US20040081984A1 - Ocular tear growth factor-like protein - Google Patents

Ocular tear growth factor-like protein Download PDF

Info

Publication number
US20040081984A1
US20040081984A1 US10/468,372 US46837203A US2004081984A1 US 20040081984 A1 US20040081984 A1 US 20040081984A1 US 46837203 A US46837203 A US 46837203A US 2004081984 A1 US2004081984 A1 US 2004081984A1
Authority
US
United States
Prior art keywords
lacritin
seq
acid sequence
nucleic acid
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/468,372
Other languages
English (en)
Inventor
Gordon Laurie
Sandhya Sanghi
Kumar Rajesh
Angela Lumsden
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
VIRGINIA UNIVERSITY OF PATENT FOUNDATION
University of Virginia UVA
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to US10/468,372 priority Critical patent/US20040081984A1/en
Assigned to VIRGINIA, UNIVERSITY OF, VIRGINIA, UNIVERSITY OF PATENT FOUNDATION reassignment VIRGINIA, UNIVERSITY OF ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: VIRGINIA, UNIVERSITY OF, LUMSDEN, ANGELA J., KUMAR, RAJESH, SANGHI, SANDHYA, LAURIE, GORDON W.
Publication of US20040081984A1 publication Critical patent/US20040081984A1/en
Priority to US11/714,538 priority patent/US7459440B2/en
Priority to US11/714,586 priority patent/US7320870B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/04—Artificial tears; Irrigation solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators

Definitions

  • the present invention is directed to a novel ocular protein, designated lacritin, and nucleic acid sequences encoding that protein.
  • compositions comprising lacritin are used to enhance corneal wound healing, and/or treat patients having deficient tear output.
  • the lacrimal acinar cells comprising the lacrimal gland are polarized and highly differentiated tear secreting cells that adhere to a complex periacinar basement membrane.
  • the bulk of the apical cell cytoplasm contains large secretory granules packed with tear proteins.
  • tear proteins include: lysozyme, which plays a prominent bacteriocidal role on the corneal surface; lactoferrin, which functions as both a bacteriocidal agent and as a potential inhibitor of complement activation; secretory component, which regulates the transcellular movement of IgA into acini lumen where it acts on the comeal surface to inhibit bacterial adhesion; and tear lipocalins (tear-specific prealbumin) and growth factors TGF ⁇ , TGF ⁇ and EGF the functions of which are not known.
  • peroxidase is a tear component which has served as a convenient marker in experimental studies. Tears not only have an important bacteriocidal role, they also keep the cornea clean and lubricated and are important for the well-being of the corneal epithelium.
  • Dry Eye also known as keratoconjunctivitis sicca [KCS]
  • KCS keratoconjunctivitis sicca
  • Lacrimal glands of Sjogren's syndrome patients contain foci of B and T lymphocytes whose pathogenic expansion, possibly due to viral insult, can destroy lacrimal acini.
  • acinar volume loss often appears insufficient relative to the theoretical overcapacity of the main lacrimal gland.
  • Estimates suggest a potential secretory output up to ten-fold greater than is required to maintain a normal aqueous tear film layer.
  • Other mechanisms therefore warrant attention, such as aberrant secretion of one or several common cytokines that may directly or indirectly alter lacrimal acinar cell function and/or lead to a decline in neural innervation.
  • Novel autocrine/paracrine factor(s) released by lacrimal acinar cells into the tear film may be required for the health of the lacrimal secretory. machinery, ductal system and corneal epithelium.
  • the periacinar basement membrane is also required for normal secretory function, in part via ‘BM180’whose apparent synergy with laminin-l promotes stimulated tear secretion. Alteration of each of these factors, together or independent of hormonal changes, could contribute to decreased secretory capacity.
  • the present invention is directed to a novel human extracellular glycoprotein termed ‘lacritin ’that is remarkably reduced in Sjogren's syndrome. Furthermore lacritin has been found to act in an autocrine manner to enhance unstimulated (but not stimulated) tear secretion. Lacritin is produced by lacrimal acinar cells and released for the most part into tear fluid—much like acinar cell-expressed TGF ⁇ 's. This glycoprotein acts like a growth factor when added in purified recombinant form to cultures of human corneal epithelial cells, and in a feedback mechanism, it also appears to act on the same lacrimal gland cells that produce it. Accordingly in one embodiment of the present invention, lacritin is included as an active agent in artificial tear products.
  • the present invention is directed to the isolation and characterization of a novel lacrimal gland protein and the nucleic acid sequences encoding that protein.
  • Purified recombinant lacritin has activity as a growth factor on both human corneal epithelial cells and on the lacrimal acinar cells that produce it.
  • a method is provided for treating Dry Eye and other disorders requiring the wetting of the eye by administering compositions comprising a lacritin polypeptide.
  • the gene promoter regulating lacritin gene expression is the most specific of any previously described lacrimal gland gene, the regulatory elements of this gene could be used to express other gene products in the eye.
  • FIG. 1 is a graphic representation that shows recombinant lacritin enhances unstimulated secretion by isolated rat lacrimal acinar cells. Enhancement of unstimulated secretion was observed in the presence of increasing amounts of lacritin on lacritin-coated wells.
  • FIG. 2A and 2B represent lacritin-induced proliferation and tyrosine phosphorylation.
  • FIG. 2A is a graphic representation of the number of human salivary gland (HSG) cells was determined four days after administering various amounts of lacritin (0 to 10 ng/ml of lacritin) to HSG cells in serum-free medium.
  • FIG. 2B is a bar graph representing the proliferation of HSG cells upon administration of BSA (lane1; 10 ng/ml) or serum (lane 2; 10%) was added for the same period of time. All experiments were performed on laminin-1-(0.05 ⁇ M) coated wells.
  • nucleic acid As used herein, “nucleic acid,” “DNA,” and similar terms also include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone.
  • nucleic acid analogs i.e. analogs having other than a phosphodiester backbone.
  • peptide nucleic acids which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.
  • peptide encompasses a sequence of 3 or more amino acids wherein the amino acids are naturally occurring or synthetic (non-naturally occurring) amino acids.
  • Peptide mimetics include peptides having one or more of the following modifications:
  • Naturally occurring amino acid residues in peptides are abbreviated as recommended by the IUPAC-IUB Biochemical Nomenclature Commission as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine is Met or M; Norleucine is Nle; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; Glycine is Gly or G, and X is any amino acid.
  • Other naturally occurring amino acids include, by way of example, 4-hydroxypro
  • Synthetic or non-naturally occurring amino acids refer to amino acids which do not naturally occur in vivo but which, nevertheless, can be incorporated into the peptide structures described herein.
  • the resulting “synthetic peptide” contain amino acids other than the 20 naturally occurring, genetically encoded amino acids at one, two, or more positions of the peptides. For instance, naphthylalanine can be substituted for trytophan to facilitate synthesis.
  • Other synthetic amino acids that can be substituted into peptides include L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, alpha-amino acids such as L-alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha.-methylalanyl, beta.-amino acids, and isoquinolyl.
  • D amino acids and non-naturally occurring synthetic amino acids can also be incorporated into the peptides.
  • Other derivatives include replacement of the naturally occurring side chains of the 20 genetically encoded amino acids (or any L or D amino acid) with other side chains.
  • a “polylinker” is a nucleic acid sequence that comprises a series of three or more different restriction endonuclease recognitions sequences closely spaced to one another (i.e. less than 10 nucleotides between each site).
  • vector is used in reference to nucleic acid molecules that has the capability of replicating autonomously in a host cell, and optionally may be capable of transferring DNA segment(s) from one cell to another.
  • Vectors can be used to introduce foreign DNA into host cells where it can be replicated (i.e., reproduced) in large quantities. Examples of vectors include plasmids, cosmids, lambda phage vectors, viral vectors (such as retroviral vectors).
  • a “gene” refers to the nucleic acid coding sequence as well as the regulatory elements necessary for the DNA sequence to be transcribed into messenger RNA (mRNA) and then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • mRNA messenger RNA
  • a “marker” is an atom or molecule that permits the specific detection of a molecule comprising that marker in the presence of similar molecules without such a marker. Markers include, for example radioactive isotopes, antigenic determinants, nucleic acids available for hybridization, chromophors, fluorophors, chemiluminescent molecules, electrochemically detectable molecules, molecules that provide for altered fluorescence-polarization or altered light-scattering and molecules that allow for enhanced survival of an cell or organism (i.e. a selectable marker).
  • a reporter gene is a gene that encodes for a marker.
  • a promoter is a DNA sequence that directs the transcription of a DNA sequence, such as the nucleic acid coding sequence of a gene.
  • a promoter is located in the 5′ region of a gene, proximal to the transcriptional start site of a structural gene. Promoters can be inducible (the rate of transcription changes in response to a specific agent), tissue specific (expressed only in some tissues), temporal specific (expressed only at certain times) or constitutive (expressed in all tissues and at a constant rate of transcription).
  • a core promoter contains essential nucleotide sequences for promoter function, including the TATA box and start of transcription.
