WO2000050447A1 - Peptides mimetiques d'epitope glucidique et utilisations correspondantes - Google Patents

Peptides mimetiques d'epitope glucidique et utilisations correspondantes Download PDF

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WO2000050447A1
WO2000050447A1 PCT/US2000/004730 US0004730W WO0050447A1 WO 2000050447 A1 WO2000050447 A1 WO 2000050447A1 US 0004730 W US0004730 W US 0004730W WO 0050447 A1 WO0050447 A1 WO 0050447A1
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peptide
carbohydrate
hnk
carbohydrate epitope
cells
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PCT/US2000/004730
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Melitta Schachner
Timothy J. Neuberger
Uri Herzberg
Maryline Simon
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Acorda Therapeutics
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Priority to CA002363601A priority patent/CA2363601A1/fr
Priority to AU28841/00A priority patent/AU2884100A/en
Publication of WO2000050447A1 publication Critical patent/WO2000050447A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to carbohydrate epitope mimic compounds, particularly peptides, to variants, analogs and active fragments thereof and to nucleic acids encoding such peptides, variants, analogs and active fragments.
  • the peptides of the invention mimic the carbohydrate epitope GlcA ⁇ l-»3Gal ⁇ 1 ⁇ 4GlcNAc or sulfate-3GlcA ⁇ 1 -3Gal ⁇ 1 GlcNAc, or the L2/HNK1 carbohydrate epitope.
  • the invention also relates to diagnostic, therapeutic and pharmaceutical compositions and uses of such compounds, particularly peptides, variants, analogs and active fragments thereof, and nucleic acids encoding such peptides, variants, analogs and active fragments, in modulating or mediating cell-cell adhesion and the processes and events mediated thereby.
  • the HNK-1 epitope is expressed predominantly on glycolipids and glycoproteins from nervous tissue (McGarry et al, (1983) Nature 306:376-378; Ilyas et al, (1984) Biochem. Biophys. Res. Comm. 122: 1206-121 1; -Kruse et al, (1984) Nature 311 :153- 155; Yuen et al, (1997) J. Biol. Chem. 272:8924-8931).
  • the expression pattern of the H ⁇ K-1 carbohydrate in both the central and peripheral nervous system is spatially and developmentally regulated (Wernecke et al, (1985) J. Neuroimmunol.
  • HNK-1 carbohydrate epitope is carried by many, but not all, neural recognition glycoproteins, and is involved in homo- and heterophilic binding of these proteins (for a review, see Schachner and Martini (1995) Trends Neurosci.
  • Rat monoclonal antibodies isolated after immunization with a fraction enriched in plasma membrane include 334 (IgM), 336 (IgG), 349 (IgM), 344 (IgM), and 392 (IgM).
  • the antibodyL2-412 (IgG) was obtained by immunization with a membrane-derived glycoprotein fraction from mouse brain (Kruse et al, 1984, Noronha et al, 1986, Schachner et al, 1989).
  • Another group of monoclonal antibodies recognizing theL2/HNK-l carbohydrate is the human IgM detected in the serum of some patients with neuropathies.
  • the IgM was shown to bind to human myelin-associated glycoprotein (MAG); and the antigenic determinant reacting with the IgM was in the carbohydrate part of the MAG molecules (llyas et al, 1984, Quarles et al, 1992).
  • the fine specificities of these human antibodies have been investigated; striking differences were seen in the structural requirements for binding: Some IgMs needed the sulfate group while others did not (llyas et al, 1990). It has been suggested that the epitope recognized by these IgM antibodies may be an important target in paraproteinemic neuropathies.
  • the L2/HNK-1 carbohydrate is found in glycolipids, glycoproteins, and proteoglycans.
  • the structure which reacts with HNK-1 antibody was first described by Chou and Jungalwala for the major antigenic glycolipid present in human peripheral nerve.
  • the compostion, sugar linkage, configuration and position of the sulfate group were characterised as sulfate-3 GlcA ⁇ (1-3) Gal ⁇ (1-4) GlcNAc ⁇ (1-3) GalNAc ⁇ (1-3) Gal ⁇ (1-4) Glc ⁇ (l-l)-ceramide for SGGL-1 and as sulfate-3 GlcA ⁇ (1-3) Gal ⁇ (1-4) GlcNAc ⁇ (1-3) Gal ⁇ (1-4) GlcNAc ⁇ (1-3) Gal ⁇ (1-4) Glc ⁇ (l-l)-ceramide for SGGL-2. (Chou et al, 1986).
  • the enzymes involved in the biosynthesis of the L2/HNK-1 carbohydrate have been studied at the biochemical level. Glycosyltransferase (Chou et al, 1996), galactosyltransferase (Chou et al, 1994), glucuronyltransferase (Chou et al, 1991) and sulfotransferase (Chou et al, 1996) have been studied using crude enzyme preparations. Two of them have been purified, i.e., an N- acetylglucosaminyltransferase (Chou et al, 1993), and a glucuronyltransferase respectively (Oka et al, 1992).
  • the mouse HNK-1 and theL2-412 antibodies have been studies using synthetic glcolipids with regard to their requirement for binding.
  • the HNK-1 antibody shows an absolute requirement for the sulfate group (lias et al, 1990).
  • theL2-412 antibody recognizes both the sulfated and the non-sulfated form of the carbohydrate structure (Schmitz et al, 1994).
  • the L2/HNK-1 carbohydrate is found on a large number of molecules both in the CNS and PNS. It has been hypothesized that molecules expressing this epitope could be involved in adhesion, although it has not yet been proven in the case of Drosophila melanogaster, zebrafish and lymphocytes. Table 1 summarizes, in a non-exhaustive list, the diversity of molecules carrying the L2/HNK-1 carbohydrate. The presence of this carbohydrate in groups as diverse as mammals, fish, and insects, may indicate the importance of this carbohydrate.
  • PI-GP 150 (Telencephaline) (Yoshihara et al, 1991; Yoshihara et al,
  • Glycolipids (SGGLs) (Chou et al, 1985; Nair et al, 1997: Nair et al, 1993)
  • the carbohydrate appears therefore on a variety of molecules. It is unclear whether the carbohydrate has the same function on different molecules or whether its function depends on the molecule carrying it at various regions and stages of development of the nervous system.
  • L2/HNK-1 epitope Although many proteins may carry the L2/HNK-1 epitope, it is difficult to determine at which developmental stage and in which brain region on a particular protein carries this epitope. In the case of N-CAM, for example, only a subpopulation of the molecules carries the L2/HNK-1 epitope (Kruse et al, 1984). This is also the case for LI (Faissner et al, 1987), MAG (Poltorak et al, 1987) and PO (Burger et al, 1990). The expression of the glycoproteins carrying the L2/HNK-1 carbohydrate has been studied in the rat (Chou et al, 1991).
  • the glycoproteins carrying the L2/HNK-1 carbohydrate are found at embryonic day 19 (ED 19) and continue to be expressed in the adult.
  • Yoshihara et al (Yoshihara et al, 1991; Yoshihara at al, 1994) have shown with their studies on the glycoprotein Pl- GP150 that the L2/HNK-1 carbohydrate moiety can be regulated independently of the expression of the protein backbone, and that its expression shows segmental differences.
  • telencephalon expresses the L2/HNK-1 epitope constitutively; in the midbrain, by contrast, its expression decreases after postnatal day 7 (PD7) and becomes completely absent in the adult myencephalon and metencephalon.
  • PD7 postnatal day 7
  • the L2/HNK-1 epitope is present not only in glycoproteins, but also in proteoglycans and glycolipids, which makes it difficult to determine the spatial expression of the L2/HNK-1 epitope in a specific molecule.
  • L2/HNK- 1 antibodies At a gross level, it was shown that in embryonic rat and mouse brain immunoreactivity with L2/HNK- 1 antibodies has a similar distribution but appears at slightly different embryonic ages.
  • the expression of the major HNK-1 -reactive glycolipids studied in rat by Schwarting (Schwarting et al, 1987) is in good correlation with the more precisely located developmental expression of the HNK-1 -reactive glycolipids in the rat (Chou et al, 1991).
  • L2/HNK-1 -carrying molecules in the mammalian nervous system may be summarized as follows: The two sulfoglucuronyl glycolipids (SGGLs) are expressed in the cerebral cortex during neonatal development, but disappear in the adult.
  • L2/HNK-1 -reactive glycoproteins continue to be expressed throughout the nervous system in adulthood (Chou et al, 1991). It should however, be noted that the rat femoral nerve was shown to be HNK-1 negative using the HNK-1 antibodies. Reactivity to other L2/HNK-1 recognizing antibodies has not been studied.
  • the integrins are a family of glycoproteins that can carry the L2/HNK-1 carbohydrate. They interact with a wide variety of ligands, including extracellular carbohydrate. They interact with a wide variety of ligands, including extracellular matrix glycoproteins such as laminin. They participate in cell-matrix and cell-cell adhesion in important processes such as embryonic development (Hynes et al, 1987). In this capacity, integrins are presumed to function in cell migration in embryos. During their migration, neural crest cells encounter various tissues and extracellular matrix molecules surrounding these tissues.
  • the HNK- 1 antibody recognizes a carbohydrate epitope on the surface of migrating neural crest cells which is closely related if not identical to the L2/HNK-1 carbohydrate.
  • the role of the carbohydrate in chicken was investigated in vivo and in vitro by treatment with HNK-1 antibody. Addition of the HNK-1 antibody to neural tube explants in tissue culture, caused neural crest cells to detach from laminin substrate and alter their morphology. Injection of the antibody into embryos caused abnormalities in neural cell migration and development. The timing appeared to be critical, indicating that the HNK-1 antibody selectively perturbs the early stages of neural crest migration (Bronner-Fraser et al, 1987).
  • L2/HNK-1 carbohydrate was shown to be involved in cell-cell adhesion
  • L2/HNK-1 carbohydrate can act as a ligand in cell adhesion (Kunemund et al, 1988), and that it is more important for cell-substrate than for cell-cell interactions. More recently, Hall and co-workers (Hall et al, 1993) showed that L2/HNK- 1 carbohydrate and heparin were using different binding sites on laminin and were thus implicated in different aspects of neural cell adhesion to laminin.
  • Selectins are a family of structurally and functionally related cell surface adhesion proteins that bind carbohydrates. They are implicated in adhesive interactions with cells of the vascular endothelium.
  • carbohydrate ligand is responsible for selectin-mediated cell adhesion, it was shown that the glycolipids carrying the L2/HNK- 1 carbohydrate are ligands for L-selectin and for P- selectin, but not E-selectin, even though all three selectins share considerable structural similarity.
  • BMECs brain microvascular origin
  • glycolipids carrying the L2/HNK-1 carbohydrate act as one of the ligands for l-selectin in inflammatory disorders of CNS/PNS, and that they regulate the attachment of activated lymphocytes and their subsequent invasion of the CNS and PNS.
  • the authors suggested that, since a number of glycoconjugates possessing the L2/HNK-1 epitope have been implicated in cellular adhesion (see section 1.5.2.), the SGGLs, through the L2/HNK-1 carbohydrate, may be involved in intercellular adhesion of BMECs for the formation of the blood-brain-barrier and may play a critical role in maintenance of the barrier function (Kanda et al, 1995).
  • Peripheral myelin glycoprotein is an example of an adhesion molecule that engages in homophilic binding (that is, it binds to itself).
  • the L2/HNK-1 carbohydrate expressed on a subset of PO molecules has been shown to be involved in this binding: Binding could be partially inhibited by antibodies to the L2/HNK-1 epitope and by L2/HNK-1 carbohydrate (but not other carobohydrates). Inhibition was also seen with polyclonal antibodies reacting with the protein backbone of PO, indicating that both protein and carbohydrate structures are involved in the binding of PO to PO, and that PO acts both as presenter and a receptor of the L2/HNK-1 carbohydrate (Griffith et al, 1992).
  • the L2/HNK-1 carbohydrate is selectively expressed on the Schwann cells and Schwann cell basement membrane of the motor branch, but is rarely found in the sensory branch (Martini et al, 1988). It persists in these locations during and after Wallerian degeneration (Martini et al, 1992).
  • Analysis of the myelin of the muscle and cutaneous branches of the adult mouse femoral nerves by immunochemical methods showed that the L2/HNK-1 carbohydrate was detectable on both SGGL-1 and SGGL-2.
  • the glycoprotein uniquely L2-412- immunoreactive in the muscle nerve was identified as MAG (Low et al, 1994).
  • the L2/HNK-1 carbohydrate also selectively promotes outgrowth of neurites from motor axons in vitro. This was demonstrated using cryostat sections of femoral nerve sensory and motor branches on which motor neurons were allowed to grow. Neurites preferentially elongate on the motor branch expressing the L2/HNK-1 as compared to the sensory branch scarcely expressing L2/HNK-1. In contrast, neurites extending from sensory neurons reached the same length on both substrates (Martini et al, 1992). In the mouse, the L2/HNK-1 carbohydrate thus selectively marks the motor pathway.
  • the femoral nerve receives sensory axons from dorsal root ganglia (DRG) and motor axons from the ventral root. Distally, it divides into a cutaneous branch (sensory axons only) and a muscle branch (both sensory and motor axons).
  • DRG dorsal root ganglia
  • the femoral nerve was deefferented by transection of the ventral root or deafferented by removal of DRG. In the deafferented muscle branch, the pattern of L2-412- immunoreactivity of Schwann cells was similar to that found in the non- deafferented control.
  • a second group (reversed group) the grafts were inserted with the cutaneous nerve grafts in the muscle branch and the muscle nerve grafts into the cutaneous branch.
  • a particular interesting pattern was observed when a cutaneous graft was introduced in the muscle branch: in the cutaneous nerve graft itself, L2/HNK-1 was poorly expressed but the muscle branch distal to the graft strongly expressed the L2/HNK-1, although the distal muscle branch was reinnervated by the same axons that had penetrated the cutaneous graft.
  • Glial cells are the decisive determinants for controlling axon regrowth. Mammalian glial cells are generally permissive for neurite outgrowth in the central nervous system during development (Silver et al. (1982) J. Comp. Neurol. 210: 10-29; Miller et al. (1985) Develop. Biol. 111:35-41; Pollerberg et al. (1985) J. Cell. Biol. 101: 1921- 1929) and in the adult peripheral nervous system (Fawcett et al. (1990) Annu. Rev. Neurosci 13:43-60).
  • glial cells of the adult mammalian peripheral nervous system can revert to some extent to their earlier neurite outgrowth-promoting potential, allowing them to foster regeneration (Kalderon (1988) J. Neurosci Res. 21:501-512; Kliot et al. "Induced regeneration of dorsal root fibres into the adult mammalian spinal cord," In: Current Issues in Neural Regeneration, New York, pp. 311-328; Carlstedt et al. (1989) Brain Res. Bull. 22:93-102). Glial cells of the central nervous system of some lower vertebrates remain permissive for neurite regrowth in adulthood (Stuermer et al. (1992) J. Neurobiol. 23:537-550). In contrast, glial cells of the central nervous system of adult mammals are not conducive to neurite regrowth following lesions.
  • neural cell adhesion molecules belonging to the immunoglobulin superfamily, and particularly to those members that mediate Ca 2+ -independent neuronal cell adhesion, of which LI, N-CAM and myelin- associated glycoprotein are particular members.
  • cell adhesion molecules which may also influence CNS neural growth include laminin, fibronectin, N-cadherin, BSP- 2/D-2 (mouse N-CAM), 224-1A6-A1, Ll-CAM, NILE (rat LI), Nr-CAM, TAG-1 (axonin-1), Ng-CAM and F3/F11/contactin.
  • laminin fibronectin
  • N-cadherin BSP- 2/D-2
  • 224-1A6-A1 Ll-CAM
  • NILE rat LI
  • Nr-CAM rat LI
  • Nr-CAM rat LI
  • TAG-1 axonin-1
  • Ng-CAM axonin-1
  • F3/F11/contactin The prominent role played in mediating neurite outgrowth by the neural adhesion molecule LI has been demonstrated
  • the homophilic binding ability of LI is enhanced by molecular association with the neural cell adhesion molecule N-CAM, allowing binding to occur through homophilic assistance (Kadmon et al. (1990a); Kadmon et al. (1990b) J. Cell Biol 110:209-218 and 1 10: 193-208; Horstkorte et al. (1993) J. Cell. Biol. 121 : 1409-1421). Besides its neurite outgrowth promoting properties, LI also participates in cell adhesion (Rathjen et al. (1984) EMBO J. 3: 1-10; Kadmon et al. (1990b) J. Cell. Biol. 110:209-218; Appel et al.
  • LI consists of six immunoglobulin-like domains and five fibronectin type III homologous repeats. LI acts as a signal transducer, with the recognition process being a first step in a complex series of events leading to changes in steady state levels of intracellular messengers. The latter include inositol phosphates, Ca 2 +, pH and cyclic nucleotides (Schuch et al. (1990) Neuron 3:13-20; von Bohlen und Halbach et al. (1992) Eur. J. Neurosci. 4:896-909; Doherty et al. (1992) Curr. Opin. Neurobiol.
  • LI protein kinases
  • protein kinases such as protein kinase C and pp ⁇ O ⁇
  • LI is also associated with a casein type II kinase and another unidentified kinase which phosphorylates LI (Sadoul et al. (1989) J. Neurochem 328:251-254).
  • LI -mediated neurite outgrowth is sensitive to the blockage of L type Ca 2+ channels and to pertussis toxin.
  • LI is also present on proliferating, immature astrocytes in culture and neurite outgrowth is promoted on these cells far better than on differentiated, LI immunonegative astrocytes (Saad et al. (1991) J Cell Biol. 115:473-484). In vivo, however, astrocytes have been found to express LI at any of the developmental stages examined from embryonic day 13 until adulthood (Bartsch et al. (1989) J.Comp. Neurol 284:451-462; and unpublished data).
  • NK and K cells are specialized lymphocytes that have been implicated in viral immunity and in defense against tumors. NK cells have also been shown to play a role in the graft- versus-host reaction and these cells may contribute to some of the skin lesions and intestinal wall damage observed. The cells make up approximately 10% of the recirculating lymphocyte population.
  • NK cells are involved in the early response to infection with certain viruses and intracellular bacteria. NK activity is stimulated by IFN-alpha, IFN-beta and IL-12. In the course of a viral infection, these cytokines rapidly rise, followed closely by a wave of NK cells that peaks in about 3 days. NK cells provide the first line of defense to virus infection, controlling viral replication during the time required for activation, proliferation and differentialtion of cytotoxic T cells (CTLs) at about day 7.
  • CTLs cytotoxic T cells
  • HIN human immunodeficiency virus
  • HTLV human T-cell lymphocyte virus
  • HIN can also infect the nervous system and is associated with AIDS-dementia.
  • Natural killer cells appear to kill tumor cells and virus infected cells by a process similar to that employed by CTLs.
  • the cytoplasm of NK cells contains numerous granules containing perform and granzymes. After an NK cells adheres to a target cell, degranulation occurs with release of perforin and granzymes at the junction of the interacting cells.
  • NK cells have also been shown to mediate target-cell destruction by apoptosis.
  • NK cells do not express antigen- specific T cell receptors or CD3 and target-cell recognition by NK cells is not MHC restricted.
  • NK cells can bind to antitumor antibodies bound to the surface of tumor cells and subsequently destroy the tumor, a process denoted antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells have been shown to secrete tumor necrosis factor (TNF).
  • TNF tumor necrosis factor
  • Chediak-Higashi syndrome an autosomal recessive disorder, is associated with an absence of NK cells and an increased incidence of lymphomas.
  • Mice with an autosomal mutation called beige lack NK cells and are more susceptible than normal mice to tumor growth following injection with live tumor cells.
  • the HNK-1 antibody has been shown to detect antigens which are heavily expressed by benign prostatic hyperplasia and carcinoma of the prostate (Lipford, G.B. and Wright, GL. Jr. Cancer Res. 51(9), 2296-3001 (1991)). This antibody also recognizes a number of human neuroblastoma lines and expression of the HNK-1 antigen on these lines can be slightly increased by retinoic acid-induced differentiation of the cells (McGarry, R.C. et al, Cancer Immunol Immunother 27(1), 47-52 (1988)).
  • Phage expressing binding peptides are selected by affinity purification with the target of interest. This sytem allows a large number of phage to be screened at one time. Since each infectious phage encodes the random sequence expressed on its surface, a particular phage, when recovered from an affinity matrix, can be amplified by another round of infection.
