁇ -D-Glc-BSA did not cause significant inhibition at the same concentrations. These results are consistent with those obtained with the freed monosaccharides.
  • the three inhibitory neoglycoproteins coprecipitate with GS-1 upon double diffusion in agar at 4°C, whereas the non-inhibitory neoglycoproteins do not.
  • Gal-/GalNAc-rich purified polysaccharides of bacterial origin were also tested for their ability to inhibit IgD-rosetting. These included pneumococcal polysaccharides S1, S4, S8, S11a, S13, S14, S15 and S29, and Klebsiella polysaccharides K11, K12, K16, K18, K21, K22, K23, K24, K25, K27, K31, K38, K41, K51, K53, K56, K74 and K83 (Heidelberger, M. et al.. supra)).
  • N-linked glycans from IgD were tested for their ability to inhibit IgD-rosetting and found active at very low concentrations.
  • the effectiveness of the glycans on a w/v basis as compared to intact IgD indicates that it alone is responsible for the rosette inhibition.
  • a change in tertiary structure could of course have contributed to the absence of inhibitory activity in DG-IgD.
  • the protein backbone structure contributes to the stabilization of IgD-IgD-R complexes after initial binding of the glycans by the receptors.
  • the present observations show that the IgD-R functions as a lectin in its interaction with IgD.
  • This has an interesting resemblance to other cell-surface lectins known in the art, including FceRII (CD23), ELAM-1
  • the IgD-R are therefore unique, since there is an absolute requirement for the presence of carbohydrate on its ligand for binding.
  • Fc -R does not have a strict requirement for glycans on IgG for its binding (Peppard, J.V. et al., Mol. Immunol. 26.495-500 (1989)).
  • FceRII exhibits a lectin-like domain in its structure important for the interaction with IgE, it does not predominantly recognize carbohydrate moieties of IgE (Bettler et al., Proc. Natl. Acad. Sci. USA 86:7118-7122 (1989)).
  • Ca ++ is required in the interaction between IgE and FceRII (Richards et al., supra), as it is for the interaction between IgD-R and IgD
  • the portion of the IgD molecule available on the surface of B cells is capable of binding to IgD-R of T cells, pointing to the possibility that Fab ⁇ -antigen complexes, released from the surface of B cells by cleavage of the IgD molecule, could function in the regulation of the immune response by upregulating IgD-R on T cells. Since the IgD idiotype is present in such complexes, a T cell-mediated idiotype- specific influence on the immune response (Bourgois, A. et al.. Eur. J. Immunol. 7:210-213 (1977)), could be an integral part of the immunoregulatory effect.
  • Human PBL were isolated from blood, and incubated with plastic dishes coated with cross-linked human IgD to upregulate human IgD-R. These cells were then rosetted with IgD-coated Ox-RBC in the presence or absence of various concentrations of potential inhibitors consisting of proteinase K digested IgD or IgG. The results, shown in Figure 10, indicate that proteinase K digested human IgD was capable of inhibiting rosette formation. The fact that this digestion was complete is shown in Figure 11.
  • Anti-CD4 and anti-CD8 antibodies each reactive with a subset of T cells, indeed induces upregula-tion of IgD-R on fewer cells than does anti-CD3.
  • the sum of percentages of IgD-RFC detected after stimulation with anti- CD4 and anti CD8 is approximately equal to that seen after stimulation with IgD.
  • the percent of CD8 cells having IgD-R is usually higher than for CD4 cells, both in young and aged individuals.
  • Anti-CD3 (18) 12.2 ⁇ 4.4 (6) 10.7 ⁇ 5.4 (5) 0.0 ⁇ 0.4 (7)

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  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Le récepteur destiné à l'IgD (IgD-R) sur les lymphocytes T présente des caractéristiques lectinoïdes et se fixe à des fractions glucidiques de l'IgD. Des glycanes associés à l'IgD se fixent à l'IgD-R. Sont donc décrits des glycanes associés à l'IgD, des néoglycanes les renfermant, et des compositions pharmaceutiques contenant ces glycanes. Les glycanes et les compositions pharmaceutiques sont utiles dans une méthode pour inhiber la fixation de l'IgD à un récepteur de celles-ci, une méthode pour inhiber une réponse immunitaire impliquant l'interaction des lymphocytes T et B chez un sujet, ainsi qu'une méthode pour traiter un sujet atteint d'une maladie d'origine immunitaire. Du fait que les glycanes peuvent réguler vers le haut l'expression de l'IgD-R, ils sont également utiles dans une méthode pour accroître le nombre des récepteurs de l'IgD sur la surface d'un lymphocyte T, et dans une méthode pour stimuler une réponse immunitaire chez un sujet immunisé avec un antigène ou un vaccin.
PCT/US1992/008724 1991-10-11 1992-10-09 Glycanes associes a l'immunoglobuline d et leurs utilisations Ceased WO1993007291A1 (fr)

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US77332891A 1991-10-11 1991-10-11
US773,328 1991-10-11

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WO1993007291A1 true WO1993007291A1 (fr) 1993-04-15

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0727216A4 (fr) * 1994-07-15 1998-05-20 Taiyo Kagaku Kk Composition medicamenteuse contenant un derive d'acide sialique

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
J. BIOL. CHEM., Volume 258, No. 19, issued 1983, MELLIS et al., "Structures of the 0-Glycosidically Linked Oligosaccharides of Human IGD", pages 11557-11563; & CHEMICAL ABSTRACTS 100(1), Abstract No. 4519g. *
JOURNAL OF IMMUNOLOGICAL METHODS, Volume 130, No. 2, issued 1990, OPPENHEIM et al., "A Rapid One Step Purification Procedure for Murine IGD Based on the Specific Affinity of Bandeiraea-Simplicifolia for Amino-Linked Carbohydrates on IGD", pages 243-250, see Abstract No. 7,707,655. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 85, No. 2, issued 1988, COICO et al., "Exposure to Crosslinked IGD Induces Receptors for IGD on T Cells In Vivo and In Vitro", pages 559-563; & CHEMICAL ABSTRACTS 108(13), Abstract No. 110499a. *
THE JOURNAL OF IMMUNOLOGY, Volume 147, No. 5, issued 01 September 1991, CAMPBELL et al., "Alpha-Chains of IGM and IGD Antigen Receptor Complexes are Differentially N-Glycosylated MB-1-Related Molecules", pages 1575-1580, See Abstract 8,647,654. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0727216A4 (fr) * 1994-07-15 1998-05-20 Taiyo Kagaku Kk Composition medicamenteuse contenant un derive d'acide sialique
US5834423A (en) * 1994-07-15 1998-11-10 Taiyo Kagaku Co., Ltd. Pharmaceutical composition containing sialic acid derivatives

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