  • a core promoter may or may not have detectable activity in the absence of specific sequences that enhance the activity or confer tissue specific activity.
  • An “enhancer” is a DNA regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.
  • the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence “A-G-T,” is complementary to the sequence “T-C-A.”
  • hybridization is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the length of the formed hybrid, and the G:C ratio within the nucleic acids.
  • lacritin polypeptide and like terms refers to peptides comprising the amino acid sequence of SEQ ID NO: 4 and biologically active fragments thereof.
  • biologically active fragments or “bioactive fragment” of an lacritin polypeptide encompasses natural or synthetic portions of the amino acid sequence MKFTTLLFLAAVAGALVYAEDASSDSTGADPAQEAGTSKPNEEI SGPAEPASPPETTTTAQETSAAAVQGTAKVTSSRQELNPLKS IVEKSILLTEQALAKAGKGM HGGVPGGKQFIENGSEFAQKLLKKFSLLKPWA (SEQ ID NO: 4) that are capable of specific binding to at least one of the natural ligands of the native lacritin polypeptide.
  • operably linked refers to a juxtaposition wherein the components are configured so as to perform their usual function.
  • control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • treating includes alleviating the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
  • the present invention is directed to a novel human growth factor-like molecule, ‘lacritin ’and compositions comprising lacritin.
  • the invention also encompasses the nucleic acid sequences encoding lacritin as well as the nucleic acid regulatory elements controlling the expression of lacritin.
  • the full length ‘lacritin ’cDNA has been cloned from a human lacrimal gland library (SEQ ID NO:2), and the corresponding genomic gene (SEQ ID NO: 1) has been cloned and sequenced, including 5.2 kb of upstream and 2.8 kb of downstream genomic sequence.
  • the present invention is directed to a purified polypeptide comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 10, a bioactive fragment of SEQ ID NO: 4, or an amino acid sequence that differs from SEQ ID NO: 4 by one or more conservative amino acid substitutions. More preferably, the purified polypeptide comprises an amino acid sequence that differs from SEQ ID NO: 4 by 20 or less conservative amino acid substitutions, and more preferably by 10 or less conservative amino acid substitutions. Alternatively, the polypeptide may comprise an amino acid sequence that differs from SEQ ID NO: 4 by 1 to 5 alterations, wherein the alterations are independently selected from a single amino acid deletion, insertion or substitution. In one preferred embodiment a composition is provided comprising a polypeptide, selected from the group consisting of SEQ ID NO: 4, or SEQ ID NO: 10, and a pharmaceutically acceptable carrier.
  • ligands that bind to the lacritin polypeptide including the natural receptor for lacritin, as well as methods for isolating such ligands.
  • the lacritin polypeptide, or bioactive fragments thereof is used to isolate ligands that bind to the lacritin polypeptide under physiological conditions.
  • the method comprises the steps of contacting the lacritin polypeptide with a mixture of compounds under physiological conditions, removing unbound and non-specifically bound material, and isolating the compounds that remain bound to the lacritin polypeptides.
  • the lacritin polypeptide will be bound to a solid support using standard techniques to allow for rapid screening of compounds.
  • the solid support can be selected from any surface that has been used to immobilize biological compounds and includes but is not limited to polystyrene, agarose, silica or nitrocellulose.
  • the solid surface comprises functionalized silica or agarose beads. Screening for such compounds can be accomplished using libraries of pharmaceutical agents and standard techniques known to the skilled practitioner.
  • a cell based assay is used to detect ligands that bind to lacritin (including lacritin's natural receptor).
  • the method comprises contacting transfected cells with lacritin and isolating the relevant genes from those cells that display lacritin-dependent calcium signaling. More particularly, in one embodiment, previously described pools of orphan G protein coupled receptor cDNA′s will be expressed in cell lines such as HEK293T and RH7777 cells, and the transfected cells will be contacted with lacritin. A transfectant that displays lacritin-dependent calcium signaling should be expressing the receptor.
  • cDNA's from a salivary ductal cell library will be transfected into 293T cells, and expressors screened by FACS with fluorescently labeled lacritin.
  • cells expressing receptors that can be activated by lacritin will be detected using a cell free system. More particularly, receptor activity will be detected via a GTP[ ⁇ 35 S] binding assay using isolated cell membranes from the transfected cells.
  • a method for detecting the lacritin receptor comprises the steps of providing a cell that has been transfected with nucleic acid sequences that encode for potential cell receptors, contacting the transfected cells with lacritin and detecting those cells that display lacritin-dependent calcium signaling. If the cells displaying lacritin-dependent calcium signaling were transfected with more than one protein encoding gene sequence, than the nucleic acid sequences encoding for the lacritin receptor will be identified by sequence analysis or other molecular technique. For example, the introduced recombinant nucleic acids will be isolated from the signaling cells and further subcloned with the resulting subclones used to transfect cells to determine the unique sequence responsible for conferring lacritin-dependent calcium signaling to a cell.
  • the present invention also encompasses nucleic acid sequences that encode the lacritin polypeptide and derivatives thereof.
  • the present invention is directed to nucleic acid sequences comprising the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or fragments thereof.
  • purified nucleic acids comprising at least 8 contiguous nucleotides (i. e., a hybridizable portion) that are identical to any 8 contiguous nucleotides of SEQ ID NO: 1 are provided.
  • the nucleic acids comprises at least 25 (contiguous) nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, or 500 nucleotides of SEQ ID NO: 1.
  • the nucleic acid sequence comprises the sequence of SEQ ID NO: 3 or a 25 bp nucleic acid sequence that is identical to a contiguous 25 bp sequence of SEQ ID NO: 3.
  • the present invention also includes nucleic acids that hybridize (under conditions defined herein) to all or a portion of the nucleotide sequence represented by SEQ ID NO: 1 or its complement.
  • the hybridizing portion of the hybridizing nucleic acids is typically at least 15 (e.g., 20, 25, 30, or 50) nucleotides in length.
  • Hybridizing nucleic acids of the type described herein can be used, for example, as a cloning probe, a primer (e.g., a PCR primer), or a diagnostic probe. It is anticipated that the DNA sequence of SEQ ID NO: 1, or fragments thereof can be used as probes to detect homologous genes from other vertebrate species.
  • Nucleic acid duplex or hybrid stability is expressed as the melting temperature or Tm, which is the temperature at which a nucleic acid duplex dissociates into its component single stranded DNAs. This melting temperature is used to define the required stringency conditions. Typically a 1% mismatch results in a 1 ° C. decrease in the Tm, and the temperature of the final wash in the hybridization reaction is reduced accordingly (for example, if two sequences having >95% identity, the final wash temperature is decreased from the Tm by 5° C.). In practice, the change in Tm can be between 0.5° C. and 1.5° C. per 1% mismatch.
  • the present invention is directed to the nucleic acid sequence of SEQ ID NO: 1 and nucleic acid sequences that hybridize to that sequence (or fragments thereof) under stringent or highly stringent conditions.
  • the invention is directed to a purified nucleic acid sequence that hybridizes to a 100 nucleotide fragment of SEQ ID NO: 1 or its complement under stringent conditions.
  • highly stringent conditions are defined as conducting the hybridization and wash conditions at no lower than ⁇ 5° C. Tm.
  • Stringent conditions are defined as involve hybridizing at 68° C. in 5 ⁇ SSC/5 ⁇ Denhardt's solution/1.0% SDS, and washing in 0.2 ⁇ SSC/0.1% SDS at 68° C.
  • Moderately stringent conditions include hybridizing at 68° C. in 5 ⁇ SSC/5 ⁇ Denhardt's solution/1.0% SDS and washing in 3 ⁇ SSC/0.1% SDS at 42° C. Additional guidance regarding such conditions is readily available in the art, for example, by Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.) at Unit 2.10.
  • nucleic acid sequences encoding the lacritin polypeptide can be inserted into expression vectors and used to transfect cells to express recombinant lacritin in the target cells.
  • the nucleic acid sequence of SEQ ID NO: 3 are inserted into a eukaryotic expression vector in a manner that operably links the gene sequences to the appropriate regulatory sequences, and lacritin is expressed in a eukaryotic host cell. Suitable eukaryotic host cells and vectors are known to those skilled in the art.