  • selector molecules immobilized on a solid support can be used to select peptides that bind to them. This procedure reveals a number of peptides that bind to the selector and that often display a common consensus amino acid sequence. Biological amplification of selected library members and sequencing allows the determination of the primary structure of the peptide(s).
  • Peptides are expressed on the tip of the filamentous phage Ml 3, as a fusion protein with the phage surface protein pilus (at the N-terminus).
  • a filamentous phage carries on its surface 3 to 5 copies of pili and therefore of the peptide.
  • no structural constraints are imposed on the N-terminus; the peptide is therefore free to adopt many different conformations, allowing for a large diversity.
  • biases in the distribution of peptides in the library may be caused by biological selection against certain of the peptides, which could reduce the diversity of peptides contained in the library. In practice, this does not appear to be a significant problem.
  • Peptide ligands identified by phage display screening frequently interact with natural binding site(s) on the target molecule, and often resemble the target's natural ligand(s). Although this system has been most often used to identify peptide epitopes recognized by antibodies, it has also been successfully used to find peptide mimics of carbohydrate molecules. Work directed towards using peptide mimics in place of carbohydrate antigens has been reviewed by Kieber-Emmons and colleagues (Kieber-Emmons et al, 1998). The demonstrated ability of a peptide to mimic a carbohydrate determinant indicates that, although mimicry is accomplished using amino acids in place of sugars, the specificity pattern can be reproduced.
  • Peptides that mimic glycosphingolipids have been found using a phage peptide library.
  • Two monoclonal antibodies that recognize lactotetraosylceramide (Lc4Cer) and its isomer neolactotetraosylceramide (nLc4Cer) were used to find peptides that mimic the carbohydrate moieties of the two glycosphingolipids. It was also shown that the peptides are biologically active, in that they could modulate the activity of ⁇ - galactosidase (Take et al, 1997).
  • the pathogen Shigella flexneri is a bacterium responsible for the endemic form of shigellosis, a dysenteric syndrome characterized by bacterial invasion of the human colonic mucosa.
  • the cell wall of this bacterium contains repeated saccharide units forming the O-antigen carbohydrate moiety of the capsular lipopolysaccharide.
  • peptide mimics of the carbohydrate epitope were isolated using phage display technology. These mimics could act as immunogenic mimics, and were capable of inducing specific anti-carbohydrate antibodies (Phalipon et al, 1997). 0/50447
  • the total synthesis requires 15 intermediate compounds and about 20 steps, of which several are very time consuming.
  • a possible solution to this problem is to mimic the carbohydrate by other compounds that are easier to prepare, e.g. peptides.
  • the most promising way to find such peptides is by use of the random peptide phage display (RPPD) technology.
  • RPPD random peptide phage display
  • the present invention encompasses an isolated peptide which mimics the carbohydrate epitope GlcA ⁇ 1 ⁇ 3Gal ⁇ 1 ⁇ 4GlcNAc or sulfate-
  • the invention extends to compounds, particularly peptides, that are capable of mimicking the L2/HNK1 carbohydrate epitope.
  • the compounds or peptides of the invention are further capable of interacting with or binding to molecules which interact with or bind to the L2/HNK1 carbohydrate epitope.
  • Such molecules are laminin, P-selectin, L-selectin, fibronectin, N-cadherin, myelin associated glycoprotein (MAG), neural cell adhesion molecules, N- CAM, BSP-2 D2 (mouse N-CAM), 224-1A6-A1, Ll-CAM, NILE (rat LI), Nr-CAM, TAG-1 (axonin-1), Ng-CAM and F3/F1 1/contactin.
  • laminin P-selectin, L-selectin, fibronectin, N-cadherin, myelin associated glycoprotein (MAG), neural cell adhesion molecules, N- CAM, BSP-2 D2 (mouse N-CAM), 224-1A6-A1, Ll-CAM, NILE (rat LI), Nr-CAM, TAG-1 (axonin-1), Ng-CAM and F3/F1 1/contactin.
  • an isolated peptide comprising an amino acid sequence Xj X 2 X 3 X 4 X 5 L/V X 6 X 7 X 8 X 9 X 10 X n ⁇ 2 X B ⁇ > wherein each residue can be independently selected as follows (SEQ ID NO: 1): x.
  • T, S, A or P ⁇ 2 L, I, V, M, F, H, W or N; x 3 T, S, H, Y, F, W, N, D or E; x 4 R, Q, K, T, S or A; x s V, I, L, M, R, Q or K; x 6 :s T, S, A, Y, F, H, W, N, L, I, V or M; x 7 s D, E, V, L, I, M, F, Y, H, W or N; x 8 s V, I, L, M, S, N T, R, Q or K;
  • an isolated peptide consisting of an amino acid sequence X ⁇ X 2 X 3 X 4 X 5 L/V X 6 X 7 X 8 X 9 X ]0 X u X 12 X !3 X 14 , wherein each residue can be independently selected as follows (SEQ ID NO: 1): X, is T, S, A or P; X 2 is L, I, V, M, F, H, W or N; X 3 is T, S, A, H, Y, F, W, N, D or E; X 4 is R, Q, K, T, S or A;
  • X 5 is V, I, L, M, R, Q or K
  • X 6 is T, S, A Y, F, H, W, N, L, I, V or M
  • X 7 is D, E, V, L, I, M, F, Y, H, W or N
  • X g is V, I, L, M, S, A, T, R, Q or K
  • X 9 is Y, F, H, W, D, E, I, V, L, M or N
  • X 10 is R, Q, K, W, Y, F, H, N, V, I, L, M or G;
  • X u is G, Y, F, H, W, N, S, A, T, I, V, L, M;
  • X 12 is R, Q, K, H, N, Y, F, W, I, V, L or M;
  • X 13 is L, V, I, M, T, S or A; and
  • X 14 is S, T, N P, G, R, Q or K; and variants, analogs and active fragments thereof.
  • the peptide comprises an amino acid sequence F L H T R L X, X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 , wherein each residue can be independently selected as follows (SEQ ID NO: 2): X j is T, S, A, Y, F, H, W, N, L, I, V or M;
  • X 2 is D, E, V, L, I, M, F, Y, H, W or N;
  • X 3 is V, I, L, M, S, A, T, R, Q or K;
  • X 4 is Y, F, H, W, D, E, I, V, L, M or N;
  • X 5 is R, Q, K, W, Y, F, H, N, V, I, L, M or G;
  • X* is G, Y, F, H, W, N, S, A, T, I, V, L, M;
  • X 7 is R, Q, K, H, N, Y, F, W, I, V, L or M;
  • X g is L, V, I, M, T, S or A;
  • Xg is S, T, A, P, G, R, Q or K; and variants, analogs and active fragments thereof.
  • the peptide consists of an amino acid sequence F L H T R L X, X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 , wherein each residue can be independently selected as follows (SEQ ID NO: 2):
  • Xj is T, S, A Y, F, H, W, N, L, I, V or M;
  • X 2 is D, E, V, L, I, M, F, Y, H, W or N;
  • X 3 is V, I, L, M, S, A T, R, Q or K
  • X 4 is Y, F, H, W, D, E, I, V, L, M or N
  • X 5 is R, Q, K, W, Y, F, H, N, V, I, L, M or G
  • X 6 is G, Y, F, H, W, N, S, A, T, I, Y, L, M
  • X 7 is R, Q, K, H, N, Y, F, W, I, V, L or M
  • X 8 is L, V, I, M, T, S or A
  • X 9 is S, T, A, P, G, R, Q or K; and variants, analogs and active fragments thereof.
  • the peptide comprises an amino acid sequence F L H T R L F V Xj X 2 X 3 X 4 X 5 X 6 X 7 , wherein each residue can be independently selected as follows (SEQ ID NO: 3):
  • X is V, I, L, M, S, A, T, R, Q or K
  • X 2 is Y, F, H, W, D, E, I, V, L, M or N
  • X 3 is R, Q, K, W, Y, F, H, N, V, I, L, M or G
  • X 4 is G, Y, F, H, W, N, S, A, T, I, V, L, M;
  • X 5 is R, Q, K, H, N, Y, F, W, I, V, L or M
  • X 6 is L, V, I, M, T, S or A
  • X 7 is S, T, A, P, G, R, Q or K; and variants, analogs and active fragments thereof.
  • a peptide is provided consisting of an amino acid sequence F L H T R L F V X j X 2 X 3 X 4 X 5 X 6 X 7 , wherein each residue can be independently selected as follows (SEQ ID NO: 3):
  • X t is V, I, L, M, S, A, T, R, Q or K;
  • X 2 is Y, F, H, W, D, E, I, V, L, M or N;
  • X 3 is R, Q, K, W, Y, F, H, N, V, I, L, M or G;
  • X 4 is G, Y, F, H, W, N, S, A, T, I, V, L, M;
  • X 5 is R, Q, K, H, N, Y, F, W, I, V, L or M;
  • X 6 is L, V, I, M, T, S or A; and
  • X 7 is S, T, A, P, G, R, Q or K; and variants, analogs and active fragments thereof.
  • the peptide comprises an amino acid sequence X ⁇ X 2 X 3 X 4 X 5 L/V X 6 X 7 X 8 Xg X 10 X_ ⁇ X J2 X 13 Xi 4 , wherein each residue can be independently selected as follows (SEQ ID NO: 4): ⁇ 2 s L or F; x 3 s T, H or E; x 4 s R or T; ⁇ 5 s V or R; ⁇ 6 s T, F or L; x 7 s D, V or F; x 8 s V, S or R;
  • a further embodiment of a peptide of the present invention comprises an amino acid sequence F L H T R L Xj X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 , wherein each residue can be independently selected as follows (SEQ ID NO: 5):
  • X, i is T, F or L;
  • X 2 i is D, V or F;
  • X 3 i is V, S or R;
  • X 4 ⁇ is Y, D, I or N;
  • X 5 i is R, W, V or G;
  • X 6 i is G, Y, S or I;
  • X 7 i is R, H, N, Y or I;
  • X 8 i is L, T or S
  • X 9 i is S, P, G or R; and variants, analogs and active fragments thereof.
  • An additional embodiment of the peptide comprises an amino acid sequence F L H T R LFVX 1 X 2 X 3 X 4 X 5 X 6 X 7 , wherein each residue can be independently selected as follows (SEQ ID NO 6):
  • X j is V, S or R;
  • X 2 isY, D, IorN;
  • X 3 is R, W, V or G
  • X 4 is G, Y, S or I;
  • X 5 is R, H, N, Y or I;
  • X 6 is L, T or S
  • X 7 is S, P, G or R; and variants, analogs and active fragments thereof.
  • the peptide comprises the amino acid sequence set out in any of SEQ ID NOS: 27-38. Still further, the peptide comprises the amino acid sequence FLHTRLFVSDWYHT (SEQ ID NO: 7). More particularly, the peptide comprises the amino acid sequence FLHTRLFV (SEQ ID NO: 8). Moreover, peptides having the amino acid sequence FLHTRLFVSDWYHT (SEQ ID NO: 7) or F L H T R L F V (SEQ ID NO: 8) are provided.
  • the peptide comprises the amino acid sequence TRLFR V/F (SEQ ID NO: 39), FLHTRLFV (SEQ ID NO: 8 ), TRLF(R)V (SEQID NO: 40) or TRLF (SEQ ED NO: 41).
  • the present invention relates to certain therapeutic methods which would be based upon the activity of the carbohydrate epitope mimic peptide(s), variants, analogs or active fragments thereof, or upon agents or other compounds determined to possess the same activity.
  • One such therapeutic method is associated with the prevention of the manifestations of conditions which can be corrected, altered or otherwise modulated by inhibition or activation of the binding activity of the carbohydrate epitope recognizing molecules, and comprises administering an agent capable of modulating the activity of the carbohydrate epitope recognizing molecules, either individually or in mixture with each other in an amount effective to prevent the development of those conditions in the host.
  • binding partners to the carbohydrate epitope recognizing molecules may be administered to inhibit or potentiate the activity of carbohydrate epitope recognizing molecules.
  • carbohydrate epitope mimic peptide may be administered to activate or otherwise modulate the activity of L2/HNK-1 recognizing molecules, as in the potentiation of neural cell adhesion molecules in CNS or PNS therapy.
  • the therapeutic method generally referred to herein could include methods for the treatment of various pathologies or other cellular dysfunctions and derangements by the administration of pharmaceutical compositions that comprise the carbohydrate epitope mimic peptide(s), variants, analogs or active fragments thereof, effective inhibitors or enhancers of activation of the carbohydrate epitope mimic peptide(s), or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention.
  • carbohydrate epitope mimic peptide(s) of the present invention may be administered to inhibit or potentiate activity of L2/HNK-1 carbohydrate epitope containing molecules or of L2/HNK-1 carbohydrate epitope recognizing molecules, as in the potentiation of neural cell adhesion molecules in CNS or PNS therapy.
  • It is also an object of the present invention to provide method for promoting neural growth and/or remyelination and/or neuroprotection in vivo in the central nervous system of a mammal comprising administering to said mammal a neural growth and/or remyelination and/or neuroprotection promoting amount of the carbohydrate epitope mimic peptide(s) of the present invention, which peptide is capable of overcoming inhibitory molecular cues found on glial cells and myelin and promoting said neural growth; variants, analogs or active fragments thereof, antagonists thereof, antibodies thereto, and secreting or expressing cells thereof.
  • the invention provides a method of promoting neural growth and/or remyelination and/or neuroprotection in vivo in the central nervous system of a mammal comprising administering to said mammal a neural growth and/or remyelination and/or neuroprotection promoting amount of the carbohydrate epitope mimic peptide(s) of the present invention, variants, analogs or active fragments thereof, antagonists thereof, antibodies thereto, and secreting or expressing cells thereof, further comprising administering to said mammal a neural growth and/or remyelination and/or neuroprotection promoting amount of a neural cell adhesion molecule.
  • the neural cell adhesion molecule is selected from the group consisting of LI, N-CAM and myelin-associated glycoprotein.
  • neural cell adhesion molecule is selected from the group consisting of laminin, fibronectin, N-cadherin, BSP-2/D2 (mouse N-CAM), 224-1A6-A1, Ll-CAM, NILE (rat LI), Nr-CAM, TAG-1 (axonin-1), Ng-CAM and F3/Fl l/contactin.
  • the present invention further relates to a method for promoting neural growth and/or remyelination and/or neuroprotection in vivo in the central nervous system of a mammal comprising administering to said mammal a neural growth promoting amount of an agent, said agent comprising a neural cell adhesion molecule, which molecule is capable of overcoming inhibitory molecular cues found on glial cells and myelin and promoting said neural growth, active fragments thereof, secreting cells thereof and soluble molecules thereof, said agent being modified by recombinant or chemical means to have the carbohydrate epitope mimic peptide(s) of the present invention, variants, analogs or active fragments thereof, attached thereto.
  • an agent comprising a neural cell adhesion molecule, which molecule is capable of overcoming inhibitory molecular cues found on glial cells and myelin and promoting said neural growth, active fragments thereof, secreting cells thereof and soluble molecules thereof, said agent being modified by recombinant
  • the neural cell adhesion molecule is selected from the group consisting of LI, N-CAM and myelin-associated glycoprotein.
  • the neural cell adhesion molecule is selected from the group consisting of laminin, fibronectin, N-cadherin, BSP-2/D2 (mouse N- CAM), 224-1A6-A1, Ll-CAM, NILE (rat LI), Nr-CAM, TAG-1 (axonin-1), Ng- CAM and F3/F11/contactin.
  • It is a further object to provide a method for enhancing memory comprising administering to the brain of a mammal in need of such enhancement, an amount of the carbohydrate epitope mimic peptide(s) of the present invention, variants, analogs or active fragments thereof effective to enhance the memory of the mammal.
  • a method for enhancing memory comprises administering to the brain of said mammal an amount of a neural cell adhesion molecule effective to enhance the memory of the mammal.
  • the method for enhancing memory comprises a method for inhibiting the onset or progression, or treating the presence or consequences of Alzheimers disease or dementia in a mammal.
  • the method for enhancing memory comprises a method for inhibiting the onset or progression, or treating the presence or consequences of Alzheimers disease or dementia in a mammal.
  • the present invention provides a method for increasing synaptic efficacy in the CNS of a mammal comprising administering to the brain of the mammal, an amount of the carbohydrate epitope mimic peptide(s) of the present invention, variants, analogs or active fragments thereof effective to increase synaptic efficacy in the brain of the mammal.
  • the increase in synaptic efficacy is demonstrated by the stabilization of long term potentiation.
  • the present invention provides a method of promoting neuroprotection and/or neuronal survival in a mammal comprising delivering to the cells of the brain of a mammal in need thereof, a vector which allows for the expression of the carbohydrate epitope mimic peptide(s) of the present invention, variants, analogs or active fragments thereof-
  • a method comprises a method for inhibiting the development or onset, or treating the presence in a mammal of a condition selected from the group consisting of apoptosis, necrosis, Alzheimers disease, dementia, Parkinsons disease, multiple sclerosis, acute spinal cord injury, chronic spinal cord injury, any of the foregoing where neurodegeneration occurs or may occur, and combinations thereof.
  • the present invention provides a method for inhibiting axonal cell death and enhancing myelination and remyelination in the central nervous system of a mammal comprising administering to said mammal a therapeutically effective amount of the carbohydrate epitope mimic peptide(s) of the present invention, which peptide is capable of overcoming inhibitory molecular cues found on glial cells and myelin and promoting said neural growth, variants, analogs or active fragments thereof, antagonists thereof, antibodies thereto, and secreting or expressing cells thereof.
  • the viral infection is the result of the human immunodeficiency virus.
  • carbohydrate epitope mimic peptide(s) whose sequences are presented in SEQ ID NOS: 1-8, 27-38, 39, 40 and 41 herein, variants, analogs, derivatives, agonists, antagonists, or active fragments thereof, could be prepared in pharmaceutical formulations for administration in instances wherein therapy to activate, inhibit or otherwise modulate L2 HNK-1 carbohydrate-recognizing molecules is appropriate, such as to promote neural growth in CNS or PNS therapy and as otherwise recited hereinabove.
  • carbohydrate epitope mimic peptide(s) hereof would make it possible to better manage the untoward effects of current CNS or PNS therapy, and would thereby make it possible to apply the carbohydrate epitope mimic peptide(s) as a general neural growth or neuroprotection promoting agent.
  • compositions for use in therapeutic methods which comprise or are based upon the carbohydrate epitope mimic peptide(s), variants, analogs, derivatives or active fragments thereof, their binding partner(s), or upon agents or compounds that control the production, or that mimic or antagonize the activities of the L2 HNK-1 carbohydrate epitope, all as aforesaid.
  • a pharmaceutical composition for promoting neural growth and/or remyelination and/or neuroprotection comprising a therapeutically effective amount of a carbohydrate epitope mimic peptide(s), variants, analogs, derivatives or active fragments thereof, and secreting or expressing cells thereof, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises a therapeutically effective amount of a neural cell adhesion molecule.
  • the neural cell adhesion molecule is selected from the group consisting of LI, N-CAM and myelin-associated glycoprotein.
  • neural cell adhesion molecule is selected from the group consisting of laminin, fibronectin, N-cadherin, BSP-2/D2 (mouse N-CAM), 224-1A6-A1, Ll-CAM, NILE (rat LI), Nr-CAM, TAG-1 (axonin-1), Ng-CAM and F3 Fl l/contactin.
  • It is an object of the present invention to provide a pharmaceutical composition for preventing, ameliorating or blocking viral infection comprising a therapeutically effective amount of the peptide of the present invention or variants, analogs, derivatives or active fragments thereof and a pharmaceutically acceptable carrier.
  • the invention encompasses derivatives of a carbohydrate epitope mimic peptide, including derivatives of variants, analogs or active fragments of such peptide.
  • derivatives encompass and include derivatives to enhance activity, solubility, effective therapeutic concentration, and transport across the blood brain barrier.
  • derivatives include the attachment of moieties or molecules which are known to contain the L2/HNK-1 carbohydrate epitope or which recognize the L2/HNK-1 carbohydrate epitope-
  • a derivative includes a derivative of the carbohydrate epitope mimic peptide(s) of the present invention, variants, analogs or active fragments thereof, capable of mimicking the carbohydrate epitope GlcA ⁇ l ⁇ 3Gal ⁇ l ⁇ 4GlcNAc, having one or more chemical moieties attached thereto.