  • nucleic acid sequences encoding lacritin may be added to a cell or cells in vitro or in vivo using delivery mechanisms such as liposomes, viral based vectors, or microinjection.
  • delivery mechanisms such as liposomes, viral based vectors, or microinjection.
  • one aspect of the present invention is directed to transgenic cell lines that contain recombinant genes that express the lacritin polypeptide of SEQ ID NO: 4.
  • the present invention is also directed to nucleic acid constructs for expressing heterologous genes under the control of the lacritin gene promoter.
  • a nucleic acid construct is provided comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 operably linked to a heterologous gene.
  • the heterologous gene is a reporter gene that encodes for a marker.
  • the marker can be any gene product that produces a detectable signal and includes proteins capable of emitting light such as Green Fluorescent Protein (GFP) (Chalfie et al., 1994, Science 11: 263:802-805) or luciferase (Gould et al., 1988, Anal. Biochem. 15: 175: 5-13), as well as proteins that can catalyze a substrate (e.g., such as ⁇ -galactosidase).
  • the marker may also comprise intracellular or cell surface proteins that are detectable by antibodies. Reporter molecules additionally, or alternatively, can be detected by virtue of a unique nucleic acid sequence not normally contained within the cell.
  • GFP refers to a member of a family of naturally occurring fluorescent proteins, whose fluorescence is primarily in the green region of the spectrum.
  • the term includes mutant forms of the protein with altered or enhanced spectral properties. Some of these mutant forms are described in Cormack, et al., 1996, Gene 173: 33-38 and Ormo, 1996, Science 273:1392-1395, the entireties of which are incorporated herein by reference.
  • the term also includes polypeptide analogs, fragments or derivatives of GFP polypeptides which differ from naturally-occurring forms by the identity or location of one or more amino acid residues, (e.g., by deletion, substitution or insertion) and which share some or all of the properties of the naturally occurring forms so long as they generate detectable signals (e.g., fluorescence). Wild type GFP absorbs maximally at 395 nm and emits at 509 nm. High levels of GFP expression have been obtained in cells ranging from yeast to human cells.
  • BFP Blue Fluorescent Protein
  • Another embodiment of the present invention comprises antibodies that are generated against the lacritin polypeptide. These antibodies can be formulated with standard carriers and optionally labeled to prepare therapeutic or diagnostic compositions. Antibodies to lacritin are generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric (i.e “humanized” antibodies), single chain (recombinant), Fab fragments, and fragments produced by a Fab expression library. These antibodies can be used as diagnostic agents for the diagnosis of conditions or diseases characterized by expression or overexpression of lacritin, or in assays to monitor patients being treated for a conditions or diseases characterized by inappropriate lacritin expression.
  • the antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics.
  • the antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a marker.
  • an antibody is provided that specifically binds to the protein of SEQ ID NO: 4, and more preferably the antibody is a monoclonal antibody.
  • the invention also encompasses antibodies, including anti-idiotypic antibodies, antagonists and agonists, as well as compounds or nucleotide constructs that inhibit expression of the lacritin gene (transcription factor inhibitors, antisense and ribozyme molecules, or gene or regulatory sequence replacement constructs), or promote expression of lacritin (e.g., expression constructs wherein the lactritin coding sequences, such as SEQ ID NO: 3 are operatively associated with expression control elements such as promoters, promoter/enhancers, etc.).
  • lacritin gene transcription factor inhibitors, antisense and ribozyme molecules, or gene or regulatory sequence replacement constructs
  • promote expression of lacritin e.g., expression constructs wherein the lactritin coding sequences, such as SEQ ID NO: 3 are operatively associated with expression control elements such as promoters, promoter/enhancers, etc.
  • the present invention also encompasses antigenic compositions for raising antibodies against lacritin.
  • an antigenic composition comprising the polypeptide of SEQ ID NO: 4 or an antigenic fragment thereof.
  • Lacritin has mitogenic activity, enhances unstimulated but not stimulated secretion, and promotes signaling in both lacrimal acinar and corneal epithelial cells.
  • Recombinant lacritin prepared in E. coli specifically and rapidly activates both human corneal epithelial cells and mouse & rat lacrimal acinar cells -the latter in an autocrine manner to enhance tear synthesis.
  • Lacritin is active at ng/ml levels, and contaminating bacterial LPS (endotoxin) is not detectable.
  • the activities of purified recombinant lacritin indicate that it acts as a growth factor on both human corneal epithelial cells and on the lacrimal acinar cells that produce it.
  • lacritin likely acts as a growth factor only in the eye, and to a lesser extent in the salivary gland.
  • compositions comprising lacritin are used to enhance corneal wound healing, and/or treat patients having deficient tear output. More particularly, lacritin is used in accordance with one embodiment to treat Dry Eye syndromes, including Sjogren′s syndrome and to enhance corneal wound healing by topical application of compositions comprising the lacritin polypeptide.
  • the composition comprises a pharmaceutically acceptable carrier and a pharmaceutically effective amount of substantially pure polypeptide comprising the amino acid sequence of SEQ ID NO: 4 is used to treat Dry Eye syndromes.
  • the lacritin compositions of the present invention can be formulated using standard ophthalmic components, and preferably the compositions are formulated as solutions, suspensions and other dosage forms for topical administration.
  • Aqueous solutions are generally preferred, based on ease of formulation, biological compatibility (especially in view of the malady to be treated, e.g., dry eye-type diseases and disorders), as well as a patient's ability to easily administer such compositions by means of instilling one to two drops of the solutions in the affected eyes.
  • the compositions may also be suspensions, viscous or semi-viscous gels, or other types of solid or semi-solid compositions.
  • compositions of the present invention may include surfactants, preservative agents, antioxidants, tonicity agents, buffers, preservatives, co-solvents and viscosity building agents.
  • Various surfactants useful in topical ophthalmic formulations may be employed in the present compositions. These surfactants may aid in preventing chemical degradation of lacritin and also prevent the lacritin from binding to the containers in which the compositions are packaged. Examples of surfactants include, but are not limited to: Cremophor.RTM. EL, polyoxyl 20 ceto stearyl ether, polyoxyl 40 hydrogenated castor oil, polyoxyl 23 lauryl ether and poloxamer 407 may be used in the compositions.
  • Antioxidants may be added to compositions of the present invention to protect the lacritin polypeptide from oxidation during storage.
  • antioxidants include, but are not limited to, vitamin E and analogs thereof, ascorbic acid and derivatives, and butylated hydroxyanisole (BHA).
  • Existing artificial tears formulations can also be used as pharmaceutically acceptable carriers for the lacritin active agent.
  • lacritin is used to improve existing artificial tear products for Dry Eye syndromes, as well as develop products to aid corneal wound healing.
  • artificial tears compositions useful as carriers include, but are not limited to, commercial products, such as Tears Naturale.RTM., Tears Naturale II.RTM., Tears Naturale Free.RTM., and Bion Tears.RTM. (Alcon Laboratories, Inc., Fort Worth, Tex.).
  • Examples of other phospholipid carrier formulations include those disclosed in U.S. Pat. No. 4,804,539 (Guo et al.), U.S. Pat. No.
  • compositions of the present invention may also be added to the ophthalmic compositions of the present invention to increase the viscosity of the carrier.
  • viscosity enhancing agents include, but are not limited to: polysaccharides, such as hyaluronic acid and its salts, chondroitin sulfate and its salts, dextrans, various polymers of the cellulose family; vinyl polymers; and acrylic acid polymers.
  • the phospholipid carrier or artificial tears carrier compositions will exhibit a viscosity of 1 to 400 centipoises (“cps”).
  • Preferred compositions containing artificial tears or phospholipid carriers will exhibit a viscosity of about 25 cps.