  • a derivative in object includes a derivative wherein at least one of said chemical moieties is a water-soluble polymer capable of enhancing solubility of said peptide. Still more particular is a derivative wherein at least one of said chemical moeities is a molecule which facilitates transfer or transport across the blood brain barrier. A further and more particular object is to provide a derivative wherein said molecule is selected from the group consisting of a biocompatible hydrophobic molecule, transferrin, ApoE or ApoJ.
  • neural cell adhesion molecule is selected from the group consisting of LI, N-CAM and myelin-associated glycoprotein.
  • neural cell adhesion molecule is selected from the group consisting of laminin, fibronectin, N- cadherin, BSP-2/D2 (mouse N-CAM), 224-1A6-A1, Ll-CAM, NILE (rat LI), Nr- CAM, TAG-1 (axonin-1), Ng-CAM and F3/F11/contactin.
  • the present invention also relates to nucleic acid sequences, or degenerate variants thereof, which encode a carbohydrate epitope mimic peptide, particularly a peptide capable of mimicking the L2/HNK-1 carbohydrate epitope.
  • a nucleic acid molecule in particular a recombinant DNA molecule, encoding the L2/HNK-1 carbohydrate epitope mimic peptide, which in a particular embodiment comprises a nucleotide sequence capable of encoding the peptide set out in any of SEQ ID NOs: 1-8, 27-38, 39, 40 or 41 or which is complementary to such a nucleotide sequence.
  • a recombinant DNA molecule (or its complement) which encodes the peptide set out in any of SEQ ID NOs: 1-8, 27-38, 39, 40 or 41.
  • Further particular examples of such a DNA sequence or recombinant DNA molecule, capable of encoding the peptide F L H T RL F V (SEQ ID NO: 8), are provided in SEQ ID NOS: 21-26.
  • Examples of such a DNA sequence or recombinant DNA molecule, capable of encoding the peptide TRLFR V F (SEQ ID NO: 39) are provided in SEQ ID NOS: 42-44 and examples capable of encoding the peptide TRLF(R)V (SEQ ID NO: 40) are provided in SEQ ID NOS: 45-47.
  • Still further particular examples of such a DNA sequence or recombinant DNA molecule, capable of encoding the peptide TRLF (SEQ ID NO: 41) are provided in SEQ ID NOS: 48-50.
  • the DNA sequences of the carbohydrate epitope mimic peptide(s) of the present invention or portions thereof, may be prepared as probes to screen for complementary sequences.
  • the present invention extends to probes so prepared that may be provided for screening phage, cDNA and genomic libraries for the carbohydrate epitope mimic peptide(s).
  • the probes may be prepared with a variety of known vectors, such as the phage ⁇ vector.
  • the present invention also includes the preparation of plasmids including such vectors, and the use of the DNA sequences to construct vectors expressing antisense RNA or ribozymes which would attack the mRNAs of any or all of the DNA sequences which are capable of encoding the peptide set out in any of SEQ ID NOS: 1-8, 27-38, 39, 40 and 41.
  • the preparation of antisense RNA and ribozymes are included herein.
  • the full DNA sequence of the recombinant DNA molecule may be operatively linked to an expression control sequence which may be introduced into an appropriate host.
  • the invention accordingly extends to unicellular hosts transformed with the recombinant DNA molecule comprising a DNA sequence encoding the present carbohydrate epitope mimic peptide(s), and more particularly, a complete DNA sequence which is capable of encoding the peptide set out in any of SEQ ID NOS: 1-8, 27-38, 39, 40 and 41.
  • A DNA capable of encoding the peptide set out in any of SEQ ID NOS : 1-8, 27-38, 39, 40 and 41;
  • B DNA sequences that hybridize to any of the foregoing DNA sequences under standard hybridization conditions;
  • the present invention naturally contemplates several means for preparation of the carbohydrate epitope mimic peptide, including as illustrated herein known peptide synthesis and recombinant techniques, and the invention is accordingly intended to cover such synthetic preparations within its scope.
  • the nucleic acid and amino acid sequences disclosed herein facilitates the reproduction of the carbohydrate epitope mimic peptide, including variants, analogs and active fragments thereof, by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the disclosed DNA sequences for expression in host systems by recombinant DNA techniques, and to the resulting transformed hosts.
  • said DNA sequence is operatively linked to an expression control sequence.
  • said expression control sequence is selected from the group consisting of the early or late promoters of S V40 or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage ⁇ , the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase and the promoters of the yeast ⁇ -mating factors, the promoters of neural cell adhesion molecules, the promoter of LI, the gFAP promoter and the promoter of myelin basic protein.
  • the invention also provides a unicellular host transformed with a recombinant DNA molecule comprising a DNA sequence or degenerate variant thereof, which encodes a carbohydrate epitope mimic peptide, including variants, analogs and active fragments thereof, selected from the group consisting of: (A) DNA capable of encoding the peptide set out in any of SEQ ID NOS :
  • the unicellular host is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeasts, CHO, Rl.l, B-W, L-M, COS 1, COS 7, BSC1, BSC40, and BMT10 cells, plant cells, insect cells, mammalian cells, human cells and neural cells in tissue culture.
  • a cloning vector which comprises the DNA sequence encoding a carbohydrate epitope mimic peptide, including variants, analogs and active fragments thereof, and a heterologous nucleotide sequence-
  • a recombinant expression system is provided to produce biologically active carbohydrate epitope mimic peptide, including variants, analogs and active fragments thereof.
  • an expression vector which comprises a DNA sequence encoding a carbohydrate epitope mimic peptide, including variants, analogs and active fragments thereof, and a heterologous nucleotide sequence.
  • the heterologous nucleotide sequence is an expression control sequence.
  • the heterologous nucleotide sequence encodes a neural cell adhesion molecule.
  • the invention includes an assay system for screening of potential drugs effective to modulate L2/HNK-1 carbohydrate epitope recognizing activity of target mammalian cells by mimicking, interrupting or potentiating the interaction or recognition of the L2/HNK-1 carbohydrate epitope.
  • the test drug could be administered to a cellular sample with the L2/HNK-1 carbohydrate epitope recognizing molecule, or an extract containing the carbohydrate epitope mimic peptide, to determine its effect upon the binding activity of the L2/HNK-1 carbohydrate epitope recognizing molecule, by comparison with a control.
  • the assay system could more importantly be adapted to identify drugs or other entities that are capable of binding to the L2/HNK-1 carbohydrate epitope recognizing molecule, thereby inhibiting or potentiating the activity of the carbohydrate epitope mimic peptide.
  • Such assay would be useful in the development of drugs that would be specific against particular cellular activity, or that would potentiate such activity, in time or in level of activity.
  • the invention contemplates antagonists of the activity of a carbohydrate epitope mimic peptide.
  • A contacting a sample in which the presence or activity of said peptide or compound is suspected with a binding partner of said peptide or compound under conditions that allow binding of said peptide or compound to said binding partner to occur;
  • the binding partner is selected from the group consisting of an antibody which recognizes GlcA ⁇ l ⁇ 3Gal ⁇ l ⁇ 4GlcNAc; an antibody which recognizes sulfate -3GlcA ⁇ l->3Gal ⁇ l-4GlcNAc; L2-412 antibody; HNK-1 antibody; a polypeptide molecule which binds or otherwise interacts with GlcA ⁇ l ⁇ 3Gal ⁇ l ⁇ 4GlcNAc or sulfate -3GlcA ⁇ l ⁇ 3Gal ⁇ l-4GlcNAc; laminin; P- selectin; L-selectin; and a neural cell adhesion molecule.
  • a method of testing the ability of a drug or other entity to mimic the carbohydrate epitope GlcA ⁇ 1 ⁇ 3 Gal ⁇ 1 ⁇ 4GlcNAc or sulfate - 3GlcA ⁇ l ⁇ 3Gal ⁇ l-4GlcNAc which comprises: a. adding CNS neurons to a cell culture system; b. adding the drug or other entity under test to the cell culture system; c. measuring the neuronal outgrowth of the CNS neurons; and d.
  • the diagnostic utility of the present invention extends to the use of the present carbohydrate epitope mimic peptide in assays to screen for L2/HNK-1 carbohydrate epitope recognizing molecules.
  • the carbohydrate epitope mimic peptide(s), including variants, analogs and active fragments thereof, and any antagonists or antibodies that may exist or be raised thereto are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay, using for example, an antibody to the carbohydrate epitope mimic peptide that has been labeled by either radioactive addition, or radioiodination.
  • a control quantity of the antagonists or antibodies thereto, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.
  • radioactive label such as the isotopes 3 H, 14 C, 32 P, 35 S, 36 C1, 51 Cr, "Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, and 186 Re
  • known currently available counting procedures may be utilized.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
  • the present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of the carbohydrate epitope mimic peptide, or to identify drugs or other agents that may mimic or block their activity.
  • the system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the carbohydrate epitope mimic peptide, their agonists and/or antagonists, and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s).
  • the invention thus provides a test kit for the demonstration of a molecule capable of binding GlcA ⁇ l ⁇ 3Gal ⁇ 4GlcNAc or sulfate -3GlcA ⁇ l-3Gal ⁇ l ⁇ 4GlcNAc in a eukaryotic cellular sample, comprising:
  • the invention further provides a test kit for demonstrating the presence of a molecule capable of binding 3GlcA ⁇ l-3Gal ⁇ l-4GlcNAc or sulfate - 3GlcA ⁇ l ⁇ 3Gal ⁇ l- , 4GlcNAc in a eukaryotic cellular sample, comprising:
  • the present invention likewise extends to the development and use of antibodies against the carbohydrate epitope mimic peptide(s), including naturally raised and recombinantly prepared antibodies.
  • Such antibodies could include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific (chimeric) antibodies, and antibodies including other functionalities suiting them for additional diagnostic use conjunctive with their capability of modulating carbohydrate epitope mimic peptide activity.
  • FIGURE 1 is a flow diagram of the phage library screening. The library was screened in three cycles, or rounds, of panning with the antibody L2-412 or the antibody HNK- 1.
  • FIGURE 2A depicts competition of the L2-412 antibody to immobilized L2/HNK-1 glycolipids by various inhibitors: positive phage (denoted phage 15-15), negative phage (denoted neg. control phage), free peptide (denoted peptide 15-15) and SO 3 - sugar.
  • L2-412 was preincubated with a stepwise 2-fold dilution series of the free peptide (starting concentration 2.2mM ), SO 3 -sugar (starting concentration 5mM), positive phage and negative phage (starting concentration 10 12 TUs/ml), and added to the coated glycolipid. After incubation and washing, the bound antibody was detected with HRP anti-rat antibody.
  • FIGURE 2B depicts the percentage inhibition of L2-412 antibody to immobilized L2/HNK-1 glycolipids by various inhibitors. The percentage is calculated for the point in Figure 2 A with the highest concentration of inhibitor. The binding of L2-412 in the absence of inhibitor is defined as 0% inhibition. Mean +/- values standard deviation from 4 experiments carried out in duplicate.
  • FIGURE 3 Depicts competition of positive phage binding to immobilized L2-412 with the 15-15 peptide coupled to BSA.
  • FIGURE 4 depicts competition of positive phage binding to immobilized laminin with the 15-15 peptide coupled to BSA.
  • FIGURE 5 depicts binding of phage 15-15 and control phage UBR2 to immobilized laminin (100 ⁇ l of 10 ⁇ g/ml used for coating). Bound phage were detected by HRP- conjugated anti-M13 antibody. The results are presented as OD 405 vs. relative concentration of the phage preparation (a relative concentration of 100 corresponds to 10 12 TUs/ml phage).
  • FIGURE 6 depicts the binding of biotinylated peptide -BSA toL2-412 in a concentration -dependent manner, biot BSA is the control biotinylated BSA.
  • FIGURE 7 depicts the binding of biotinylated peptide -BSA to immobilized laminin in a concentration -dependent manner, biot BSA is the control biotinylated BSA.
  • FIGURE 8 is a diagramatic representation of outgrowth of neurites from chick motor neurons on substrate consisting of collagen mixed with the peptide -BSA conjugates or BSA as control.
  • FIGURE 9A-9C shows outgrowth of neurites from motor neurons (network) cultured on substrate consisting of: (A) 8 amino acid peptide coupled to BSA 15 amino acid peptide coupled to BSA; (B) scrambled 8 amino acid peptide coupled to BSA scrambled 15 amino acid peptide coupled to BSA; and (C) BSA.
  • the bar represents 20 ⁇ m.
  • FIGURE 10 depicts the average length of the longest neurite and average length of all neurites when cultured in the presence of: the 8 amino acid peptide; the L2-HNK-1 glycolipid; the 15 amino acid peptide; the scrambled 8 amino acid peptide; the scrambled 15 amino acid peptide; and BSA.
  • FIGURE 11 depicts the degree of polarity, calculated as the ratio of the mean length of the longest neurite divided by the average length of all neurites, of motor neurons cultured in the presence of: 8 amino acid peptide;L2/HNK-l glycolipid; 15 amino acid peptide; scrambled 8 amino acid peptide; scrambled 15 amino acid peptide; and BSA.
  • FIGURE 12A-12F depicts the outgrowth of neurites from dorsal root ganglion neurons cultured on substrate consisting of: (A) BSA; (B) 8 amino acid peptide coupled to BSA; (C) 15 amino acid peptide coupled to BSA; (D) scrambled 15 amino acid peptide coupled to BSA; (E) BSA; and (F)L2/HNK-1 glycolipid.
  • the bar represents 20 ⁇ m.
  • FIGURE 13A-13C shows staining of motor neurons by: (A) biotinylated 8 amino acid peptide coupled to BSA; (B) biotinylated scrambled 8 amino acid peptide coupled to BSA; and (C) biotinylated BSA. Detection was done with streptavidin-HRP. The bar represents 20 ⁇ m.
  • FIGURE 14 depicts binding of HNK-1 selected phage 15H92 and 15H233, L2-412 selected 15-15 phage, and controls UBR2 and UBH to bound L2-412 antibody, IgG, HNK-1 antibody, and IgM. Detection was done with HRP-coupled anti-phage antibody.
  • FIGURE 15 depicts comparative binding of various phage clones to bound antibody L2-412 and antibody HNK-1.
  • L2-412 selected phage clones are 15-90, 15-91, 15-92, 15-93, 15-94 and 15-95.
  • HNK-1 selected phage clones are 15H92, 15H94, 15H86, 15H85, 15H78, 15H36, 15H34 and 15H26.
  • K91Kan, UB412 and UB HNK-1 are controls. Detection was done with HRP-coupled anti-phage antibody.
  • FIGURE 16 depicts comparative binding of various phage clones to bound antibody L2-412 and antibody HNK-1.
  • L2-412 selected phage clones are 15cho4, 15-94, 15-15 and 15phl.
  • HNK-1 selected clones are 15H212, 15H207, 15H208, 15H26, 15H78, 15H233, 15H136 and 15H92.
  • UBR2 and UBH are unbound phage controls. Detection was done with HRP-coupled anti-phage antibody.
  • FIGURE 17 depicts comparative binding of phage 15-15, 15H92 and unbound phage UBR2 and UBH to bound antibodies L2-412 and HNK-1. Detection was done with HRP-coupled anti-phage antibody. The vertical axis indicating absorbance at OD 405nm. The sequences of the 15-mer phage inserts of 15-15 (SEQ ID NO: 28) and 15H92 (SEQ ID NO: 34) are also shown, with the homologous (consensus) amino acids in bold.
  • FIGURE 18 depicts L2 glycolipid binding to CD4 peptide in a concentration-dependent manner.
  • FIGURE 19 depicts competition of L2 glycolipid binding to immobilized laminin with the CD4 peptide.
  • FIGURE 20 depicts fluorescence microscopy of cultures treated with gpl20 alone or with HNK-1 epitope mimic peptide.
  • A (Upper left hand panel): Culture was not treated with either the HNK-1 epitope mimic peptide or gpl20. RIP positive oligodendrocytes were observed in control wells, but only minimal membrane deposition onto the substrate was seen.
  • B (Upper right hand panel): Culture was treated with lOnM HNK-1 epitope mimic peptide. Numerous mature RIP positive oligodendrocytes with extensive membrane sheaths were observed.
  • C (Bottom left hand panel): Culture was treated with lnM gpl20. Mature RIP positive oligodendrocytes with intact sheaths of membrane were not observed.
  • RIP positive oligodendrocytes observed in these cultures were immature oligodendrocytes, lacking membrane sheaths and RIP positive oligodendrocytes with collapsed processes, i.e., degenerating mature oligodendrocytes.
  • D (Bottom right hand panel): Culture was treated with lnM gpl20 that was preincubated with luM HNK-1 epitope mimic peptide. Mature RIP positive cells were indistinguishable from mature RIP positive cells observed in cultures treated with the HNK-1 epitope mimic peptide only. Oligodendrocytes were observed elaborating extensive sheaths of membrane.
  • the present invention encompasses carbohydrate epitope mimic peptides, which peptides mimic the structure and/or activity of carbohydrate epitopes.
  • the present invention is particularly exemplified in L2/HNK1 carbohydrate epitope mimic peptides, capable of mimicking the structure and/or activity of the L2 and/or HNK1 epitope, particularly the carbohydrate epitope GlcA ⁇ l- 3Gal ⁇ l ⁇ 4GlcNAc or sulfate - 3GlcA ⁇ l-3Gal ⁇ l-4GlcNAc .
  • carbohydrate epitope mimic peptides mimic or can otherwise replace, interact with, block, or facilitate particular carbohydrate epitopes which participate in carbohydrate-protein and protein-protein interactions. These carbohydrate epitopes and carbohydrate epitope containing molecules interact with themselves and/or carbohydrate epitope recognizing molecules.
  • L2/HNK1 carbohydrate epitope mimic peptides which particularly mimic the carbohydrate epitope GlcA ⁇ l ⁇ 3Gal ⁇ l ⁇ 4GlcNAc or sulfate- 3GlcA ⁇ 1 -*3Gal ⁇ 1 -4GlcNAc.
  • L2/HNK1 carbohydrate epitope mimic peptides comprising the amino acid sequences set out in any of SEQ ID NOS: 1-8, 27-38, 39,
  • carbohydrate epitope mimic peptide(s) may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including peptides which mimic the structure of a carbohydrate epitope, thereby mimicking, modulating or otherwise facilitating the activity of the carbohydrate epitope or ligand.
  • Carbohydrate epitope mimic peptide(s) are particularly exemplified herein in the peptides of the present invention which mimic the carbohydrate epitope GlcA ⁇ l ⁇ 3Gal ⁇ l ⁇ 4GlcNAc or sulfate-3GlcA ⁇ l-3Gal ⁇ l-'4GlcNAc-
  • the carbohydrate epitope mimic peptide(s) particularly exemplified herein mimic the L2/HNK1 epitope and comprise peptides having the amino acid sequences described herein and presented in SEQ ID NOS: 1-8, 39, 40 and 41 and in TABLE 2 and TABLE 4, and the profile of activities and characteristics set forth herein and in the Claims.
  • carbohydrate epitope mimic peptide(s) “carbohydrate epitope mimic”, “carbohydrate epitope peptidomimetic” and “peptidomimetic” are intended to include within their scope those peptides specifically recited herein as well as all variants, analogs and active fragments thereof, including substantially homologous variants and analogs.
  • L2/HNK1 carbohydrate epitope mimic peptide(s) may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including peptides, and extends to those peptides having the amino acid sequences described herein and presented in SEQ ID NOS: 1-8, 39, 40 and
  • peptides displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through screening for carbohydrate epitope mimic peptide(s)using the methods and assays provided and described herein.
  • L2/HNK1 carbohydrate epitope mimic peptide(s) are intended to include within their scope those peptides specifically recited herein as well as all variants, analogs and active fragments thereof, including substantially homologous variants and analogs.
  • amino acid residues may be changed or modified to include, for example, active fragments such as deletions containing less than all of the residues specified for the peptide, variants wherein one or more residues are replaced or substituted by other residues or wherein one or more amino acid residues are added to a terminal or medial portion of the peptide, and analogs wherein one or more residues are replaced or substituted with unnatural amino acids, L-amino acids, various "designer" amino acids (for example ⁇ -methyl amino acids, C ⁇ -methyl amino acids, and N ⁇ -methyl amino acids), nonclassical amino acids or synthetic amino acids. Analogs further encompass cyclic peptides, which can be generated by any of recognized methods in the art.
  • amino acid residues described herein are preferred to be in the "L" isomeric form.
  • residues in the "D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin-binding is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino- terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
  • the above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.
  • Synthetic peptide prepared using the well known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids.
  • Amino acids used for peptide synthesis may be standard Boc (N ⁇ -amino protected N ⁇ -t-butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or the base- labile N ⁇ -amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and Han (1972, J. Org. Chem. 37:3403-3409).
  • polypeptide of the invention may comprise D-amino acids, a combination of D- and L- amino acids, and various "designer" amino acids (e.g., ⁇ -methyl amino acids, C ⁇ - methyl amino acids, and N ⁇ -methyl amino acids, etc.) to convey special properties.
  • Synthetic amino acids include ornithine for lysine, fluorophenylalanine for phenylalanine, and norleucine for leucine or isoleucine. Additionally, by assigning specific amino acids at specific coupling steps, ⁇ -helices, ⁇ turns, ⁇ sheets, ⁇ -turns, and cyclic peptides can be generated.
  • the peptides may comprise a special amino acid at the C-terminus which incorporates either a CO 2 H or CONH 2 side chain to simulate a free glycine or a glycine-amide group. Another way to consider this special residue would be as a D or L amino acid analog with a side chain consisting of the Unker or bond to the bead.
  • the pseudo-free C-terminal residue may be of the D or the L optical configuration; in another embodiment, a racemic mixture of D and L- isomers may be used.
  • pyroglutamate may be included as the N-terminal residue of the peptide.
  • pyroglutamate is not amenable to sequence by Edman degradation, by limiting substitution to only 50% of the peptides on a given bead with N-terminal pyroglutamate, there will remain enough non-pyroglutamate peptide on the bead for sequencing.
  • this technique could be used for sequencing of any peptide that incorporates a residue resistant to Edman degradation at the N-terminus. Other methods to characterize individual peptides that demonstrate desired activity are described in detail infra.
  • peptides that have more well defined structural properties, and the use of peptidomimetics, and peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties.
  • a peptide may be generated that incorporates a reduced peptide bond, i.e., R ! -CH 2 -NH-R 2 , where R ! and R 2 are amino acid residues or sequences.
  • a reduced peptide bond may be introduced as a dipeptide subunit.
  • Such a molecule would be resistant to peptide bond hydrolysis, e.g., protease activity.
  • Such peptides would provide ligands with unique function and activity, such as extended half-lives in vivo due to resistance to metabolic breakdown, or protease activity. Furthermore, it is well known that in certain systems constrained peptides show enhanced functional activity (Hruby, 1982, Life Sciences 31 : 189-199; Hruby et al., 1990, Biochem J. 268:249-262); the present invention provides a method to produce a constrained peptide that incorporates random sequences at all other positions.
  • a constrained, cyclic or rigidized peptide may be prepared synthetically, provided that in at least two positions in the sequence of the peptide an amino acid or amino acid analog is inserted that provides a chemical functional group capable of cross-linking to constrain, cyclise or rigidize the peptide after treatment to form the cross-link. Cyclization will be favored when a turn-inducing amino acid is incorporated.
  • Examples of amino acids capable of cross-linking a peptide are cysteine to form disulfide, aspartic acid to form a lactone or a lactase, and a chelator such as ⁇ -carboxyl-glutamic acid (Gla) (Bachem) to chelate a transition metal and form a cross-link.
  • Protected ⁇ -carboxyl glutamic acid may be prepared by modifying the synthesis described by Zee-Cheng and Olson (1980, Biophys. Biochem. Res. Commun. 94: 1128-1132).
  • a peptide in which the peptide sequence comprises at least two amino acids capable of cross-linking may be treated, e.g., by oxidation of cysteine residues to form a disulfide or addition of a metal ion to form a chelate, so as to cross-link the peptide and form a constrained, cyclic or rigidized peptide-
  • the present invention provides strategies to systematically prepare cross-links. For example, if four cysteine residues are incorporated in the peptide sequence, different protecting groups may be used (Hiskey, 1981, in 77--e Peptides: Analysis, Synthesis, Biology, Vol. 3, Gross and Meienhofer, eds., Academic Press: New York, pp. 137- 167; Ponsanti et al., 1990, Tetrahedron 46:8255-8266). The first pair of cysteine may be deprotected and oxidized, then the second set may be deprotected and oxidized. In this way a defined set of disulfide cross-links may be formed. Alternatively, a pair of cysteine and a pair of collating amino acid analogs may be incorporated so that the cross-links are of a different chemical nature-
  • non-classical amino acids may be incorporated in the peptide in order to introduce particular conformational motifs: l,2,3,4-tetrahydroisoquinoline-3- carboxylate (Kazmierski et al., 1991, J. Am. Chem. Soc. 113:2275-2283); (2S,3S methyl-phenylalanine, (2S,3R)-methyl-phenylalanine, (2R,3 S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine (Kazmierski and Hruby, 1991, Tetrahedron Lett.); 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis, 1989, Ph.D. Thesis,
  • LL-Acp LL-3-amino- 2-propenidone-6-carboxylic acid
  • ⁇ -turn inducing dipeptide analog Kemp et al., 1985, J. Org. Chem. 50:5834-5838
  • ⁇ -sheet inducing analogs Kemp et al., 1988, Tetrahedron Lett. 29:5081-5082
  • ⁇ -turn inducing analogs Kemp et al., 1988, Tetrahedron Lett.
  • the present invention further provides for modification or derivatization of the polypeptide or peptide of the invention.
  • Modifications of peptides are well known to one of ordinary skill, and include phosphorylation, carboxymethylation, and acylation. Modifications may be effected by chemical or enzymatic means.
  • glycosylated or fatty acylated peptide derivatives may be prepared. Preparation of glycosylated or fatty acylated peptides is well known in the art.
  • Fatty acyl peptide derivatives may also be prepared. For example, and not by way of limitation, a free amino group (N-terminal or lysyl) may be acylated, e.g., myristoylated.
  • an amino acid comprising an aliphatic side chain of the structure - (CH 2 ) n CH 3 may be incorporated in the peptide.
  • This and other peptide-fatty acid conjugates suitable for use in the present invention are disclosed in U.K. Patent GB- 8809162.4, International Patent Application PCT/AU89/00166, and reference 5, supra.
  • Chemical Moieties For Derivatization Derivatives of the peptides (including variants, analogs and active fragments thereof) of the present invention are further provided. Such derivatives encompass and include derivatives to enhance activity, solubility, effective therapeutic concentration, and transport across the blood brain barrier.
  • compositions of the present invention include the attachment of moieties or molecules which are known to contain the L2/HNK-1 carbohydrate epitope or which recognize the L2/HNK-1 carbohydrate epitope.
  • the chemical moieties may be N-terminally or C-terminally attached to the peptides of the present invention.
  • Chemical moieties suitable for derivatization may be, for instance, selected from among water soluble polymers.
  • the polymer selected can be water soluble so that the component to which it is attached does not precipitate in an aqueous environment, such as a physiological environment.
  • the polymer will be pharmaceutically acceptable.
  • the polymer may be branched or unbranched.
  • the desired polymer based on such considerations as whether the polymer/component conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations. For the present component or components, these may be ascertained using the assays provided herein.
  • the water soluble polymer may be selected from the group consisting of, for example, polyethylene glycol, copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohol.
  • Polyethylene glycol propionaldenhyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight is between about 2kDa and about lOOkDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
  • Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
  • polymer molecules so attached may vary, and one skilled in the art will be able to ascertain the effect on function.
  • One may mono-derivative, or may provide for a di-, tri-, tetra- or some combination of derivatization, with the same or different chemical moieties (e.g., polymers, such as different weights of polyethylene glycols).
  • the proportion of polymer molecules to component or components molecules will vary, as will their concentrations in the reaction mixture.
  • the optimum ratio in terms of efficiency of reaction in that there is no excess unreacted component or components and polymer
  • the desired degree of derivatization e.g., mono, di-, tri-, etc.
  • the molecular weight of the polymer selected whether the polymer is branched or unbranched, and the reaction conditions.
  • polyethylene glycol molecules should be attached to the component or components with consideration of effects on functional or antigenic domains of the protein.
  • attachment methods available to those skilled in the art, e.g., EP 0 401 384 herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al, 1992, Exp. Hematol 20:1028-1035 (reporting pegylation of GM-CSF using tresyl chloride).
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • amino acid residues having a free amino group include lysine residues and the - terminal amino acid residues; those having a free carboxyl group include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue.
  • Sulfhydrl groups may also be used as a reactive group for attaching the polyethylene glycol molecule(s). Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
  • the invention provides derivatives wherein at least one of said attached chemical moieties is a molecule which facilitates transfer or transport across the blood-brain barrier, particularly molecules that naturally cross the blood-brain barrier.
  • molecules include a biocompatible hydrophobic molecule, transferrin or apolipoprotein. Transferrin has been shown to facilitate transfer, even or larger peptides, as for example, nerve growth factor (Friden, P.M. et al, Science 259, 373- 377 (1993), Kordower, J.H. et al, Proc Natl Acad Sci USA 91, 9077-9080 (1994)).
  • Apolipoprotein E Apolipoprotein E
  • ApoJ apolipoproetin J
  • the present invention provides derivatives which are fusion proteins comprising the peptides of the present invention or fragments thereof.
  • peptides of the present invention and fragments thereof can be "modified” i.e., placed in a fusion of chimeric peptide or protein, or labeled, e.g., to have an N-terminal FLAG- tag.
  • a peptide can be modified by linkage or attachment to a marker protein such as green fluorescent protein as described in U.S. Patent No. 5,625,048 filed April 29, 1997 and WO 97/26333, published July 24, 1999 (each of which are hereby incorporated by reference herein in their entireties).
  • a chimeric peptide can be prepared, e.g., a glutathione- S- transferase (GST) fusion protein, a maltose-binding (MPB) protein fusion protein, or a poly-histidine-tagged fusion protein, for expression in a eukaryotic cell.
  • GST glutathione- S- transferase
  • MPB maltose-binding
  • poly-histidine-tagged fusion protein for expression in a eukaryotic cell.
  • Expression of the peptide of the present invention as a fusion protein can facilitate stable expression, or allow for purification based on the properties of the fusion partner.
  • GST binds glutathione conjugated to a solid support matrix
  • MBP binds to a maltose matrix
  • poly-histidine chelates to a Ni-chelation support matrix.
  • the fusion protein can be eluted from the specific matrix with appropriate buffers, or by treating with a protease specific for a cleavage site usually engineered between the peptide and the fusion partner (e.g., GST, MBP, or poly-His).
  • a protease specific for a cleavage site usually engineered between the peptide and the fusion partner (e.g., GST, MBP, or poly-His).
  • the chimeric peptide may contain the green fluorescent protein, and be used to determine the intracellular localization of the peptide in the cell.
  • the neural cell adhesion molecule is selected from the group consisting of LI, N-CAM and myelin-associated glycoprotein.
  • the neural cell adhesion molecule can be selected from the group consisting of laminin, fibronectin, N-cadherin, BSP-2/D2 (mouse N- CAM), 224-1A6-A1, Ll-CAM, NILE (rat LI), Nr-CAM, TAG-1 (axonin-1), Ng- CAM and F3/F11/contactin.
  • the invention also includes derivatives wherein at least one of the attached chemical moieties is a molecule having multiple sites for peptide attachment and capable of binding at least two of said peptides simultaneously to generate a multimeric peptide structure.
  • This derivative has the effect of increasing the available local concentration of the carbohydrate epitope mimic peptide(s) of the present invention.
  • moieties can function in providing a stable scaffold to retain the peptide in place for activity, thereby reducing or preventing diffusion or degradation. More particularly, such molecule is selected from the group of BSN ovalbumin, human serum allbumin, polyacrylamide, beads and synthetic fibers (biodegradable and non-biodegradable).
  • the carbohydrate epitope mimic peptide of the present invention may be prepared and utilized as monomers, dimers, multimers, heterodimers, heteromultimers, etc.
  • the use of multimers is particularly attractive in view of the activity of carbohydrate epitopes in homophilic and cell-cell interactions. Presentation or administration of the carbohydrate epitope mimic peptide in multimeric form may result in enhanced activity or otherwise increased modulation of the activity mediated by the carbohydrate epitopes, including the activity of carbohydrate epitope recognizing molecules.
  • the carbohydrate epitope mimic peptide monomer could be produced in a variety of ways.
  • the carbohydrate epitope mimic peptide of the present invention can be synthesized using a protein synthesizer and utilizing methods well known in the art and as described hereinabove, incorporating amino acid modifications, analogs, etc. as hereinabove described.
  • the DNA sequence of the peptide can be inserted into an expression vector such as pSE (Invitrogen) or pCDNA3 (Invitrogen) for production in bacterial or mammalian cell expression systems. Insect or yeast expression systems could also be used. Purification of the peptide could be facilitated by the addition of a tag sequence such as the 6-Histidine tag which binds to Nickel- NTA resins. These tag sequences are often easily removed by the addition of a protease specific sequence following the tag.
  • Dimers can be produced using a variety of methods in the art.
  • Dimers and multimers can also be generated using crosslinking reagents such as Disuccinimidyl suberate (DSS) or Dithoiobis (succinimidyl propionate) (DSP). These reagents are reactive with amino groups and could crosslink the carbohydrate epitope mimic peptide through free amine groups at the arginine residues and the free amine group at the N-terminus. Dimers and multimers can also be formed using affinity interactions between biotin and avidin, Jun and Fos, and the Fc region of antibodies. The purified arbohydrate epitope mimic peptide can be biotinylated and mixed with factors that are known to form strong protein-protein interactions.
  • the peptidomimetic could be linked to the regions in Jun and Fos responsible for dimer formation using crosslinkers such as those mentioned above or using molecular techniques to create a carbohydrate epitope mimic peptide- Jun/Fos molecule- When the Jun and Fos carbohydrate epitope mimic peptide hybrids are mixed, dimer formation would result. In addition, production of a carbohydrate epitope mimic peptide-Fc hybrid could also be produced. When expressed in mammalian cells, covalent disulfide bonds form through cysteines in the Fc region and dimer formation would result-
  • Heterodimers and heteromultimers of the carbohydrate epitope mimic peptide could also be produced. This would generate possible multifunctional molecules where parts of the whole molecule are responsible for producing a multitude of effects, such as neuroprotection and neurite outgrowth. The same technologies as those listed above could be used to generate these multifunctional molecules.
  • Molecular techniques could be used to insert the carbohydrate epitope mimic peptide into a protein at the DNA level. This insertion could take place at the N- or C- terminus, or in the middle of the protein molecule.
  • Heterodimers could be formed using carbohydrate epitope mimic peptide/Fc or carbohydrate epitope mimic peptide/June or Fos hybrid molecules.
  • a "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a "vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogs thereof, such as phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNN DNA- RNA and RNA-RNA helices are possible.
  • nucleic acid molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. This, this term includes double-stranded and single-stranded DNA or RNA molecules.
  • a "DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double- stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • linear DNA molecules e.g., restriction fragments
  • viruses e.g., plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine- Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is "under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA which is then translated into the protein encoded by the coding sequence-
  • a “signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
  • oligonucleotide as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
  • the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains about 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the primers herein are selected to be “substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” by exogenous or heterologous. DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term “operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • standard hybridization conditions refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65 °C for both hybridization and wash.
  • Standard hybridization conditions are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like. Also important in the determination of “standard hybridization conditions” is whether the two sequences hybridizing are RNA-RNN DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20°C below the predicted or determined T m with washes of higher stringency, if desired.
  • Two DNA sequences are "substantially homologous" when at least about 80% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Nols. I & II, supra; Nucleic Acid Hybridization, supra.
  • two polypeptide sequences are "substantially homologous" when at least about 80% (preferably at least about 90%, and most preferably at least about 95%) of the amino acids are either identical or contain conservative changes, as herein defined, over the defined length of the polypeptide sequences.
  • the similar or homologous sequences are identified by alignment using sequence alignment or search programs and methods known to the skilled artisan-
  • the similar or homologous sequences are identified by alignment using the GCG pileup program (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison Wisconsin), using the default parameters-
  • the term "about” refers to approximately or close to, usually within (i.e., +/-) 10% of the given value or quantity.
  • an oligonucleotide of about 10 nucleotides encompasses between 9 and 11 nucleotides.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNN or RNA when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al, 1989, supra). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.
  • low stringency hybridization conditions corresponding to a T m of 55 °C, can be used, e.g., 5x SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5%) SDS).
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g., 40% formamide, with 5x or 6x SCC.
  • High stringency hybridization conditions correspond to the highest T m , e.g., 50% formamide, 5x or 6x SCC.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
  • the relative stability corresponding to higher T- of nucleic acid hybridizations decreases in the following order: RNA:RNN DNA:RNN DNA:DNA.
  • equations for calculating T m have been derived (see Sambrook et al, 1989, supra, 9.50-0.51).
  • a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; more preferably at least about 15 nucleotides; most preferably the length is at least about 20 nucleotides.
  • DNA sequences capable of encoding the peptides set out in SEQ ED NOS: 1-8, 27-38, 39, 40 or 41 but which are degenerate to the particular exemplary such DNA sequences ie; those degenerate to any of SEQ ID NOS: 9-26 and SEQ ID NOS: 42-50.
  • degenerate to is meant that a different three-letter codon is used to specify a particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:
  • Aspartic Acid Aspartic Acid (Asp or D) GAU or GAC Glutamic Acid (Glu or E) GAA or GAG Cysteine (Cys or C) UGU or UGC Arginine (Arg or R) CGU or CGC or CGA or CGG orAGA orAGG Glycine (Gly or G) GGU or GGC or GGA or GGG
  • Trp Tryptophan (Trp or W) UGG Termination codon UAA (ochre) or UAG (amber) or UGA (opal)
  • codons specified above are for RNA sequences.
  • the corresponding codons for DNA have a T substituted for U.
  • Mutations can be made in the DNA sequences of the present invention such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. Additionally, alterations or mutations can be made directly in the amino acid sequence of the peptide(s) of the present invention. This is particularly straightforward in that the particular exemplified peptides and active fragments thereof are of a size which makes them readily synthesized, using methods as previously described and well known in the art.
  • a substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping), thereby generating a non-conserved variant, or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping), thereby generating a non-conserved variant.
  • Such a conservative change generally leads to less change in the structure and function of the resulting protein.
  • a non-conservative change is more likely to alter the structure, activity or function of the resulting protein.
  • the present invention should be considered to include such variants containing conservative changes or non-conservative changes which do not significantly alter the activity or binding or epitope mimicking characteristics of the resulting peptide.
  • amino acids with nonpolar R groups Alanine Valine Leucine Isoleucine Proline Phenylalanine Tryptophan Methionine
  • Another grouping may be those amino acids with phenyl groups: Phenylalanine Tryptophan Tyrosine
  • Another grouping may be according to molecular weight (i.e., size of R groups): Glycine 75 Alanine 89
  • Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property.
  • a Cys may be introduced a potential site for disulfide bridges with another Cys.
  • a His may be introduced as a particularly "catalytic" site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis).
  • Pro may be introduced because of its particularly planar structure, which induces ⁇ -turns in the protein's structure-
  • Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.
  • a "heterologous" region of the D ⁇ A construct is an identifiable segment of D ⁇ A within a larger D ⁇ A molecule that is not found in association with the larger molecule in nature.
  • the heterologous region encodes a mammalian gene
  • the gene will usually be flanked by D ⁇ A that does not flank the mammalian genomic D ⁇ A in the genome of the source organism-
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cD ⁇ A where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene).
  • heterologous nucleotide sequence is a nucleotide sequence that is added to a nucleotide sequence of the present invention by recombinant methods to form a nucleic acid which is not naturally formed in nature.
  • nucleic acids can encode chimeric and/or fusion proteins.
  • heterologous nucleotide sequence can encode peptides and/or proteins which contain regulatory and/or structural properties.
  • the heterologous nucleotide can encode a protein or peptide that functions as a means of detecting the peptide encoded by the nucleotide sequence of the present invention after the recombinant nucleic acid is expressed.
  • the heterologous nucleotide can function as a means of detecting a nucleotide sequence of the present invention.
  • a heterologous nucleotide sequence can comprise non-coding sequences including restrictions sites, regulatory sites, promoters and the like.
  • an “antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope.
  • the term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567.