  • Topical ophthalmic products are typically packaged in multidose form. Preservatives are thus required to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, chlorobutanol, benzododecinium bromide, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium, sorbic acid, polyquaternium-1, or other agents known to those skilled in the art. Such preservatives are typically employed at a level of from 0.001 to 1.0% w/v. Unit dose compositions of the present invention will be sterile, but typically unpreserved. Such compositions, therefore, generally will not contain preservatives.
  • lacritin is produced by the lacrimal gland (large amounts), salivary gland (moderate), the basal cells of the corneal epithelium (based on inmmunostaining of human cornea by anti-lacritin antibodies; and ELISA detection of lacritin in human corneal epithelial cell cultures) and possibly in the thyroid, but not elsewhere.
  • Lacritin enhances unstimulated but not stimulated secretion, has mitogenic activity and promotes signaling in both lacrimal acinar and corneal epithelial cells.
  • This glycoprotein has a highly restricted glandular distribution, and this highly restricted expression pattern in combination with its functional attributes are evidence for its putative autocrine/paracrine differentiative role in the lacrimal gland and neighboring ocular system.
  • the gene promoter regulating lacritin gene expression is the most specific of any previously described lacrimal gland gene, the regulatory elements of this gene could be used to express other gene products in the eye.
  • the lacritin gene promoter can be operably linked to a wide variety of exogenous genes to regulate the expression of the gene products to the lacrimal gland and/or used as gene therapy to treat Dry Eye syndromes.
  • recombinant constructs comprising the lacritin promoter can be used to transform host cells in vitro as a means of screening for agonist and antagonist of lacritin function.
  • the lacritin gene promoter is linked to a heterologous gene and reintroduced into a patient to provide gene therapeutic treatment of Dry Eye syndromes.
  • the promoter could be used to artificially drive the synthesis and secretion of tear proteins in patients for which the normal gene control of these proteins may have been lost.
  • Lacritin stimulates calcium signaling in human corneal epithelial cells and in mouse lacrimal acinar cells. It stimulates tyrosine phosphorylation in rat lacrimal acinar and human salivary ductal cells, and it enhances the quantity of tear proteins released from the same acinar cells that produce it.
  • Duplicate filters containing plaques (5 ⁇ 104 per filter) from each of ten sublibraries of a human lacrimal gland cDNA library were prehybridized at 42° C. for 4 hr in 5 ⁇ Denhardt's, 6.76 ⁇ SSC, 10 mM sodium phosphate, 1 mM EDTA, 0.5% SDS and 182 ⁇ g/ml salmon sperm DNA, and then hybridized overnight at 42° C.
  • oligonucleotides ‘s1’ [AGCTGGGGCACAGGCACCCGCAC; SEQ ID NO: 11 ] and ‘S2’ [GGGGTTCTGGGGCTGCAGCTGGG; SEQ ID NO: 12]) that had been end-labeled with [32P]gATP 7000 Ci/mmole (ICN, Irvine Calif.) and purified.
  • Final wash conditions were 2 ⁇ SSC (45° C.), corresponding to 29.5° C. less than the S1 or S2 Tm (74.5° C. in 2 ⁇ SSC for both). Plaques positive in both filters were picked and rescreened three times in duplicate with each oligonucleotide, giving rise to forty-seven clones.
  • Lacritin insert was subsequently used to screen a human P1 genomic library (carried out by Genome Systems Inc; St. Louis MO) and three identical clones were obtained, as determined by restriction digestion and Southern analysis. The largest lacritin-positive fragment (12.4 kb) was subcloned intact into pBluescript and both strands were completely sequenced. Alignment and analyses (Kumar et al, 2000) of cDNA and genomic sequence was primarily with Unix-based (Gelstart, Gap) and web-based (FASTA, BestFit, Gap) Genetics Computer Group (Madison WI) software using default settings and E values (FASTA) restricted to 5 or less.
  • Genomic exon searching and identification of splice sites was facilitated by the Baylor College of Medicine Human Genome Sequencing Center web site. All nucleotide sequences have been submitted to the GenBank/EBI Data Bank with accession numbers af238867 (cDNA) and ay005150 (genornic).
  • RNA integrity was initially determined by electrophoresis of ethidium bromide-complexed RNA samples in a gel containing 0.22M formaldehyde. Samples that did not show prominent 28S and 18S rRNA bands in a 1:1-2:1 ratio under UV light were rejected. For blotting, RNA (5 ⁇ g/lane) was separated on a 0.8% agarose gel under denaturing conditions (Laurie et al, 1989) and transferred to nitrocellulose.
  • Blots were hybridized with [32P]-labeled lacritin insert, washed in 0.1 ⁇ SSC, 0.1% SDS (Northern) or 2 ⁇ SSC, 0.1% SDS (dot blot) at 55° C., and exposed to X-ray film. Dot blots were then quantitated using NIH Image by measurement of pixel gray values of individual dots.
  • Lacrimal acinar cells are polarized exocrine secretory cells containing some mRNA′s that are remarkably under-represented in gene data banks and may code for a rich array of differentiation factors—a presumption underlying the paired oligonucleotide screening of a little used human lacrimal gland cDNA library.
  • SEQ ID NO: 2 a novel cDNA sequence represented by several independent clones and corresponding to a 760 bp transcript and the corresponding amino acid sequence (SEQ ID NO: 4).
  • the secreted gene product of this lacrimal gland-specific transcript was designated ‘lacritin’.
  • the lacritin nucleic acid sequence contains a 417 bp open reading frame that predicts a 14.3 kDa hydrophilic protein core with a 19 amino acid signal peptide giving rise to a mature secreted core protein of 12.3 kDa with an isoelectic point of 5.
  • Noteworthy is a moderately high level of glycosylation with six putative O-glycosylation sites between residues 52 and 64, and a single N-glycosylation site near the C-terminus, indicating that lacritin is a moderately well-glycosylated core protein much like the neuroglycan C glycosaminoglycan binding domain and fibulin-2 amino globular domain to which lacritin bears partial homology.
  • Northern Blot analysis indicates a high level of lacrimal gland specificity.
  • Neuroglycan C (af059274) is a component of brain extracellular matrix (anchored by transmembrane domain; Yasuda et al, 1998).
  • lacritin coding sequence was subcloned into pET-28b and pcDNA3.1/myc-His(+)C. to generate recombinant bacterial and mammalian (293-T cell) lacritin, respectively. Both forms of lacritin displayed anomalous migration in SDS PAGE.
  • Anti-bacterial lacritin antiserum was subsequently prepared in rabbits (Covance Research Products, Denver PA), and assessed by ELISA (1/1000 dilution) using recombinant bacterial lacritin (4 ⁇ g/ml) as coat and preimmune serum (1/1000) as control.
  • ELISA ELISA 1/1000 dilution
  • recombinant bacterial lacritin 4 ⁇ g/ml
  • preimmune serum (1/1000) as control.
  • sections of zinc formalin-fixed, paraffin-embedded human tissues and a human tissue microarray were deparaffinized and rehydrated, and microwave heated (20 min in 10 mM citrate buffer, pH 6.0) to expose antigen.
  • rat lacrimal acinar cells and HSG (human salivary gland) ductal and HCE (human corneal epithelial) cell lines were used to study lacritin function.
  • HSG human salivary gland
  • HCE human corneal epithelial
  • rat acinar cells were plated serum-free overnight on wells co-coated with 0.05 ⁇ M laminin 1 (to ensure adhesion) and 0 to 20 ⁇ M lacritin, or alternatively with laminin-1 (0.05 ⁇ M) and treated the next day with serum-free medium containing 0 to 162 ng/ml of soluble lacritin for four hours.
  • Unstimulated and stimulated (carbachol 10-4M/VIP10-8M) secretions were then collected, assessed (peroxidase assay) and normalized to ⁇ g cellular DNA.
  • To study tyrosine phosphorylation overnight serum-free cultures of both rat lacrimal acinar and HSG cells were washed and treated with 10 ng/ml of soluble lacritin for 0.5, 2.5, 10 and 30 min. Py(20) anti-phosphotyrosine antibody immunoprecipitation of cell lysates was then examined in Western blots of 7% SDS PAGE gels using Py(20) and ECL for detection.
  • HCE cells were grown to confluency on glass coverslips in keratinocyte media (Life Technologies, Rockville Md.) containing bovine pituitary extract (30 ⁇ g/ml), EGF (0.1 ng/ml) and penicillin/streptomycin, and rendered quiescent 18 hrs before loading with Fluo-3AM (2 ⁇ M; Molecular Probes, Eugene Oreg.) at 37° C. for 30 min.
  • Fluo-3AM 2 ⁇ M; Molecular Probes, Eugene Oreg.
  • Binding studies were carried out in 96 well plates coated with 10 ⁇ g/well of collagen IV, laminin-1, entactin/nidogen-1, collagen I, fibronectin, vitronectin, EGF, heparin or BMS (Matter & Laurie, 1994). Wells were washed, blocked (PBS-T), incubated with 0-30 nM lacritin (in PBS-T containing 1% BSA) for 1 hr (4° C.), washed and detected with anti-lacritin antibody (1/1000) by ELISA.