  • an "antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • antibody molecule in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab 1 , F(ab') 2 and F(v), which portions are preferred for use in the therapeutic methods described herein.
  • Fab and F(ab') 2 portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous et al.
  • Fab' antibody molecule portions are also well- known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.
  • An antibody containing intact antibody molecules is preferred herein.
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen.
  • a monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the S phase activity of a target cellular mass, or other feature of pathology such as for example, elevated blood pressure, fever or white cell count as may attend its presence and activity.
  • the present invention concerns the identification of carbohydrate epitope mimic compound(s), particularly peptide(s).
  • carbohydrate epitope mimic compound(s) particularly peptide(s).
  • Such compounds or peptides particularly mimic the carbohydrate epitope GlcA ⁇ l ⁇ 3Gal ⁇ l ⁇ 4GlcNAc or sulfate- 3GlcA ⁇ l-3Gal ⁇ l-4GlcNAc.
  • the compounds or peptides are capable of mimicking the L2/HNK1 carbohydrate epitope.
  • the present invention relates peptides comprising the amino acid sequence set out in any of SEQ ID NOS: 1-8, 27-38, 39, 40 and 41.
  • Particularly preferred are peptides comprising the amino acid F L H T R L F V S D W Y H T (SEQ ID NO: 7), F L H T R L F V (SEQ ID NO: 8), TRLFR(V/F) (SEQ ID NO: 39), TRLF(R)V (SEQ ED NO: 40) or TRLF (SEQ ID NO: 41).
  • the present invention also relates to a recombinant DNA molecule, or a degenerate variant thereof, which encodes a carbohydrate epitope mimic peptide, variant, analog or active fragment thereof, that possesses an amino acid sequence set forth in any of SEQ ID NOS: 1-8, 27-38, 39, 40 and 41, preferably a nucleic acid molecule, in particular a recombinant DNA molecule.
  • exemplary nucleic acid sequences are those of SEQ ID NOS: 9-26 and SEQ ID NOS: 42-50. Sequences complementary to or degenerate to the DNA sequences of any of SEQ ID NOS: 9-26 and SEQ ID NOS: 42-50 are readily contemplated.
  • DNA sequences disclosed herein may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.
  • Such operative linking of a DNA sequence of this invention to an expression control sequence includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.
  • a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.
  • phage DNAS e.g., the numerous derivatives of phage ⁇ , e.g., NM989, and other phage DNA e.g., Ml 3 and filamentous single stranded phage
  • any of a wide variety of expression control sequences sequences that control the expression of a DNA sequence operatively linked to it — may be used in these vectors to express the DNA sequences of this invention.
  • useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the ERR system, the major operator and promoter regions of phage ⁇ , the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast ⁇ -mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof-
  • a wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention.
  • These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture. It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention.
  • Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.
  • carbohydrate epitope mimic peptide variants, analogs and active fragments may be prepared from nucleotide sequences of the peptide derived within the scope of the present invention. Active fragments, may be produced, for example, by proteolytic (e.g., pepsin) digestion of the peptide material, or by direct or chemical synthesis of parts or fragments of the described peptide sequence(s). Variants such as muteins, can be produced by standard site-directed mutagenesis of peptide coding sequences.
  • Analogs exhibiting "carbohydrate epitope mimic activity" such as small molecules or peptides incorporating non-peptide chemical components or unnatural or non-classical amino acids, whether functioning as promoters or inhibitors, may be identified by known in vivo and/or in vitro assays including the assays and methods as described and demonstrated herein.
  • a DNA sequence encoding the carbohydrate epitope mimic peptide(s) can be prepared synthetically rather than cloned.
  • the DNA sequence can be designed with the appropriate codons for the carbohydrate epitope mimic peptide amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223: 1299 (1984); Jay et al., J. Biol Chem., 259:6311 (1984).
  • Synthetic DNA sequences allow convenient construction of genes which will express carbohydrate epitope mimic peptide variants or "muteins".
  • DNA encoding such variants or muteins can be made by site-directed mutagenesis of nucleotide sequences capable of encoding the carbohydrate epitope mimic peptide(s), and muteins can be made directly using conventional polypeptide synthesis.
  • the present invention extends to the preparation of antisense oligonucleotides and ribozymes that may be used to interfere with the expression of the carbohydrate epitope mimic peptide(s) at the translational level.
  • This approach utilizes antisense nucleic acid and ribozymes to block translation of a specific mRNA either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme. This might be particularly applicable in interfering with the expression of a carbohydrate epitope mimic peptide from an expression vector.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule. (See Weintraub, 1990; Marcus-Sekura, 1988.) In the cell, they hybridize to that mRNA forming a double stranded molecule. The cell does not translate an m-RNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein. Oligomers of about fifteen nucleotides and molecules that hybridize to the AUG initiation codon will be particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules when introducing them into carbohydrate epitope mimic peptide(s)-producing cells. Antisense methods have been used to inhibit the expression of many genes in vitro (Marcus-Sekura, 1988; Hambor et al., 1988).
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these RNAs, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988.). Because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • Tetrahymena-type and "hammerhead”-type Tetrahymena-type ribozymes recognize four-base sequences, while "hammerhead”-type recognize eleven- to eighteen-base sequences. The longer the recognition sequence, the more likely it is to occur exclusively in the target mRNA species. Therefore, hammerhead-type ribozymes are preferable to Tetrahymena-type ribozymes for inactivating a specific mRNA species, and eighteen base recognition sequences are preferable to shorter recognition sequences.
  • DNA sequences described herein may thus be used to prepare antisense molecules against, and ribozymes that cleave mRNAs for carbohydrate epitope mimic peptide(s) and their ligands.
  • carbohydrate epitope mimic peptide(s) derive from the fact that the carbohydrate epitopes appear to participate in direct and causal carbohydrate-protein and protein- protein interaction between the carbohydrate epitope containing molecules and carbohydrate epitope recognizing molecules.
  • various aspects of cell-cell adhesion and cell-cell interactions involved in cell signaling, cell migration, cell recognition and cell activation are mediated via recognition of or binding to carbohydrate epitopes, particularly the L2/HNK-1 carbohydrate epitope.
  • the present invention contemplates pharmaceutical intervention in the cascade of reactions in which the carbohydrate epitope, particularly the L2/HNK-1 carbohydrate epitope, is implicated, to modulate the activity initiated by carbohydrate epitope containing molecules and carbohydrate epitope recognizing molecules.
  • carbohydrate epitope containing molecules and carbohydrate epitope recognizing molecules this could be remedied by the introduction of the carbohydrate epitope mimic peptide(s) of the present invention, variants, analogs, active fragments and the like.
  • carbohydrate epitope mimic peptide(s) or inhibitors or antagonists thereof could be introduced to block the interaction of carbohydrate epitope containing molecules and carbohydrate epitope recognizing molecules.
  • Carbohydrate epitopes can exert activating or inhibiting activities by and among carbohydrate epitopes, carbohydrate epitope containing molecules and carbohydrate epitope recognizing molecules. In as much as these activities are mediated by carbohydrate-protein, carbohydrate-carbohydrate or protein-protein interactions, including homophilic interactions, the amount or effective local concentration or degree of cell surface expression of a carbohydrate epitope can influence its activity as activating or inhibitory. Carbohydrate epitopes which are stimulatory can actually become inhibitory at high concentrations. This phenomenon would be expected to be similarly seen for the carbohydrate epitope mimic peptides of the present invention-
  • carbohydrate epitope mimic peptides of the present invention derive from the various aspects of cell-cell adhesion and cell-cell interactions involved in cell signaling, cell migration, cell recognition and cell activation which are mediated via recognition of or binding to carbohydrate epitopes, particularly the L2/HNK-1 carbohydrate epitope. Certain of these particular activities are particularly exemplified in the Examples provided herein. Additional therapeutic applications and uses, particulary of the L2/HNK-1 carbohydrate epitope, will be apparent to the skilled artisan by virtue of the recognized roles of the L2/HNK1 epitope in physiological processes, recognition phenomena and cell-cell interactions, including those outlined and specifically contemplated herein.
  • the carbohydrate epitope mimic peptides can be utilized in enhancing, activating or otherwise modulating the surveillance and clearance of tumors and virus-infected cells.
  • the carbohydrate epitope mimic peptides of the present invention may also have use in the protection of cells, particularly neural cells from chemotherapeutic agents.
  • the inventors treated embryonic neural cell cultures with a combination of the neural cell adhesion molecule LI and chemotherapeutic agents, including cisplatin and vincristin.
  • the cell cytopathic effects of the chemotherapeutic agents were reduced in te presence of LI.
  • LI expresses the HNK-1 epitope and a good portion of LI homophilic binding is HNK-1 mediated.
  • the L2/HNK-1 epitope mimic peptide of the present invention can be utilized in the protection of cells, particularly neural cells from chemotherapeutic agents. Untoward cellular cytotoxic effects and problematic symptoms associated therewith are a recognized and limiting side effect of chemotherapy, inherently limiting the dose of the agents which can be administered.
  • the carbohydrate epitope mimic peptides can be utilized in enhancing, activating or otherwise modulating the surveillance and clearance of HIN virus or HIN virus-infected cells.
  • the carbohydrate epitope mimic peptides of the present invention can be utilized in the prevention, amelioration or blocking of HIV infection, both in the immune system, particularly in lymphocytes, and in the nervous system and nervous system cells.
  • the CD4 protein contains a consensus HNK-1 epitope binding sequence and binds L2/HNK-1 carbohydrate. Having now recognized an L2/HNK-1 epitope binding sequence, the skilled artisan can readily identify and/or isolate other L2/HNK-1 interacting molecules containing a homologous or otherwise related L2/HNK-1 epitope binding sequence.
  • the infections, pathologies or alterations can be inhibited, reduced or prevented by administration or expression of the carbohydrate epitope mimic peptides.
  • van den Berg and colleagues investigated the binding of the gpl20 glycoprotein of HIV to neural glycolipids and glycoproteins by ELIS A.
  • the neuropathy and inflammatory response generated by the HIN envelope glycoprotein gpl20 is blocked or reduced in the presence of the L2/H ⁇ K-1 epitope mimic peptide of the present invention.
  • the present invention also demonstrates that the cellular effects of gpl20 on mature oligodendrocytes is blocked by the L2/HNK-1 epitope mimic peptide of the present invention.
  • the Examples provided herein demonstrate that gpl20 induced inflammation and peripheral neuropathy is blocked by preincubation with the L2/HNK- 1 epitope mimic peptide of the present invention.
  • the peptides of the present invention may therefore be utilized in treatment and prevention of neuropathies associated with viral or immune-mediated disease or resulting form injury to the nervous system, for instance spinal cord injury, head injury or trauma.
  • Patients with MS and PNS neuropathies have been shown to have IgM and/or IgG against peripheral myelin lipids, for instance.
  • Anti-sulfoglucuronyl paragloboside IgM antibodies have also been identified in ALS patients (Ben Younes-Chennoufi A et al (1995) J Neuoimmunol 57(1-2)111-115).
  • HCMV human cytomegalovirus
  • SGGLs sulfated glucuronyl glycosphingolipids
  • 3GalBl-4GlcNAcl- 3GalBl-4GlcNAcl-
  • HNK- 1 antibody partially inhibited plaque formation by HCMV
  • inhibition or prophylaxis against viral infections, particularly wherein the surface virus proteins interact or otherwise associate with host cells via L2/ITNK-1 epitope interactions is contemplated by this invention.
  • an L2/HNK1 carbohydrate epitope mimic peptide may be administered to activate or otherwise modulate the activity of L2/HNK-1 recognizing molecules, as in the potentiation or inhibition of neural cell adhesion molecules in CNS or PNS therapy.
  • the L2/HNK1 carbohydrate epitope mimic peptides may inhibit the inhibitory effects of extracellular matrix molecules such as chondroitin sulfate proteoglycan (CSPG), NG2, Neurocan, Tenascin-C, Tenascin-R etc. which are inhibitory for neurite outgrowth.
  • the present invention includes therapeutic methods for modulating, activating or inhibiting L2/HNK-1 epitope containing or recognizing molecules, particularly neural cell adhesion molecules.
  • Such methods include methods for promoting neural growth and/or remyelination and/or neuroprotection in vivo in the central nervous system of a mammal comprising administering to said mammal a neural growth and/or remyelination and/or neuroprotection promoting amount of the carbohydrate epitope mimic peptide(s) of the present invention, which peptide is capable of overcoming inhibitory molecular cues found on glial cells and myelin and promoting said neural growth, and derivatives, variants, analogs or active fragments thereof, antagonists thereof, antibodies thereto, and secreting or expressing cells thereof.
  • Such methods can further incorporate a neural growth and/or remyelination and/or neuroprotection promoting amount of a neural cell adhesion molecule, including a molecule selected from the group of LI, N-CAM, myelin-associated glycoprotein, laminin, fibronectin, N-cadherin, BSP-2/D2 (mouse N-CAM), 224-1 A6- Al, Ll-CAM, NILE (rat LI), Nr-CAM, TAG-1 (axonin-1), Ng-CAM and F3/Fl l/contactin.
  • a neural cell adhesion molecule including a molecule selected from the group of LI, N-CAM, myelin-associated glycoprotein, laminin, fibronectin, N-cadherin, BSP-2/D2 (mouse N-CAM), 224-1 A6- Al, Ll-CAM, NILE (rat LI), Nr-CAM, TAG-1 (axonin-1), Ng-CAM and F3/Fl l/
  • Method for enhancing memory comprising administering to the brain of a mammal in need of such enhancement, an amount of the carbohydrate epitope mimic ⁇ eptide(s) of the present invention, variants, analogs or active fragments thereof effective to enhance the memory of the mammal, partticularly for inhibiting the onset or progression, or treating the presence or consequences of Alzheimers disease or dementia in a mammal.
  • methods for increasing synaptic efficacy are contemplated.
  • Further therapeutic methods include promoting neuroprotection and/or neuronal survival in a mammal, particularly for inhibiting the development or onset, or treating the presence in a mammal of a condition selected from the group consisting of apoptosis, necrosis, Alzheimers disease, dementia, Parkinsons disease, multiple sclerosis, acute spinal cord injury, chronic spinal cord injury, any of the foregoing where neurodegeneration occurs or may occur, and combinations thereof.
  • methods are contemplated for inhibiting axonal cell death and enhancing myelination and remyelination in the central nervous system or peripheral nervous system.
  • Methods are contemplated for preventing, ameliorating or blocking viral infection of a mammal comprising administering to said mammal an effective amount of the carbohydrate epitope mimic peptide, variants thereof, analogs thereof, active fragments thereof or derivatives thereof.
  • the viral infection is the result of the human immunodeficiency virus.
  • Any of such therapeutic methods can utilize any of or any combination of the carbohydrate epitope mimic peptide(s), its derivatives, variants, analogs, active fragments, nucleic acids or DNA molecules capable of encoding such peptides, or vectors or host cells capable of expressing or otherwise presenting such peptides.
  • the carbohydrate epitope mimic peptide(s) or other agents exhibiting either mimicry or antagonism to the carbohydrate epitope mimic peptide(s) or control over their production may be prepared in pharmaceutical compositions, with a suitable carrier and at a strength effective for administration by various means to a patient experiencing an adverse medical condition associated with specific carbohydrate epitope containing molecules and/or carbohydrate epitope recognizing molecules for the treatment thereof.
  • a variety of administrative techniques may be utilized, among them parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, catheterizations and the like. Average quantities of the carbohydrate epitope mimic peptide(s) or their subunits may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian.
  • the therapeutic method of the present invention could include the method for the treatment of various pathologies or other cellular dysfunctions and derangements by the administration of pharmaceutical compositions that comprise the carbohydrate epitope mimic peptide(s), derivatives, variants, analogs or active fragments thereof, effective inhibitors or enhancers of activation of the carbohydrate epitope mimic peptide(s), or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with the present invention.
  • carbohydrate epitope mimic peptide(s) of the present invention may be administered to inhibit or potentiate activity of L2/HNK-1 carbohydrate epitope containing molecules or of L2/HNK-1 carbohydrate epitope recognizing molecules, as in the potentiation of neural cell adhesion molecules in CNS or PNS therapy.
  • carbohydrate epitope mimic peptide(s) whose sequences are presented in SEQ ID NOS: 1-8, 39, 40 and 41 and SEQ ID NOS: 27-38 herein, variants, analogs, derivatives, agonists, antagonists, or active fragments thereof, could be prepared in pharmaceutical formulations for administration in instances wherein therapy to activate, inhibit or otherwise modulate L2/HNK- 1 carbohydrate-recognizing molecules is appropriate, such as to promote neural growth in CNS or PNS therapy.
  • carbohydrate epitope mimic peptide(s) hereof would make it possible to better manage the untoward effects of current CNS or PNS therapy, and would thereby make it possible to apply the carbohydrate epitope mimic peptide(s) as a general neural growth or neuroprotection promoting agent.
  • present invention provides the carbohydrate epitope mimic peptide(s), variants, analogs, derivatives or active fragments thereof, in purified form, that exhibits certain characteristics and activities associated with the L2/HNK-1 carbohydrate epitope or L2 HNK-1 carbohydrate epitope containing molecules for the promotion or modulation of the activity of L2/HNK-1 carbohydrate epitope recognizing molecules.
  • a subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a carbohydrate epitope mimic peptide, variant, analog or active fragment thereof, as described herein as an active ingredient.
  • the composition comprises the peptide(s) as set out in any of SEQ ED NOS: 1-8, 27-38, 39, 40 and 41.
  • the composition further comprises a carbohydrate epitope recognizing molecule or a carbohydrate epitope containing molecule, particularly a neural cell adhesion molecule.
  • neural cell adhesion molecules for use in these compositions include LI, N-CAM, myelin-associated glycoprotein, laminin, fibronectin, N-cadherin, BSP-2/D2 (mouse N- CAM), 224-1A6-A1, NILE (rat LI), Nr-CAM, TAG-1 (axonin-1), Ng-CAM and F3/Fl l/contactin.
  • compositions for preventing, ameliorating or blocking viral infection comprising a therapeutically effective amount of the carbohydrate epitope mimic peptide or variants, analogs, derivatives or active fragments thereof and a pharmaceutically acceptable carrier.
  • compositions which contain peptides, variants, analogs or active fragments as active ingredients are well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
  • a peptide, variant, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the therapeutic peptide-, variant-, analog- or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • quantity to be administered depends on the subject to be treated, capacity of the subject's neural or immune system to utilize the active ingredient, and degree of activation or modulation of carbohydrate epitope mimic peptide binding capacity desired.
  • Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual.
  • suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration.
  • Suitable regimes for initial administration and further dosing are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration.
  • continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood or similarly appropriate concentrations in the CNS are contemplated.
  • the therapeutic compositions may further include an effective amount of the carbohydrate epitope mimic peptide(s), variant, analog, active fragment or antagonist thereof, and one or more of the following active ingredients: a neural cell adhesion molecule, a growth factor, a synthetic carbohydrate, an antibiotic, a steroid.
  • active ingredients a neural cell adhesion molecule, a growth factor, a synthetic carbohydrate, an antibiotic, a steroid.
  • Intravenous Formulation III Ingredient mg/ml gentamicin (charged as sulfate) 40.0 carbohydrate epitope mimic peptide 10.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1.0 ml Intravenous Formulation IN
  • carbohydrate epitope mimic peptide 10.0 dextrose USP 45.0 sodium bisulfite USP 3.2 edetate disodium USP 0.1 water for injection q.s.a.d. 1.0 ml
  • the component or components of a therapeutic composition of the invention may be introduced parenterally, transmucosally, e.g., orally, nasally, pulmonarilly, or rectally, intrathecally or transdermally.
  • administration is parenteral, e.g., via intravenous injection, and also including, but is not limited to, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration.
  • Oral or pulmonary delivery may be preferred to activate mucosal immunity; since pneumococci generally colonize the nasopharyngeal and pulmonary mucosa, mucosal immunity may be a particularly effective preventive treatment.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • the active compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317- 327; see generally ibid).
  • a liposome see Langer, Science 249: 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317- 327; see generally ibid).
  • the therapeutic compound can be delivered in a controlled release system.
  • the polypeptide may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
  • a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald et l, Surgery 88:507 (1980); Saudek et al, N. Engl. J. Med. 321 :574 (1989)).
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol Sci. Rev. Macromol Chem. 23:61 (1983); see also Levy et al, Science 228: 190 (1985); During et al, Ann. Neurol 25:351 (1989); Howard et al, J. Neurosurg. 71 :105 (1989)).
  • a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
  • a controlled release device is introduced into a subject in proximity of the site of inappropriate immune activation or a tumor.
  • Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).
  • pulmonary delivery of the peptide of the present invention which acts as carbohydrate epitope mimic peptide (or derivatives thereof).
  • the carbohydrate epitope mimic peptide (or derivative) is delivered to the lungs of a mammal, where it can interfere with bacterial, i.e., streptococcal, and preferably pneumococcal binding to host cells.
  • carbohydrate epitope mimic peptide inhibitory agent or derivative
  • each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvant and/or carriers useful in therapy.
  • liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.
  • Chemically modified carbohydrate epitope mimic peptide inhibitory agent may also be prepared in different formulations depending on the type of chemical modification or the type of device employed.
  • Formulations suitable for use with a nebulizer will typically comprise epitope mimic peptide inhibitory agent (or derivative) dissolved in water at a concentration of about 0.1 to 25 mg of biologically active carbohydrate epitope mimic peptide per ml of solution.
  • the formulation may also include a buffer and a simple sugar (e.g. , for carbohydrate epitope mimic peptide stabilization and regulation of osmotic pressure).
  • the nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the carbohydrate epitope mimic peptide caused by atomization of the solution in forming the aerosol.
  • Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the carbohydrate epitope mimic peptide (or derivative) suspended in a propellant with the aid of a surfactant.
  • the propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1, 1, 1,2-tetrafluoroethane, or combinations thereof.
  • Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.
  • the liquid aerosol formulations contain carbohydrate epitope mimic peptide and a dispersing agent in a physiologically acceptable diluent.
  • the dry powder aerosol formulations of the present invention consist of a finely divided solid form of carbohydrate epitope mimic peptide and a dispersing agent.
  • the formulation must be aerosolized. That is, it must be broken down into liquid or solid particles in order to ensure that the aerosolized dose actually reaches the mucous membranes of the nasal passages or the lung.
  • aerosol particle is used herein to describe the liquid or solid particle suitable for nasal or pulmonary administration, i.e., that will reach the mucous membranes.
  • the mass median dynamic diameter will be 5 micrometers or less in order to ensure that the drug particles reach the lung alveoli [Wearley, L.L. (1991) Crit. Rev. in Ther. Drug Carrier Systems 8:333].
  • an aerosol formulation of the present invention can include other therapeutically or pharmacologically active ingredients in addition to carbohydrate epitope mimic peptide, such as but not limited to an antibiotic, a steroid, a non-steroidal anti-inflammatory drug, etc.
  • the present invention provides aerosol formulations and dosage forms for use in treating subjects suffering from bacterial, e.g., streptococcal, in particularly pneumococcal, infection.
  • dosage forms contain carbohydrate epitope mimic peptide in a pharmaceutically acceptable diluent.
  • Pharmaceutically acceptable diluents include but are not limited to sterile water, saline, buffered saline, dextrose solution, and the like.
  • a diluent that may be used in the present invention or the pharmaceutical formulation of the present invention is phosphate buffered saline, or a buffered saline solution generally between the pH 7.0-8.0 range, or water.
  • the liquid aerosol formulation of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, surfactants and excipients.
  • the formulation may include a carrier.
  • the carrier is a macromolecule which is soluble in the circulatory system and which is physiologically acceptable where physiological acceptance means that those of skill in the art would accept injection of said carrier into a patient as part of a therapeutic regime.
  • the carrier preferably is relatively stable in the circulatory system with an acceptable plasma half life for clearance.
  • Such macromolecules include but are not limited to Soya lecithin, oleic acid and sorbitan trioleate, with sorbitan trioleate preferred.
  • the formulations of the present embodiment may also include other agents useful for pH maintenance, solution stabilization, or for the regulation of osmotic pressure.
  • agents include but are not limited to salts, such as sodium chloride, or potassium chloride, and carbohydrates, such as glucose, galactose or mannose, and the like.
  • the present invention further contemplates liquid aerosol formulations comprising carbohydrate epitope mimic peptide and another therapeutically effective drug, such as an antibiotic, a steroid, a non-steroidal anti-inflammatory drug, etc.
  • Aerosol Dry Powder Formulations It is also contemplated that the present aerosol formulation can be prepared as a dry powder formulation comprising a finely divided powder form of carbohydrate epitope mimic peptide and a dispersant.
  • Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing carbohydrate epitope mimic peptide (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
  • the carbohydrate epitope mimic peptide (or derivative) should most advantageously be prepared in particulate form with an average particle size of less than 10 mm (or microns), most preferably 0.5 to 5 mm, for most effective delivery to the distal lung.
  • the dry powder formulation can comprise a finely divided dry powder containing carbohydrate epitope mimic peptide, a dispersing agent and also a bulking agent.
  • Bulking agents useful in conjunction with the present formulation include such agents as lactose, sorbitol, sucrose, or mannitol, in amounts that facilitate the dispersal of the powder from the device.
  • the present invention further contemplates dry powder formulations comprising carbohydrate epitope mimic peptide and another therapeutically effective drug, such as an antibiotic, a steroid, a non-steroidal anti-inflammatory drug, etc.
  • Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets.
  • liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673).
  • Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556).
  • the formulation will include the component or components (or chemically modified forms thereof) and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.
  • oral dosage forms of the above derivatized component or components may be chemically modified so that oral delivery of the derivative is efficacious.
  • the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine.
  • the increase in overall stability of the component or components and increase in circulation time in the body examples include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline.
  • the location of release may be the stomach, the small intestine (the duodenum, the jejunem, or the ileum), or the large intestine.
  • the stomach the small intestine (the duodenum, the jejunem, or the ileum), or the large intestine.
  • One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine.
  • the release will avoid the deleterious effects of the stomach environment, either by protection of the protein (or derivative) or by release of the biologically active material beyond the stomach environment, such as in the intestine.
  • a coating impermeable to at least pH 5.0 is essential.
  • examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PNAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings may be used as mixed films.
  • a coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow.
  • Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic i.e. powder; for liquid forms, a soft gelatin shell may be used.
  • the shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.
  • the peptide therapeutic can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1mm.
  • the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets.
  • the therapeutic could be prepared by compression.
  • Colorants and flavoring agents may all be included.
  • the protein (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
  • diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextran and starch.
  • Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride.
  • Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
  • Disintegrants may be included in the formulation of the therapeutic into a solid dosage form.
  • Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used.
  • Another form of the disintegrants are the insoluble cationic exchange resins.
  • Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
  • Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.
  • MC methyl cellulose
  • EC ethyl cellulose
  • CMC carboxymethyl cellulose
  • PVP polyvinyl pyrrolidone
  • HPMC hydroxypropylmethyl cellulose
  • Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoro ethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.
  • Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added.
  • the glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
  • surfactant might be added as a wetting agent.
  • Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride.
  • nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.
  • Additives which potentially enhance uptake of the polypeptide (or derivative) are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
  • Pulmonary Delivery also contemplated herein is pulmonary delivery of the present polypeptide (or derivatives thereof).
  • the polypeptide (or derivative) is delivered to the lungs of a mammal while inhaling and coats the mucosal surface of the alveoli.
  • Other reports of this include Adjei et al. (1990) Pharmaceutical Research 7:565-569; Adj ei et al. (1990) International Journal of Pharmaceutics 63 : 135 - 144 (leuprolide acetate); Braquet et al. ( 1989) Journal of Cardiovascular Pharmacology,
  • Formulations suitable for use with a nebulizer will typically comprise polypeptide (or derivative) dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution.
  • the formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure).
  • the nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.
  • Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the polypeptide (or derivative) suspended in a propellant with the aid of a surfactant.
  • the propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1 ,2-tetrafluoroethane, or combinations thereof.
  • Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.
  • Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing polypeptide (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation-
  • the protein (or derivative) should most advantageously be prepared in particulate form with an average particle size of less than 10 mm (or microns), most preferably 0.5 to 5 mm, for most effective delivery to the distal lung.
  • Nasal Delivery Nasal or nasopharyngeal delivery of the polypeptide (or derivative) is also contemplated. Nasal delivery allows the passage of the polypeptide directly over the upper respiratory tract mucosal after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung.
  • Formulations for nasal delivery include those with dextran or cyclodextran.
  • antibodies including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of the carbohydrate epitope mimic peptide(s) and/or their subunits may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring conditions such as neural damage, remyelination, demyelination, viral infection or the like.
  • the carbohydrate epitope mimic peptide(s) or variants, analogs or active fragments thereof may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells.
  • antibodies such as 22-4X2 and HNK- 1 may be utilized.
  • small molecules that mimic or antagonize the activity(ies) of the carbohydrate epitope mimic peptide(s) of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols.
  • known L2/HNK-1 carbohydrate epitope recognizing molecules such as laminin, selectin, N-CAM, LI, etc., may be utilized.
  • Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., "Hybridoma Techniques” (1980); Hammerling et al., “Monoclonal Antibodies And T- cell Hybridomas” (1981); Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,911; 4,472,500; 4,491,632; 4,493,890.
  • Panels of monoclonal antibodies produced against carbohydrate epitope mimic peptide(s) can be screened for various properties; i.e., isotope, epitope, affinity, etc.
  • monoclonal antibodies that neutralize the activity of the carbohydrate epitope mimic peptide(s) or its subunits can be readily identified in carbohydrate epitope mimic peptide activity assays.
  • High affinity antibodies are also useful when immunoaffinity purification of native or recombinant carbohydrate epitope mimic peptide(s) is possible.
  • the anti-peptide antibody used in the diagnostic methods of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb).
  • mAb monoclonal antibody
  • the anti-peptide antibody molecules used herein be in the form of Fab, Fab', F(ab') 2 or F(v) portions of whole antibody molecules.
  • the diagnostic method of the present invention comprises examining a cellular sample or medium by means of an assay including an effective amount of a carboyhdrate epitope recognizing molecule, such as an anti-peptide antibody, L2-412 antibody, HNK-1 antibody, laminin, selectin, LI, or N-CAM, preferably an affinity-purified polyclonal antibody, and more preferably a mAb.
  • a carboyhdrate epitope recognizing molecule such as an anti-peptide antibody, L2-412 antibody, HNK-1 antibody, laminin, selectin, LI, or N-CAM, preferably an affinity-purified polyclonal antibody, and more preferably a mAb.
  • the anti-carbohydrate epitope or anti-peptide antibody molecules used herein be in the form of Fab, Fab', F(ab') 2 or F(v) portions or whole antibody molecules.
  • patients capable of benefitting from this method include those suffering from cancer, a pre-cancerous lesion, a viral infection or other like pathological derangement.
  • Methods for isolating the peptide and inducing anti-peptide antibodies and for determining and optimizing the ability of anti- carbohydrate epitope antibodies to assist in the examination of the target cells are all well-known in the art.
  • Methods for producing polyclonal anti-polypeptide antibodies are well-known in the art. See U.S. Patent No. 4,493,795 to Nestor et al.
  • a monoclonal antibody typically containing Fab and/or F(ab') 2 portions of useful antibody molecules, can be prepared using the hybridoma technology described in Antibodies -A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, New York (1988), which is incorporated herein by reference. Briefly, to form the hybridoma from which the monoclonal antibody composition is produced, a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with a carbohydrate epitope mimic peptide or synthetic carbohydrate. Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000.
  • PEG polyethylene glycol
  • Fused hybrids are selected by their sensitivity to HAT.
  • Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the present paptide and their ability to inhibit specified binding activity in target cells or to target substrates.
  • a monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity.
  • the culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium.
  • the antibody- containing medium is then collected.
  • the antibody molecules can then be further isolated by well-known techniques.
  • Media useful for the preparation of these compositions are both well-known in the art and commercially available and include synthetic culture media, inbred mice and the like.
  • An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol 8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mm glutamine, and 20% fetal calf serum.
  • DMEM Dulbecco's minimal essential medium
  • An exemplary inbred mouse strain is the Balb/c.
  • the present invention also relates to a variety of diagnostic applications, including methods for detecting the presence of carbohydrate epitope mimic peptides, by reference to their ability to elicit or competitively inhibit the activities which are mediated by the present carbohydrate epitope mimic peptides.
  • antibodyries) to the carbohydrate epitope mimic peptides can be produced and isolated by standard methods including the well known hybridoma techniques-
  • the antibody(ies) to the carbohydrate epitope mimic peptides will be referred to herein as A ⁇ and antibody(ies) raised in another species as Ab 2 .
  • carbohydrate epitope mimic peptide(s) or of carbohydrate epitope recognizing molecules or of carbohydrate epitope containing molecules in cells or in a sample can be ascertained by the usual immunological procedures applicable to such determinations.
  • a number of useful procedures are known. Three such procedures which are especially useful utilize either the carbohydrate epitope mimic peptide(s) labeled with a detectable label, antibody Ab 1 labeled with a detectable label, or antibody Ab 2 labeled with a detectable label.
  • the carbohydrate epitope mimic peptide forms complexes with one or more antibody(ies) or binding partners (e.g., carbohydrate epitope containing or recognizing molecules) and one member of the complex is labeled with a detectable label.
  • one or more antibody(ies) or binding partners e.g., carbohydrate epitope containing or recognizing molecules
  • one member of the complex is labeled with a detectable label.
  • the fact that a complex has formed and, if desired, the amount thereof, can be determined by known methods applicable to the detection of labels.
  • Ab 2 will react with Abj. This is because Ab t raised in one mammalian species has been used in another species as an antigen to raise the antibody Ab 2 .
  • Ab 2 may be raised in goats using rabbit antibodies as antigens. Ab 2 therefore would be anti-rabbit antibody raised in goats.
  • Ab will be referred to as a primary or anti-carbohydrate epitope antibody, and Ab 2 will be referred to as a secondary or anti-A ⁇ antibody.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, dyes, chemicals which fluoresce when exposed to ultraviolet light, and others.
  • a number of fluorescent materials are known and can be utilized as labels.
  • labels include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow, Green Fluorescent Protein (GFP), horse radish peroxidase (HRP) and beta-galactosidase.
  • GFP Green Fluorescent Protein
  • HRP horse radish peroxidase
  • beta-galactosidase examples include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow, Green Fluorescent Protein (GFP), horse radish peroxidase (HRP) and beta-galactosidase.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • the peptide or its binding partner(s) can also be labeled with a radioactive element or with an enzyme.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3 H, 14 C, 32 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, I31 I, and 186 Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like.
  • a particular assay system developed and utilized in accordance with the present invention is known as a receptor assay.
  • the material to be assayed is appropriately labeled and then certain cellular test colonies are inoculated with a quantity of both the labeled and unlabeled material after which binding studies are conducted to determine the extent to which the labeled material binds to the cell receptors. In this way, differences in affinity between materials can be ascertained.
  • a purified quantity of the carbohydrate epitope mimic peptide may be radiolabeled and combined, for example, with antibodies or other inhibitors thereto, after which binding studies would be carried out. Solutions would then be prepared that contain various quantities of labeled and unlabeled uncombined carbohydrate epitope mimic peptide, and cell samples would then be inoculated and thereafter incubated. The resulting cell monolayers are then washed, solubilized and then counted in a gamma counter for a length of time sufficient to yield a standard error of ⁇ 5%. These data are then subjected to Scatchard analysis after which observations and conclusions regarding material activity can be drawn. While the foregoing is exemplary, it illustrates the manner in which a receptor assay may be performed and utilized, in the instance where the cellular binding ability of the assayed material may serve as a distinguishing characteristic.
  • an assay useful and contemplated in accordance with the present invention is known as a "cis/trans” assay. Briefly, this assay employs two genetic constructs, one of which is typically a plasmid that continually expresses a particular receptor of interest when transfected into an appropriate cell line, and the second of which is a plasmid that expresses a reporter such as luciferase, under the control of a receptor/ligand complex.
  • one of the plasmids would be a construct that results in expression of the receptor in the chosen cell line, while the second plasmid would possess a promoter linked to the luciferase gene in which the response element to the particular receptor is inserted.
  • the compound under test is an agonist for the receptor
  • the ligand will complex with the receptor, and the resulting complex will bind the response element and initiate transcription of the luciferase gene.
  • the resulting chemiluminescence is then measured photometrically, and dose response curves are obtained and compared to those of known ligands.
  • the foregoing protocol is described in detail in U.S. Patent No. 4,981,784 and PCT International Publication No. WO 88/03168, for which purpose the artisan is referred.
  • kits suitable for use by a medical specialist may be prepared to determine the presence or absence of predetermined carbohydrate epitope mimic peptide activity or predetermined carbohydrate epitope recognizing activity capability in suspected target cells or sample-
  • one class of such kits will contain at least the labeled carbohydrate epitope mimic peptide or its binding partner, for instance an antibody specific thereto or a carbohydrate recognizing molecule (such as laminin), and directions, of course, depending upon the method selected, e.g., "competitive,” “sandwich,” “DASP” and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc-
  • test kit may be prepared for the demonstration of the presence or capability of cells for predetermined carbohydrate epitope mimicking activity, comprising:
  • the diagnostic test kit may comprise: (a) a known amount of the carbohydrate epitope mimic peptide as described above (or a binding partner) generally bound to a solid phase to form an immunosorbent, or in the alternative, bound to a suitable tag, or plural such end products, etc. (or their binding partners) one of each; (b) if necessary, other reagents; and
  • test kit may be prepared and used for the purposes stated above, which operates according to a predetermined protocol (e.g. "competitive,” “sandwich,” “double antibody,” etc.), and comprises:
  • an assay system for screening potential drugs effective to modulate the activity of the carbohydrate epitope mimic peptide may be prepared.
  • the carbohydrate epitope mimic peptide may be introduced into a test system, and the prospective drug may also be introduced into the resulting test system, and the system thereafter examined to observe any changes in the carbohydrate epitope mimic peptide activity therein, due either to the addition of the prospective drug alone, or due to the effect of added known quantities of the carbohydrate epitope mimic peptide.
  • the L2/HNK-1 carbohydrate occurs in biologically active glycoproteins and glycolipids in the immune and nervous systems and has been recognized as an important ligand in various cell-cell and cell-substrate interactions.
  • the carbohydrate may contribute to the preferential reinnervation of motor nerve by regenerating motor axons in vivo.
  • the carbohydrate is recognized by the so-called HNK-1 monoclonal antibody. It is likely that the trisaccharide sulfate-3GlcA ⁇ l ⁇ 3Gal ⁇ 4GlcNAc represents the minimal structure necessary for HNK-1 recognition, with the sulfate group required for binding to HNK-1 antibody.
  • the monoclonal antibodyL2-412 by contrast, binds to both sulfated and non-sulfated forms of the carbohydrate.
  • Screening of phage-displayed random peptide libraries represents a powerful means of identifying peptide ligands for targets of interest. Based on this method, we have isolated a collection of phages expressing peptides that bind to theL2-412 antibody. These peptides share a consensus sequence of 8 amino acids.
  • the selected peptide can compete with the interaction between the L2-412 antibody and glycolipids or glycoproteins carrying the L2/HNK-1 carbohydrate. Phages bearing the selected peptide of interest promote neurite outgrowth from motor neurones in vitro.
  • the development of a highly complex network such as the nervous system requires the controlled outgrowth of neurites and the formation of the correct synaptic connections.
  • the extension of the neurites depends on the interaction of receptor molecules with the extracellular matrix and with the cell surfaces of surrounding neuronal or non-neuronal cells.
  • carbohydrates carried by cell surface and extracellular matrix glycoproteins or by glycolipids, are involved in the recognition processes that determine the interaction of neural cells with their environment (Schachner and Martini, 1995).
  • the L2/HNK-1 carbohydrate is expressed on recognition molecules, for instance of the immunoglobulin superfamily and on extracellular matrix glycoproteins and integrins (Schachner and Martini, 1995) It specifically binds to certain isoforms of laminin (Hall, H. et al, Eur. J. Neurosci.5, 34-42 (1993)).
  • the L2/HNK-1 carbohydrate epitope is also the target for autoimmune IgM antibodies in demyelinating neuropathies of the peripheral nervous system in humans (for a review, see Steck, 1993), llyas, A.A. et al. Proc. Natl. Sci. USA 81, 1225-9 (1984)). It has been recently observed that these antibodies from human patients cause demyelination in chicken, confirming their involvement in damaging nervous tissue (Tatum, 1993).