  • Antibodies prepared against bacterial lacritin were applied to sections of human lacrimal and salivary glands and to tissue microarrays containing formalin-fixed, paraffin embedded sections of 75 different human tissues and organs (see Table I). Immunoreactivity was clearly observed in secretory granules of acinar cells in lacrimal and major and minor salivary glands, but was not apparent in other epithelia or stroma. Presence in thyroid was equivocal (Table I). Frequency of acinar cell staining was high in lacrimal gland, whereas only scattered salivary acinar cells were reactive. Immunoreactivity was also apparent in secretions within lumens of lacrimal and salivary ducts.
  • Lacritin function was assessed in serum-free cultures of lacrimal acinar, salivary ductal and corneal epithelial cells using secretion (acinar), proliferation (ductal), tyrosine phosphorylation (acinar, ductal) and calcium signaling (corneal epithelial) assays.
  • Freshly isolated rat lacrimal acinar cells were plated on increasing amounts of lacritin (with a constant small amount of laminin 1 to ensure adherence), or on laminin-l-coated wells in which lacritin was added to the medium. Both coated and soluble lacritin enhanced unstimulated secretion in a dose-dependent manner (see FIG.
  • Exon 3 contains sequence for all putative 0-glycosylation sites.
  • the predicted N-glycosylation site is formed at the exon 4/exon 5 splice junction.
  • Exon 5 includes 53 bp of 3′ untranslated sequence.
  • Three potential polyadenylation sites are detected 367, 474 and 534 bp downstream of exon 5, the first of which would be in keeping with a 760 bp transcript.
  • Sequences at exon-intron boundaries all conform to predicted splice donors or acceptors, with the exception of the exon 4 splice acceptor.
  • Intronic sequences revealed common intronic repeat elements. Also independently discovered on a separate genomic fragment was a lacritin pseudogene lacking 38 bp of 5′ exon 1 sequence.
  • RT-PCR was used with submandibular or lacrimal gland cDNA as template and forward and reverse primers from exons 1 and 5, respectively, each including untranslated flanking sequence.
  • a single PCR product was detected in both organs whose size (449 bp) was in keeping with transcription from all five exons without alternative splicing.
  • FISH revealed that the lacritin gene is located on chromosome 12, a result confirmed by double labeling with a probe for 12q15. Measurement often specifically labeled chromosomes located the lacritin gene approximately 16% of the distance from the centromere to the telomere of 12q, an area that corresponds to 12q13.
  • Lacrimal acinar cells displayed enhanced unstimulated (but not stimulated) secretion and rapid tyrosine phosphorylation of a 48 kDa protein.
  • Ductal cells phosphorylated the same 48 kDa band and were proliferative.
  • a rapid and sustained calcium transient was noted in comeal epithelial cells.
  • all cell types contributing to or benefiting from lacritin outflow appear to be lacritin-inducible, whereas controls were negative and there was no evidence of contaminating bacterial lipopolysaccharide (known to be proliferative in immune cell cultures).
  • lacritin acts remains to be elucidated.
  • a common receptor(s) is mediatory, ligation of which may be jointly linked to tyrosine phosphorylation and calcium release as in neural retina where tyrosine kinases have been associated with capacitative calcium entry and inositol-3-phosphate induced release of intracellular calcium stores.
  • lacritin signaling in the three cell types may differ.
  • Lacrimal acinar, ductal and corneal epithelial cells perform strikingly different functions. Although some intracellular signaling machinery may be common, others are unique, and some common machinery may be put to different use.
  • Inr elements at +1,+220 also TATA-box at +190 bp
  • +316 bp intra-tet al.
  • Tasheva ES Conrad AH
  • Conrad GW Conrad GW.
  • transcription could begin at ⁇ 62 bp, as suggested by ‘CorePromoter’. Primer extension and RNA ligase-mediated 5′-RACE will resolve this question.
  • LacP83 20-mer reverse primer
  • Primer extension advantage will be taken of a 20-mer reverse primer (‘LacP83’) designed by ‘Prime’ (GCG, Madison Wis.) which is complementary to nucleotides 64 to 83 bp of lacritin mRNA.
  • LacP83 will be end-labeled by phosphorylation with T4 polynucleotide kinase in the presence of [g 32 P]ATP, annealed with total lacrimal RNA (100 fmol primer per 10 ⁇ g RNA) for 20 min at 58° C., cooled, and then incubated for 30 min at 41° C.
  • Primer extension controls will include replacement of lacrimal RNA with total yeast RNA (or no RNA), and use of an RNA prepared by in vitro transcription with accompanying primer (Promega, Madison Wis.) for which primer extension conditions have been previously established.
  • RNA ligase-mediated 5′-RACE (‘GeneRacer’- 2 ; Invitrogen) will be utilized. This is a powerful PCR-based modification of primer extension. For this purpose, 1-5 ⁇ g of total human lacrimal RNA will be treated with calf intestinal phosphatase (1 U per 10 ⁇ l reaction mix) to remove 5′ phosphates from degraded RNA and non-mRNA contaminants.
  • amplifications will be attempted in the absence of LacP83 or GeneRacer RNA Oligo or without template, and if banding or smearing is observed further PCR optimization will be carried out (ie. use of less template or fewer PCR cycles or do nested PCR to increase amplicon amount, or use touchdown PCR).
  • a luciferase reporter constructs will also be generated and transfection-based regional mapping of lacritin gene regulatory elements will be initiated. It is hypothesized that Bayesian alignment of human and mouse lacritin genes will provide an excellent foundation for interpretation of reporter construct activity, and that evolutionary conservation similarly will make feasible utilization of a rabbit lacrimal acinar cell line as transfection host-the only immortalized cell line from lacrimal gland of any species. This exploratory approach will lay the conceptual groundwork for more detailed studies both in vitro and in vivo.
  • aA-crystallin gene for example, is governed by a transcription complex of CREB/CREM, aA-CRYBP1, Pax 6, TBP, USF, AP-1 (context of AP-1 important for tissue specificity) and L-maf that nucleates on the 150 bp aA-crystallin promoter.
  • Transfected plasmid constructs that artificially position luciferase or chloramphenicol acetyltransferase expression under the control of intact or progressively 5′ shortened (or mutated) promoter regions, has been used previously to identify cis-acting regulatory region of a promoter.
  • the versatile and sensitive ‘Dual-Luciferase Reporter Assay System’(Promega) for example sequentially assays both the transfected gene promoter under investigation (as manifested by the level of expressed firefly luciferase) and a co-transfected internal positive HSV-TK control promoter designed to independently drive expression of a synthetic sea pansy luciferase with distinct substrate properties at a constant baseline level (see below).
  • upstream genomic constructs spanning ⁇ 2435 to ⁇ 10 bp (‘Lacrgen2.4’), ⁇ 1619 to ⁇ 10 bp (‘Lacrgen1.6’) or ⁇ 856 to ⁇ 10 bp (‘Lacrgen0.9’) could include all or most elements necessary for tissue specific and elevated expression.
  • Preparation of each will take advantage of parent amplicon ‘LacrgenInit’( ⁇ 2960 to ⁇ 10 bp) to be generated by PCR from the 12.4 kb lacritin genomic fragment using reverse primer ‘LacP ⁇ 10/Xho I’( ⁇ 10 to ⁇ 31 bp) with an Xho I site incorporated, and forward primer ‘LacP ⁇ 2960’( ⁇ 2960 to ⁇ 2942 bp). Primer pairs are designed by ‘Prime’ (GCG, Madison Wis.).
  • a new rabbit lacrimal acinar cell line (Nguyen et al, ‘99), that has been cultured for twelve months without difficulty will be used for the transfection studies.
  • the cells display a strong epithelial morphology and synthesize secretory component, transferrin and transferrin receptor. Importantly, they also express lacritin and are readily transfectable.
  • HCE-T Araki-Sasaki human corneal epithelial cells
  • HSG human salivary cells both secrete lacritin
  • Optimal lacrimal LipofectAMINE transfection, and ‘Bright-Glo’ luciferase assay conditions (Promega) will take advantage of the pGL3-Control vector whereby transfected cells benefit from luc+expression under SV40 promoter and enhancer control.
  • transfection efficiency will be 75 ⁇ 90%, and that one of Lacrgen2.4, Lacrgen1.6 or Lacrgen0.9—likely Lacrgenl.6 or LacrgenO.9—will best define the minimal sequence required for lacritin promoter activity.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Ophthalmology & Optometry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicinal Preparation (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
US10/468,372 2001-02-20 2002-02-20 Ocular tear growth factor-like protein Abandoned US20040081984A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US10/468,372 US20040081984A1 (en) 2001-02-20 2002-02-20 Ocular tear growth factor-like protein
US11/714,538 US7459440B2 (en) 2001-02-20 2007-03-06 Ocular tear growth factor-like protein
US11/714,586 US7320870B2 (en) 2001-02-20 2007-03-06 Ocular tear growth factor-like protein

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US26990001P 2001-02-20 2001-02-20
US60269900 2001-02-20
PCT/US2002/004971 WO2002065943A2 (en) 2001-02-20 2002-02-20 Ocular tear growth factor-like protein
US10/468,372 US20040081984A1 (en) 2001-02-20 2002-02-20 Ocular tear growth factor-like protein

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US11/714,586 Division US7320870B2 (en) 2001-02-20 2007-03-06 Ocular tear growth factor-like protein
US11/714,538 Division US7459440B2 (en) 2001-02-20 2007-03-06 Ocular tear growth factor-like protein

Publications (1)

Publication Number Publication Date
US20040081984A1 true US20040081984A1 (en) 2004-04-29

Family

ID=23029096

Family Applications (3)

Application Number Title Priority Date Filing Date
US10/468,372 Abandoned US20040081984A1 (en) 2001-02-20 2002-02-20 Ocular tear growth factor-like protein
US11/714,538 Expired - Lifetime US7459440B2 (en) 2001-02-20 2007-03-06 Ocular tear growth factor-like protein
US11/714,586 Expired - Lifetime US7320870B2 (en) 2001-02-20 2007-03-06 Ocular tear growth factor-like protein

Family Applications After (2)

Application Number Title Priority Date Filing Date
US11/714,538 Expired - Lifetime US7459440B2 (en) 2001-02-20 2007-03-06 Ocular tear growth factor-like protein
US11/714,586 Expired - Lifetime US7320870B2 (en) 2001-02-20 2007-03-06 Ocular tear growth factor-like protein

Country Status (8)

Country Link
US (3) US20040081984A1 (ja)
EP (1) EP1395278B1 (ja)
JP (1) JP4288070B2 (ja)
AU (1) AU2002252014A1 (ja)
CA (1) CA2438334C (ja)
DE (1) DE60232967D1 (ja)
ES (1) ES2330087T3 (ja)
WO (1) WO2002065943A2 (ja)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006129867A2 (en) * 2005-06-01 2006-12-07 Senju Pharmaceutical Co., Ltd. ENHANCED EXPRESSION OF LACTOFERRIN mRNA BY LACRITIN
US20100183572A1 (en) * 2007-02-28 2010-07-22 Takeshi Nakajima Cell capable of expressing lacritin at high level
US20110008891A1 (en) * 2008-03-19 2011-01-13 Senju Pharmaceutical Co., Ltd. Partial peptide of lacritin
US20120190627A1 (en) * 2009-07-16 2012-07-26 L'oreal Cosmetic use of lacritin-type polypeptides
US20130196926A1 (en) * 2011-07-26 2013-08-01 University Of Southern California Controlled release of ocular biopharmaceuticals using bioresponsive protein polymers