  • GlcNAc ⁇ (l-3) Gal ⁇ (l-4) Glc ⁇ (l-l)-ceramide More recently, the structure of an HNK-1 -reactive carbohydrate epitope of bovine peripheral myelin glycoprotein (PO) has been elucidated (Voshol, 1996). It contains the same terminal trisaccharide as in the glycolipid determined by Chou and Jungalwala suggesting that this structure is sufficient for its immunoreactivity. Thus, the carbohydrate epitope present on various L2/HNK-1 antibody reaction cells and in various L2/HNK-1 epitope containing molecules corresponds in core structure to GlcA ⁇ l- ⁇ 3Gal ⁇ - ⁇ 4GlcNAc.
  • a possible solution to this problem is to mimic carbohydrates by other compounds that are easier to prepare, e.g., peptides.
  • the most promising way to find such peptides is by use of the random peptide phage display technology.
  • peptides or proteins are expressed on the tip of a filamentous phage, as a fusion protein with the phage surface protein pilus (Devlin, 1992), (Cwirla, S.E. et al, Proc. Natl. Acad. Sci. USA, 87, 6378-6882 (1990)).
  • Screening phage-displayed random peptide libraries represents a powerful means of identifying peptides ligands for targets of interest.
  • Phage expressing binding peptides are selected by affinity purification with the target of interest. Peptide ligands identified in the manner frequently interact with natural binding site(s) on the target molecule and often resemble the target's natural ligand(s). Although this system is most often used to identify peptide epitopes, it has also been successfully applied to the carbohydrate binding site of the pectin concanavalin A (Scott, 1992). Peptides that mimic the binding of methyl a-D-mannopyranoside to ConA were identified by screening a phage-displayed random hexa-or decapeptide library (Scott, 1992), (Oldenburg, R.K. et al, Proc. Natl Acad. Sci.
  • the peptide can be produced in much larger amounts than the naturalL2/HNK-l carbohydrate. It may therefore be tested for example, in applications directed towards promoting regeneration of the peripheral nervous system, particularly of motor axons.
  • the library consisting of 2 x 10 8 original clones, was screened in three cycles of panning with the antibody L2-412, elution with pH shift and amplification as shown in Figure 1. Increasing number of binders were observed in successive rounds of screening (10 "4 % to 0.1%) suggesting that selective phages enrichment was occurring.
  • 96 clones were tested on plates coated withL2-412 or rat IgG in an ELIS A System. Then 20 clones, chosen from those binding toL2-412 but not to rat IgG, were sequenced as described in Materials and Methods.
  • Clone 15(UBR2) (SEQ ID NO: 33) is an unbound control phage.
  • the complete sequence was initially selected because: 1) it contains the FLHTRLFV (SEQ ED NO: 8) consensus sequence and 2) since it was found 12 times, the rest of the sequence might be of potential importance for stabilizing or presenting correctly the consensus sequence to the mAbL2-412.
  • a randomized form of the 15-15 sequence was also synthesized (Table 3). In both cases the peptides were freshly synthesized and coupled to BSA.
  • FIG. 3 shows an experiment in which different concentrations of the peptide coupled to BSA could indeed inhibit the binding of the positive phage to the immobilized L2-412.
  • Figure 4 shows that the peptide coupled to BSA was able to compete with the binding of the positive phage to laminin, a binding partner of the L2/HNK-1 carbohydrate.
  • the positive phage was also shown to bind to laminin in a concentration dependent manner (FIGURE 5).
  • Figure 7 shows the concentration-dependent binding of biotinylated complex to immobilized laminin, a binding partner of the HNK-1 carbohydrate. Binding of the biotinylated BSA used as a control, was never observed.
  • the peptide sequence FLHTRLFVSDWYHT (SEQ ID NO: 7) was synthesized and assayed for its ability to inhibit the binding of the L2/HNK-1 carbohydrate to its natural binding partner laminin or the binding of mAb L2-412 to HNK-1 glycolipids.
  • Our peptide- shows an inhibitory effect comparable to that of the SO 3 -sugar, which shows that the peptide behaves like the carbohydrate in these particular experimental conditions.
  • the phage bearing another peptide used as a negative control never showed any inhibitory effect.
  • the lack of complete inhibition of the mAb binding to the L2/HNK-1 glycolipids may be due to a multivalency-monovalency problem.
  • the antibody is bivalent and the free peptide in monovalent.
  • the peptide coupled to BSA might still act as a monovalent unit in this particular situation compared to the antibody.
  • FLHTRLFVSDWYHT SEQ ID NO: 7
  • peptide bind to the antigen combining site of the antibody, mimicking the L2/HNK-1 epitope recognized by L2-412. This conclusion was also confirmed with both the direct binding of the bioitynilated peptide-BSA complex to the L2-412 and to laminin and with the functional studies on neurite outgrowth from chicken motor neurons in vitro.
  • a more detailed understanding of the molecular nature of protein-carbohydrate interactions could influence the development of new therapeutic agents.
  • Binding to the L2/HNK-1 carbohydrate recognizing antibodies provides a good model to study the properties of mimics for biologically active carbohydrates- Peptides that compete effectively with the binding of the natural L2/HNK-1 carbohydrate to the antibody could also represent a step towards finding neutralizing compounds, which could prevent damage to nervous tissue by HNK-1 autoantibodies present in some neuropathies of the peripheral nervous system (Giese, K.P. et al, Cell 71, 565-576 (1992); Montag, D. et al. Neuron 13, 229-246 (1994)). Furthermore, HNK-1 binds to the P- and L- selections which are implicated in leukocyte-endothelial cell interactions also outside the nervous system. These interactions have been shown to play a role in immunopathological responses.
  • the peptide FLHTRLFVSDWYHT (SEQ ID NO: 7) (and a subfragment of it, FLHTRLFV (SEQ ID NO: 8) (see Example 2)), have been shown to at least in some respects mimic the L2/HNK-1 carbohydrate. Since the peptides are accessible through organic synthetic procedures, as well as in nucleic acids encoding such peptides, modified variants with altered amino acid sequences and analogs, including even unnatural amino acids, could be produced. Introduction of a sulfate group (perhaps on the N-terminal phenylalanine) might be a relevant modification, since this moiety is an important element of the natural HNK-1 carbohydrate. Testing carriers other than BSA might also lead to altered, improved or enhanced biological activity.
  • the phage peptides show a consensus sequence FLHTRLFV (SEQ ID NO: 8).
  • SEQ ID NO: 8 the short 8 amino acid consensus sequence and a corresponding randomized form were also synthesized for comparison, both coupled to BSA (ANAWA AG, Switzerland). These were tested in combination with the 15-mer peptide and its corresponding randomized form, as shown in Table 3 below.
  • the neurites extended from motor neurons cultured on the short 8 amino acid sequence were significantly longer than the neurites obtained on control culture (BSA randomized peptides). Similar results were obtained with the positive control,
  • L2/HNK-1 glycolipid L2/HNK-1 glycolipid.
  • the neurites extended in response to the 15 amino acid peptide sequence were shorter than those extended in response to the 8mer peptide, but still significantly longer than those obtained with the control consisting of BSA alone.
  • the neurites extended from motor neurons cultured on the randomized BSA- peptide conjugate were no longer than those on BSA alone.
  • the network formed by the neurites also appeared less dense than that seen with the active peptides (FIGURE 9A-9C).
  • neuronal polarity Another important point raised in this study is the possible role of the peptide in neuronal polarity.
  • the concept of neuronal polarity implies that axons and dendrites are different. Chada and coworkers (Chada et al, 1997) have described differences between axon-like and dendrite-like processes, defining the axonal/dendritic polarity of the forebrain neurons.
  • the cytoplasm of dendrite-like processes contained abundant polysomes throughout their length; in contrast, polysomes were not detected in the long axon-like processes in regions further than about 75 ⁇ m away from the soma.
  • the dendrite-like processes showed a lower density of micro tubules and neurofilaments than the axon-like processes. It has been shown for chick forebrain neurons (Chada et al, 1997) and for chick sensory neurons (Prochiantz et al, 1995) that mechanical tension initiates neurite elongation and that the addition to the culture medium of neurotrophic factors influences the development of fully polarized neurons (Lein et al, 1995; Prochiantz et al, 1995).
  • mice maintained on substrate containing tenascin were shown to develop a more polarized phenotype (Domes et al, 1996; Lafont et al, 1994; Faissner at al, 1997; Lein et al, 1991; Lein et al, 1989). Together, these results highlight the importance of combinatorial effects.
  • the polarization index was calculated based on the data provided by the IB AS analysis system.
  • the degree of polarity was defined as the mean length of the longest neurite divided by the average length of all neurites, where the average length of all neurites was obtained by dividing the average total length by the average number of neurites.
  • TheL2-412 antibody which was used as positive control for the immunostaining procedure (but not for the binding of the pept/BSA), gave strong staining of the ventral roots and of the motor branch of the femoral nerve. This also means that theL2/HNK-l carbohydrate was present on these sections. It is possible that the amount of receptor was too low to be detected by binding of peptide, or that the receptor was already saturated with endogenous L2/HNK-1 carbohydrate. Alternatively, the peptide concentration or affinity was not high enough. This might be ameliorated with peptides coupled to other multivalent carriers.
  • HNK-1 antibody is biotinylated with a coupling agent incorporating a disulfide bridge.
  • the biotinylated antibody is pre- reacted with the streptavidin-coated tube, unbound antibody is washed off, and the immunotube is used for screening.
  • phage are reacted with the biotinylated antibody in solution, and then the biotinylated complex is allowed to react with an immunotube coated with streptavidin.
  • FIGURE 14 Comparative binding of the HNK-1 selected phage/positive clones versus the L2-412 selected 15-15 phage to bound L2-412 antibody, IgG, HNK-1, and IgM is shown FIGURE 14.
  • Supernatant (100 ⁇ l) from an overnight culture of bacteria secreting phade were incubated with coated antibodies (100 ⁇ l of 1 ⁇ g/ml useded for coating) and detected with HRP-coupled anti-phage antibody.
  • Both of the HNK-1 selected phage 15H92 and 15H233 bind L2-412 and HNK-1, nearly to the same extent.
  • the 22-4X2 antibody selected 15-15 phage does not bind HNK-1 antibody to a significant extent versus IgG or IgM.
  • FIGURES 15 and 16 A further comparison of HNK-1 antibody selected clones versus L2-412 antibody selected clones is shown in FIGURES 15 and 16.
  • the HNK-1 selected clones (all designated 15H#) all bind both HNK-1 and L2-412 antibodies to a significant extent, in some cases to an approximately comparable extent.
  • FIGURE 17 depicts a direct comparison of 15-15, 15H92 and the controls UBR2 and UBH in binding 412 and HNK-1 antibodies. Control phage were picked at random from the unbound fraction after the first round of screening.
  • TRLFR V/F (SEQ ID NO: 39) is found in eight of the phage clones.
  • sequence shows similarity and homology to the consensus 8 mer of the L2-412 phage, FLHTRLFV (SEQ ID NO: 8), particularly containing the sequence TRLF(R)V (SEQ ED NO: 40) and TRLF (SEQ ID NO: 41) which is conserved universally and compressed in many of the 122-4X2 and HNK-1 binders.
  • Laminin is a self-aggregating, multifunctional glycoprotein, consisting of three polypeptide chains o-l, ⁇ l and ⁇ 1.
  • Laminin is known to recognize the L2/HNK- 1 carbohydrate, and such carbohydrate is implicated in cell-to-laminin adhesion.
  • Cell-to- laminin adhesion is mediated by direct binding of the L2/HNK-1 carbohydrate to the G2 domain of the terminal globular domain of the laminin cl chain.
  • Hall et al has reported testing a variety of G2 domain-derived synthetic peptides for their ability to inhibit L2/HNK-1 binding to laminin, and has isolated the competitive binding to a single peptide (Hall et al, Glycobiology 5, 435-441 (1995).
  • This peptide, KGVSSRSYVGCEKNLEISRST (SEQ ID NO: 51) bound to the L2/HNK-1 carbohydrate in a concentration-dependent manner and inhibited HNK-1 -mediated neural cell adhesion to laminin.
  • the laminin peptide sequence was used to search the publicly available sequence database and a group of proteins possessing homologous sequences were identified (SEQ ID NOS: 52-57) (TABLE 5). A consensus L2 binding protein sequence was also determined (SEQ ID NO: 58)(TABLE 5).
  • the proteins include already recognized L2/HNK-1 interacting molecules, including merosin, L-selectin, and PO.
  • the CD4 protein present on lymphocytes and recognized as interacting with HIV virus and required for HIV infection of human cells, contains a homologous sequence at amino acids 244-264, corresponding to RASSSKSWITFDLKNKEVSVK (SEQ ID NO: 56). This amino acid sequence is located in the extracellular domain of the CD4 protein, between the C-like domain and the transmembrane domain.
  • CD-4 peptide was immobilized in water at different concentrations and dried onto microtiter plates overnight. The microtiter plates were blocked with 1% BSA in PBS, incubated with 3ug/ml L2 glycolipid for 1.5 hours at room temperature, and washed with PBS. Bound L2 glycolipid was detected with L2-412 antibody followed by HRP-linked secondary antibody. The resits (not shown) demonstrated that CD-4 peptide binds L2 glycolipid in a concentration dependent manner.
  • CD4 peptide was coated onto microtiter plates by coating with serial dilutions of CD4 peptide (1 mg/ml 1 : 1 in EtOH) and allowed to dry overnight. The wells were incubated with L2-glycoli ⁇ id (3 ⁇ g/ml) for 1 hour at RT, L2-412 antibody (1:1000) then added and further incubated for 3 hours at RT. Goat anti-rat antibody was then added, incubated 2 hours at RT and detected by HRP-linked secondary antibody. As shown in FIGURE 18, L2 glycolipid binds to CD4 peptide in a concentration-dependent manner.
  • CD-4 peptide was conjugated to Ovalbumin (by similar methods as for BSA conjugation described in Materials and Methods) to generate CD4-ON, and binding experiments were performed to assess binding of conjugated peptide to laminin and L2 glycolipid. In particular, it was necessary to confirm that CD4 peptide and laminin compete directly in binding to L2/H ⁇ K-1 glycolipids. In this set of experiments, microtiter plates were coated with serial dilutions of either CD4-ON or laminin (each lO ⁇ g/ml 1 : 1 in EtOH, allowed to dry overnight).
  • the coated plates were then incubated in the following combinations: (a) CD4-ON coated plates with L2 glycolipid (3 ⁇ g/ml, 1 hr, RT); (b) laminin coated plates with L2 glycolipid (3 ⁇ g/ml.1 hr, RT); and (c) laminin coated plates with CD-4-ON (10 ⁇ g/ml) and L2 glycolipids (3 ⁇ g/ml) for 1 hr at RT. Similar to the previous set of experiments 122-4X2 antibody was added
  • the 15-mer peptide library and the E.coli K91 Kan cells used were kindly provided by G. Smith, Division of Biological Sciences, University of Missouri, Columbia.
  • the 15- mer library was constructed in the vector fUSE5, a derivative of the filamentous phage fd-tet (Scott et al, 1990).
  • This vector carries a tetracycline resistance gene allowing for selection.
  • the filamentous phage do not kill their host; thus the infected cells become tetracycline resistant, continue to grow and secrete progeny particles.
  • the E.coli strain K91Kan also from G.
  • Smith is a lambda " derivative of K38 (Lyons et al, 1972), has a chromosomal genotype thi and carries a kanamycin-resistance gene (mkh) (Smith et al, 1993; Yu et al, 1996).
  • L2/HNK-1 glycolipids were purified from beef cauda equina by B. Becker in our laboratory. Sulfated sugars, SO 3 -GlcA-Gal-allyl, were kindly provided by N. Nifant'ev, Zelinsky Institutre of Organic Chemistry, Russian Academy of Sciences, Moscow.
  • HNK-1 antibody Characterization and purification of the monoclonal antibody (mAb 1X2-4X22), raised in rats and recognizing the HNK-1 carbohydrate has been described by Noronha, A. et al, Brain Res. 385, 237-244 (1986)).
  • the L2-412 antibody has been deposited with the DSMZ - Deutsche Sammlung Von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Germany, under the Budapest Treaty, and is designated .
  • HNK-1 antibody is available as TEB200 from the American
  • HRP horseradish peroxidase
  • LB medium g/L Bacto-Tryptone, 5 g/L NAcl, 5 g/L yeast extract
  • kanamycin 100 ⁇ g/ml kanamycin
  • a IL flask containing 100 ml of Terrific Broth was prepared (12 g Bacto-Tryptone, 24 g yeast extract, 5.04 g glycerol (4ml) added to 900 ml of water and autoclaved in 90 ml portions; 10 ml of potassium phosphate buffer (0.17M KH 2 PO 4 , 0.72M K 2 HPO 4 , no pH adjustment required) were added to each 90 ml portion bef ore use).
  • the 100 ml Terrific Broth were inoculated with 1 ml of the overnight culture of K91kan cells and shaken vigorously until the OD 600 of a 1 : 10 dilution reached 0.2. Shaking was then slowed down for 10 min to allow F-pili to regenerate and 10 ⁇ l of the starting library was added to the flask; slow shaking was continued to allow for adsorption. The culture was then transferred to 1 L of LB containing 0.22 ⁇ g/ml tetracycline and allowed to shake vigorously for 35 minutes at 37°C. The tetracycline concentration was adjusted to 20 ⁇ g/ml, and an aliquot was taken for determination of the titer.
  • the phage were titered (recovered titer) by plating infected cells on tetracycline medium and counting the number of tetracycline resistant colonies.
  • An infectious unit defined in this way is called a transforming unit (TU) and the infectivity is the ratio of number of TU's to number of physical particles.
  • TU transforming unit
  • an aliquot of 50 ⁇ l of the culture was removed and diluted with LB containing 0.2 ⁇ g/ml tetracycline (dilution range was 10 3 -10 5 ).
  • TBS 50 mM Tris-HCl pH7.5, 150 mM NaCl
  • the pellet obtained after centrifugation (14'500 x g, SA600, 10 min, 4°C) was redissolved in 10 ml TBS and transferred into a tared vessel containing 4.83 g CsCl.
  • the vessel was retared and TBS was added to a net weight of 10.75 g. This should give 12 ml of a 31% w/v solution of CsCl (density 1.30 g/ml); the solution was centrifuged 48 hrs at 150O00 x g at 5°C in a SW41 rotor (Beckman).
  • a faint bluish non-flocculent band (containing the amplified phages) was visible above a narrow flocculent opaque white band (probably deriving from PEG).
  • the phage band was collected by first aspirating slowly the fluid overlying the phage band and then, using a pipette, the phage band was withdrawn avoiding as much as possible the flocculent band underneath.
  • the phage band was then delivered to a 26 ml polycarbonate centrifuge bottle, which was filled to the shoulder with TBS and centrifuged in a Ti70 rotor (279O00 x g, 4h, 5°C) and resuspended in 2 ml TBS per 1 L of culture. Phages can be stably stored in this form in a refrigerator.
  • the amplified library was then titered (final titer) as follows: several dilutions of phage were prepared in TBS/gelatine (0.1 g gelatin in 100 ml TBS) covering the dilution range from 10 7 to 10 10 . Then 10 ⁇ l of each of these dilutions were used to infect 10 ⁇ l of K91kan cells prepared as described at the beginning of this section and each dilution mixture was incubated 15 min at room temperature (RT) to allow phage to infect the concentrated cells. One ml of LB containing 0.2 ⁇ g/ml tetracycline was added and incubated 30 min at 37°C in a shaker-incubator. The infected cells were then spread (200 ⁇ l) on an agar plate containing 40 ⁇ g/ml tetracycline and 100 ⁇ g/ml kanamycin as described above (recovered titer).
  • TBS/gelatine 0.1 g
  • the phage library was panned using Immunotubes (Nunc, Maxisorb) coated with mAbL 2 -412.
  • the tubes were coated by incubating overnight at 4°C with antibodyL2- 412 at 10 ⁇ g/ml protein in PBS (1 ml total volume) for the first round and 1 ⁇ g/ml for the second and third round of screening.
  • 10 11 transforming units (in 250 ⁇ l volume) of the phage library per immunotube were allowed to bind 1 hour at 37° C in a rotating chamber.
  • the phages were preincubated 1 hour with 100 ⁇ g/ml of rat IgG before being added to the immunotube, in order to decrease the number of non-specific binders.
  • the tubes were washed 10 times with PBS-0.05% (v/v) Tween 20 and eluted with 0.1 M Glycine pH 2.2 (0.5-1 ml total volume), 10 min. at 4° C.
  • Eluted phages were neutralized with 1.5M Tris pH9 and then used to infect 0.5-1 ml of log phase E. coli K91 Kan cells 15 min at room temperature.
  • the infected bacteria were transferred to 20 ml of LB containing 0.2 ⁇ g/ml tetracycline, and after removing an aliquot for determination of the titer (recovered titer), allowed to grow overnight as described in the previous section.
  • the amplified eluate was then twice centrifuged (10 min, 3600 x g and 10 min, 14' 500 x g, SA600) and the final supernatant was precipitated with 0.15 volume of PEG/NaCl overnight at 4°C.
  • the phage was pelleted (15 min. 14'500 x g, SA600) and dissolved in 1 ml PBS by pipetting and vortexing, microcentrifuged 1 min.
  • the phage were titered (final titer) as described.
  • the colonies were counted on the next day and the yield of the screening was calculated by dividing the recovered titer by the titer (input) of the previous round.
  • the HNK-1 antibody was biotinylated as described below using NHS-SS-biotin.
  • NHS-SS-Biotin links the biotin to the protein via a disulfide bridge, in order to allow the biotin group to be subsequently removed by incubation with dithiothreitol (DTT).
  • DTT dithiothreitol
  • TheL2-412 antibody was similarly biotinylated as described below.
  • procedure A the biotinylated antibody is first allowed to bind to a streptavidin coated immunotube, which is then subsequently used to pan the phage input.
  • procedure B the biotinylated antibody is preincubated with the phage in solution, and the reaction mixture is allowed to bind (a few minutes) to the streptavidin-coated immunotube.
  • the immunotubes were coated with 10 ⁇ g/ml streptavidin in PBS, 1 ml total volume (wet the entire surface of the tube), overnight at 4°C on a rotator. Streptavidin was discarded and the tube was filled with blocking solution, PBS containing 0.5% (w/v) BSN for 2 hrs at 4°C. After washing 6 times with PBS-0.05% (v/v) Tween 20 (PBS-T), the biotinylated antibody was added. Typically, 3 ⁇ g of the biotinylated HNK-1, or 5 ⁇ g of the biotinylatedL2-412 antibody were added in 400 ⁇ l of the blocking solution.
  • the antibody was allowed to bind for at least 2 hrs (or overnight) at 4°C on the rotator. After washing 6 times with PBS-T, 10 10 phages from the 15-mer starting library, in 400 ⁇ l of blocking solution, were allowed to bind to the /50447
  • the absorbance of the colored reaction product was determined at 405 nm in a Multiscan TitertekPlus (Flow, Switzerland). In parallel, each clone was also tested on 96-well plates coated with rat IgG, (lOO ⁇ l, 1 ⁇ g/ml in PBS and identically blocked for 2 hours). Bacteria producing the selected binding clones (named positive phage), that were positive binders for the mAb L2 -412 but did not bind to rat IgG were streaked on an agar plate containing LB medium with 40 ⁇ g/ml tetracycline and lOO ⁇ g/ml kanamycin. Two individual colonies were picked and re-assayed for positivity towards mAb L2-412. Positive single colonies were stored in 40% glycerol at -80° C. R. Competition Binding
  • Microtiter plates (Nunc) were coated with theL2/HNK-l glycolipids (50 ⁇ l, 1 ⁇ g/ml, dissolved in EtOH) and allowed to dry overnight. While blocking the wells for 2 hours with 0.5% (w/v) fatty-acid-free BSA in PBS, a limiting concentration of L2 -412, previously determined, was pre-incubated with successive 2-fold dilutions of the inhibitor, starting at a concentration of 2.2 mM for the free peptide, 5mM for the SO 3 sugar and 10 12 positive and negative phages (the negative phages were cloned from the unbound fraction of the first round of screening).
  • the pre-incubated mixture was then added to the well in lOO ⁇ l and incubated for 1 hour at RT. After washing 5 times with PBS-0/05% (v/v) Tween 20, the binding of mAb L2-412 was detected by incubation with HRP-conjugated goat anti-rat IgG for 1 hour, followed by the color reaction described earlier. The percentage of inhibition of the binding of mAb L2-412 to the substrate in the presence of the inhibitor was calculated with reference to the control value obtained in the absence of inhibitor (0% of inhibition).
  • Microtiterplates were coated overnight at 4° C with laminin (Gibco/BRL), (10 ⁇ g/ml,100 ⁇ l), or mAb L2 -412 (1 ⁇ g/ml, lOO ⁇ l) in PBS. All the following reaction steps were carried out at room temperature. After blocking with PBS + 0.5% (w/v) BSA, 50 ⁇ l of successive 2-fold dilutions of peptide coupled to BSA (ANAWA Ag, Switzerland) starting at a concentration of 30 ⁇ M was added for 1-2 hours at RT. Then a limiting number of phages bearing the peptide of interest, previously determined, was added and incubated for another hour.
  • laminin Gibco/BRL
  • mAb L2 -412 (1 ⁇ g/ml, lOO ⁇ l
  • the bound phages were detected with HRP/anti-M13 antibody as described in the ELIS A screening section.
  • the analogous experiment was done with immobilized L2-412 instead of laminin, the peptide coupled to BSA competing with the binding of positive phages to the antibody L2-412.
  • Microtiter plates were coated with lOO ⁇ l of mAb L2-412 or laminin as described above and lOO ⁇ l of biotinylated peptide coupled to BSA was added starting at a concentration of 30 ⁇ M, incubated 2 hours at room temperature, and detected with HRP-streptavidin.
  • Biotinylation of the HNK-1 antibody, BSA and the peptide coupled to BSA was done using Sulfo-NHS -biotin (Pierce) according to the manufacturer's instructions. A molar ratio of 10 to 1 was used for the antibody and 5 to 1 for BSA or the peptides coupled to BSA. The biotinylated product was dialysed overnight against PBS at 4°C. Neurite Outgrowth Experiments Preparation and Culture of Motor Neurons
  • Cover slips were sterilized by baking them overnight at 160°C and coated by an overnight incubation with polyornithine (Sigma, 1.5 ⁇ g/ml in water) at 4°C. The cover slips were then washed 3 times with water and further coated with test substances as follows: 1) The BSA-peptide conjugates were dissolved at 100 ⁇ g/ml in PBS, sonicated 1 min with a table sonicator and centrifuges in a microfuge for 20 min at maximum speed. The protein concentration of the supernatant was determined each time by the method of Bradford (Bradford et al, 1976).
  • 120 ⁇ l complex was mixed with 280 ⁇ l of collagen solution (20 ⁇ g/ml collagen in PBS) and 100 ⁇ l were applied on each cover slip overnight at 4°C; 2) As a negative control, untreated BSA was used in place of the peptide-BSA complex; 3) The glycolipids carrying the L2/HNK-1 carbohydrate were dissolved in ethanol at a concentration of 10 ⁇ g/ml, and 80 ⁇ l were added to 1 ml of the collagen solution described above. A volume of 100 ⁇ l was used for coating. Cover slips were placed in quadruplicate in a 24-well plate (NUNC), and finally washed 3 times before the cells were plated (the cover slips were never allowed to dry).
  • NUNC 24-well plate
  • Motor neuronal cells were prepared as described by Arakawa (1990) from spinal cord of 6-day old chick embryos dissociated in 1 ml of ice cold solution containing 0.05% DNAse 1 (Sigma), 0.1% BSA in L-15 medium (Life Technologies). Cells were layered on 2 ml of 6.8% Metrizamide (Fluka) in L-15 and centrifuged 15 minutes at 500 x g, 4° C. Cells collected from the Metrizamide/medium interface were diluted in 5 ml L-15 and loaded on a 4 ml cushion of BSA (4% BSA in L-15) and centrifuged 10 minutes at 300 x g, 4° C.
  • the pellet was resuspended in 0.5-1 ml of complete medium ((22 mM NaHCO 3 , 22 mM glucose, 1% of penicillin and streptomycin (Gibco) in L-15 supplemented with 1% N2 supplement (Gibco) and 15 ⁇ g/ml chicken muscle extract (3.5 mg/ml).
  • complete medium ((22 mM NaHCO 3 , 22 mM glucose, 1% of penicillin and streptomycin (Gibco) in L-15 supplemented with 1% N2 supplement (Gibco) and 15 ⁇ g/ml chicken muscle extract (3.5 mg/ml).
  • 30,000 cells were plated on poly-ornithine/collagen coated cover slips in the presence or absence of the peptide coupled to BSA and incubated in a humidified chamber at 37° C and 5% CO 2 .
  • the length and number of neurites were measured and counted for isolated neurons that were not in contact with other cells and with at least one process
  • Dorsal root ganglia neurons were isolated from embryonic-day 11 chicken eggs. The ganglia were transferred into 1 ml of digestion solution (0.05% Trypsin, 0.01% DNAse 1 in HBSS medium) and incubated 15 min. at 37°C with resuspending every 2-5 min. The ganglia were then dissociated in 1 ml of ice cold dissociation solution (0.05% DNAse 1, 0.1% BSN in L15 medium), loaded on 3 ml of a 4% BSA cushion in a 15 ml Falcon tube and centrifuged at 4°C, 600 x g for 20 min.
  • digestion solution 0.05% Trypsin, 0.01% DNAse 1 in HBSS medium
  • the ganglia were then dissociated in 1 ml of ice cold dissociation solution (0.05% DNAse 1, 0.1% BSN in L15 medium), loaded on 3 ml of a 4% BSA cushion in a 15 ml Falcon tube and centrifuged at 4
  • the cells were resuspended in 0.5 ml of the complete medium described in the previous section. 20,000 cells were added to wells containing one cover slip, and allowed to grow for 18 hrs in a humidified chamber at 37°C and 5% CO 2 . Fixing and analysis of neurite outgrowth was performed as described in the preceding section. Immunohistology and Immunocytology
  • Cryosections of femoral nerve from a 4-month-old mouse were used to look for binding of peptide-BSA complex.
  • the sections were treated for 1 hr with 1% H 2 O 2 , 0.5% bovine serum albumin (BSA), and 10% goat serum in PBS, in order to reduce the endogenous peroxidase activity.
  • the sections were then incubated overnight at 4°C with peptide-BSA complex or BSA (1 mg/ml in PBS, 150 ⁇ l/cover slips), and then washed 4 times with PBS-0.01% Tween 20.
  • anti-BSA antibody Sigma, 1 : 16 dilution, 150 ⁇ l/cover slips
  • HRP-coupled goat anti rabbit serum was added (1 :2000), for 1 hr in a volume of 150 ⁇ l per cover slip.
  • the color reaction was developed using a 5% dilution of a 4 mg/ml stock solution of 9-amino-3-ethylcarbazol (AEC, Fluka) in N,N ' -dimethylformamide in 0.1 M sodium acetate buffer, pH 4.8, containing 0.1% H 2 O 2 .L2-412 antibody and HRP-coupled goat anti-rat antibody were used for the positive control.
  • a similar experiment was performed using biotinylated BSA-peptide conjugate. A concentration of 50 ⁇ g/ml was used for the overnight incubation and HRP-coupled streptavidin (1 :2000) was added for 1 hr.
  • the color reaction was developed as described above.
  • Cover slips were coated with polyornithine (1.5 ⁇ g/ml) then with collagen (20 ⁇ g/ml,) and 40,000 cells were allowed to grow for 40 hrs at 37°C under 5% CO 2 as described above.
  • the fixed cover slips were then blocked in 5% non-fat dry milk powder in PBS for 2 hrs.
  • biotinylated BSA- peptide conjugate was added at a concentration of 50 ⁇ g/ml for 4 hrs.
  • detection was done using HRP-coupled streptavidin, 1 :500, for 1 hr. Color detection was as described above for immunohistology.
  • the fixed neurons were photographed at 40 X magnification.
  • the images presented were processed for enhanced color rendition using Adobe Photoshop. EXAMPLE 10
  • tissue was harvested from postnatal day five rat pups, dissociated and plated on poly-lysine coated 24 well cluster plates in Neural basal medium plus B27 supplement, 1% FBS and penicillin and streptomycin.
  • HNK-1 epitope mimic peptide 8mer FLHTRLFN (SEQ ID ⁇ O:8) (ImM, lOOnM or lOnM) was added to wells at the time of plating.
  • lnM gpl20 was preincubated with ImM, lOOnM or lOnM HNK-1 peptidomimetic for 1 hour at 37oC.
  • gpl20 or gpl20 plus HNK-1 peptidomimetic was added to mixed cerebellar cultures in sets of 6 replicates. Four days later, cultures were fixed and immunostained with the oligodendrocyte specific antibody marker, RIP. Data was analyzed by counting the number of RIP positive cells in twenty 20X-microscope fields. In addition, the number of RIP positive cells, mature oligodendrocytes with extensive intact membrane sheaths were counted. The data is presented in TABLE 6 below. The cells are depicted in FIGURE 20. HNK-1 carbohydrate epitope mimic peptide increases the number of mature oligodendrocytes and blocks myelin destruction associated with gpl20 treatment.
  • 0x42 (also called MAC1) is a general marker for immune/inflammatory activation of the central nervous system (expressed by microglia - when those proliferate and/or extend their processes). If 0x42 marker is present two weeks, and particularly four weeks, after initial neural injury, this indicates a chronic problem. An ongoing inflammatory process in the CNS is correlative of neuropathy. TNF is a cytokine indicating an inflammatory process, and in this case neuronal degeneration.
  • GFAP is a marker for a general activation of astrocytes in the spinal cord and is expressed following damage to the CNS.
  • the left sciatic nerve was isolated by blunt dissection, and wrapped with oxidized cellulose (Oxycel, Becton Dickinson) saturated with 250 ul sterile saline containing 400ng of recombinant gpl20 protein alone, or in the presence of 43 ng of the 8-mer
  • L2/HNK-1 mimic peptide (corresponding to a 10X excess of gpl20 on a mole-per- mole basis), utilizing the Oxycel delivery method of Eliav et al (Eliav et al (1999) Pain
  • Sections of 40um thickness from the lumbar enlargement from each animal were thaw mounted onto gelatized slides and stained immunohisto chemically for Ox-42 (MACl),
  • TNF and GFAP TNF and GFAP
  • Faissner A. Neuroscience Letters 83, 327-332 (1987). Faissner, A. Cell Tissue Research 290, 331-341 (1997).

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Abstract

L'invention concerne des composés mimétiques d'épitope glucidique, notamment des peptides, et des analogues et variants dérivés. Plus précisément, les composés et les peptides de la présente invention imitent l'épitope glucidique GlcAβ1→3Galβ1→4GlcNAc ou le sulfate - 3GlcAβ1→3Galβ1→4GlcNAc, ou L2/HNK1 épitope glucidique. Ladite invention concerne un peptide isolé contenant une séquence d'acide aminé d'un peptide mimétique d'épitope glucidique, dans lequel la séquence d'acide aminé est exposée dans n'importe quelles des SEQ ID NOS: 1-8, 27-38, 39, 40 et 41, y compris les variants, les analogues et les fragments actifs correspondants. Cette invention concerne, en outre, un acide nucléique isolé codant un peptide contenant une séquence d'acide aminé d'un peptide mimétique d'épitope glucidique. Ladite invention a trait à des compositions pharmaceutiques et à des méthodes diagnostiques et thérapeutiques d'utilisation des polypeptides isolés et des acides nucléiques, notamment de modulation et de médiation de l'adhésion intercellulaire et de l'infection virale, et aux processus et aux faits ainsi médiés. Cette invention porte également sur des dosages pour composés qui imitent, modifient ou inactivent les polypeptides de la présente invention, et qui sont utilisés en thérapie.
PCT/US2000/004730 1999-02-24 2000-02-24 Peptides mimetiques d'epitope glucidique et utilisations correspondantes WO2000050447A1 (fr)

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AU28841/00A AU2884100A (en) 1999-02-24 2000-02-24 Carbohydrate epitope mimic peptides and uses thereof

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WO2002004007A2 (fr) * 2000-07-06 2002-01-17 Georgetown University Formes (fixes) stables de proteines virales capsidiales et de proteines virales capsidiales hybrides, de preference les proteines l1 du papillomavirus, et leurs utilisations
EP1279732A2 (fr) * 2001-07-18 2003-01-29 Nemod Immuntherapie AG Procédé pour l'isolation de grandes diversités de molécules spécifiques pour une molécule cible d'une banque de gènes du type phagemide
EP2248530A1 (fr) 2009-05-08 2010-11-10 Universitätsklinikum Hamburg-Eppendorf Nouvelles compositions pour le traitement d'une infection pseudomonique
CN103059101A (zh) * 2012-12-21 2013-04-24 南昌大学 黄曲霉毒素b1的抗原模拟表位及其应用
CN104311634A (zh) * 2014-05-26 2015-01-28 南昌大学 黄曲霉毒素b1的抗原模拟表位am-1及其应用
EP2622091B1 (fr) * 2010-09-23 2019-03-13 Precision Biologics, Inc. Peptidomimétiques du cancer du côlon et du pancréas
US10662212B2 (en) 2014-03-13 2020-05-26 Universitat Basel Carbohydrate ligands that bind to IGM antibodies against myelin-associated glycoprotein
US11091591B2 (en) 2015-09-16 2021-08-17 Universität Basel Carbohydrate ligands that bind to antibodies against glycoepitopes of glycosphingolipids

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US5817748A (en) * 1995-03-17 1998-10-06 The Research Foundation Of State University Of New York Mimotopes of human Platelet glycoprotein Ib/IX
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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002004007A2 (fr) * 2000-07-06 2002-01-17 Georgetown University Formes (fixes) stables de proteines virales capsidiales et de proteines virales capsidiales hybrides, de preference les proteines l1 du papillomavirus, et leurs utilisations
WO2002004007A3 (fr) * 2000-07-06 2002-04-18 Univ Georgetown Formes (fixes) stables de proteines virales capsidiales et de proteines virales capsidiales hybrides, de preference les proteines l1 du papillomavirus, et leurs utilisations
EP1279732A2 (fr) * 2001-07-18 2003-01-29 Nemod Immuntherapie AG Procédé pour l'isolation de grandes diversités de molécules spécifiques pour une molécule cible d'une banque de gènes du type phagemide
EP1279732A3 (fr) * 2001-07-18 2003-10-29 Nemod Immuntherapie AG Procédé pour l'isolation de grandes diversités de molécules spécifiques pour une molécule cible d'une banque de gènes du type phagemide
US9056081B2 (en) 2009-05-08 2015-06-16 Universitätsklinikum Hamburg-Eppendorf Compositions for treating pseudomonas infection
EP2248530A1 (fr) 2009-05-08 2010-11-10 Universitätsklinikum Hamburg-Eppendorf Nouvelles compositions pour le traitement d'une infection pseudomonique
EP2622091B1 (fr) * 2010-09-23 2019-03-13 Precision Biologics, Inc. Peptidomimétiques du cancer du côlon et du pancréas
US10533040B2 (en) 2010-09-23 2020-01-14 Precision Biologics, Inc. Colon and pancreas cancer peptidomimetics
US11078245B2 (en) 2010-09-23 2021-08-03 Precision Biologics, Inc. Colon and pancreas cancer peptidomimetics
CN103059101A (zh) * 2012-12-21 2013-04-24 南昌大学 黄曲霉毒素b1的抗原模拟表位及其应用
CN103059101B (zh) * 2012-12-21 2014-12-03 南昌大学 黄曲霉毒素b1的抗原模拟表位及其应用
US10662212B2 (en) 2014-03-13 2020-05-26 Universitat Basel Carbohydrate ligands that bind to IGM antibodies against myelin-associated glycoprotein
US11220523B2 (en) 2014-03-13 2022-01-11 Universität Basel Carbohydrate ligands that bind to IgM antibodies against myelin-associated glycoprotein
CN104311634A (zh) * 2014-05-26 2015-01-28 南昌大学 黄曲霉毒素b1的抗原模拟表位am-1及其应用
CN104311634B (zh) * 2014-05-26 2017-07-28 南昌大学 黄曲霉毒素b1的抗原模拟表位am‑1及其应用
US11091591B2 (en) 2015-09-16 2021-08-17 Universität Basel Carbohydrate ligands that bind to antibodies against glycoepitopes of glycosphingolipids

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