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2849383B1 (fr) * 2002-12-26 2005-09-30 Jean Noel Thorel Composition trophique en milieu aqueux, et ses applications, notamment en ophtalmologie
CA2566607C (en) * 2004-05-13 2014-04-15 University Of Virginia Patent Foundation Use of lacritin in promoting ocular cell survival
WO2007058383A2 (en) * 2005-11-17 2007-05-24 Senju Pharmaceutical Co., Ltd. Lipid secretion promoter containing lacritin or compound having lacritin activity
US20090312252A1 (en) * 2006-09-14 2009-12-17 University Of Virginia Patent Foundation Antimicrobial Activity in Variants of Lacritin
US7932227B1 (en) 2007-09-17 2011-04-26 University Of Virginia Patent Foundation Lacritin-syndecan fusion proteins
EP2478010B1 (en) 2009-09-16 2014-08-06 Senju Pharmaceutical Co., Ltd. Partial peptide of lacritin
WO2011037196A1 (ja) * 2009-09-25 2011-03-31 千寿製薬株式会社 ラクリチン遺伝子の転写調節因子のスクリーニング方法
JP6612247B2 (ja) 2014-03-12 2019-11-27 ユニバーシテイ・オブ・バージニア・パテント・フアウンデーシヨン 眼の感染症および疾患を処置するための組成物および方法
AU2018225485B2 (en) 2017-02-21 2021-05-27 Tearsolutions, Inc. Stable peptide compositions
KR102337935B1 (ko) * 2019-04-18 2021-12-10 코스맥스 주식회사 라크리틴을 포함하는 피부장벽 개선 및 보습 증진용 화장료 조성물

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US102604A (en) * 1870-05-03 Improved sash-holder
US164669A (en) * 1875-06-22 Improvement in lamps

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0958360A2 (en) * 1996-12-18 1999-11-24 Genetics Institute, Inc. Secreted proteins and polynucleotides encoding them
AUPO500997A0 (en) 1997-02-07 1997-03-06 Macquarie Research Limited Diagnosis of disease using tears
US7053190B2 (en) * 1997-03-07 2006-05-30 Human Genome Sciences, Inc. Secreted protein HRGDF73
JP3320342B2 (ja) 1997-09-12 2002-09-03 インターナショナル・ビジネス・マシーンズ・コーポレーション 印刷をプリンタ・システムに実行させる方法、印刷実行方法、コンピュータ、及びプリンタ・システム
US20020102604A1 (en) * 1999-12-08 2002-08-01 Milne Edwards Jean-Baptiste Dumas Full-length human cDNAs encoding potentially secreted proteins

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US102604A (en) * 1870-05-03 Improved sash-holder
US164669A (en) * 1875-06-22 Improvement in lamps

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006129867A2 (en) * 2005-06-01 2006-12-07 Senju Pharmaceutical Co., Ltd. ENHANCED EXPRESSION OF LACTOFERRIN mRNA BY LACRITIN
WO2006129867A3 (en) * 2005-06-01 2007-04-12 Senju Pharma Co ENHANCED EXPRESSION OF LACTOFERRIN mRNA BY LACRITIN
US20100183572A1 (en) * 2007-02-28 2010-07-22 Takeshi Nakajima Cell capable of expressing lacritin at high level
US20110008891A1 (en) * 2008-03-19 2011-01-13 Senju Pharmaceutical Co., Ltd. Partial peptide of lacritin
US8383130B2 (en) 2008-03-19 2013-02-26 Senju Pharmaceutical Co., Ltd. Partial peptide of lacritin
US20120190627A1 (en) * 2009-07-16 2012-07-26 L'oreal Cosmetic use of lacritin-type polypeptides
US9168215B2 (en) * 2009-07-16 2015-10-27 L'oreal Cosmetic use of lacritin-type polypeptides
EP2453838B1 (en) * 2009-07-16 2020-02-12 L'Oréal Cosmetic use of lacritin-type polypeptides
US20130196926A1 (en) * 2011-07-26 2013-08-01 University Of Southern California Controlled release of ocular biopharmaceuticals using bioresponsive protein polymers
US9102763B2 (en) * 2011-07-26 2015-08-11 University Of Southern California Controlled release of ocular biopharmaceuticals using bioresponsive protein polymers

Also Published As

Publication number Publication date
ES2330087T3 (es) 2009-12-04
DE60232967D1 (de) 2009-08-27
JP2004536570A (ja) 2004-12-09
US20070167372A1 (en) 2007-07-19
AU2002252014A1 (en) 2002-09-04
CA2438334C (en) 2012-07-24
US7459440B2 (en) 2008-12-02
CA2438334A1 (en) 2002-08-29
US20070167371A1 (en) 2007-07-19
EP1395278B1 (en) 2009-07-15
US7320870B2 (en) 2008-01-22
EP1395278A4 (en) 2005-08-10
EP1395278A2 (en) 2004-03-10
WO2002065943A3 (en) 2003-12-11
WO2002065943A2 (en) 2002-08-29
JP4288070B2 (ja) 2009-07-01

Similar Documents

Publication Publication Date Title
US7320870B2 (en) Ocular tear growth factor-like protein
US7648964B2 (en) Use of lacritin in promoting ocular cell survival
JPH11507332A (ja) 結合組織成長因子
JP2003159083A (ja) 新規な神経栄養因子
JPH09510103A (ja) 線維芽細胞成長因子−１０
US20100105625A1 (en) Product and Methods for Diagnosis and Therapy for Cardiac and Skeletal Muscle Disorders
JPH06503949A (ja) 新規インシュリン様成長因子結合タンパク質ｉｇｆｂｐ―５
JP2003512304A (ja) 医薬組成物および分泌フリズル化関連タンパクを用いる方法
CN1747744B (zh) 使用otx2基因再生和新生视网膜感光细胞
US20060286117A1 (en) Neuronally expressed stem cell factor modulates angiogenesis and neural stem cell migration to areas of brain injury
US6372890B1 (en) Water-soluble polypeptides
EP1648494A2 (fr) Nouvel agent anti-angiogenique et son utilisation, notamment dans le cadre du traitement des cancers
US20040014947A1 (en) Novel human ependymin-like protein
EP1191096A1 (en) Novel protein and dna thereof
US5866693A (en) DNA encoding human MAD proteins
JP4175680B2 (ja) 新規タンパク質およびそのｄｎａ
EP0837127A2 (en) Hereditary multiple exostose gene, Ext2
JPH11103867A (ja) Ｅｐｏ一次応答遺伝子１、ｅｐｒｇ１
US20060116338A1 (en) Mammalian early developmental regulator gene
JP2005151826A (ja) Ｃ１ｑｔｎｆ５の用途
WO1998029547A1 (en) Modulators of radial glia-astrocyte differentiation and transformation, and diagnostic and therapeutic uses thereof

Legal Events

Date Code Title Description
AS Assignment

Owner name: VIRGINIA, UNIVERSITY OF, VIRGINIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LAURIE, GORDON W.;SANGHI, SANDHYA;KUMAR, RAJESH;AND OTHERS;REEL/FRAME:014911/0330;SIGNING DATES FROM 20020214 TO 20020422

Owner name: VIRGINIA, UNIVERSITY OF PATENT FOUNDATION, VIRGINI

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LAURIE, GORDON W.;SANGHI, SANDHYA;KUMAR, RAJESH;AND OTHERS;REEL/FRAME:014911/0330;SIGNING DATES FROM 20020214 TO 20020422

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION