Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
• • 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
  • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents

Definitions

• the present invention generally relates to lactoferrin related glycoproteins. More specifically, the present invention relates to lactoferrin variants and portions thereof, their production and uses.
• Lactoferrin is a member of the transferrin family of no ⁇ -heme iron binding glycoproteins (Aisen et al. , 1980, Ann. Rev. Biochem. 49:357-393), which includes transferrin, the major iron-transport protein in blood (MacGillivray et al. , 1983, J. Biol. Chem. 25.8:3543-3553), ovotransferrin, an avian egg white protein (Jeltsch et al , 1982, Eur. J. Biochem. 122:291-295) and melanotransferrin, a membrane bound form of this family found in human melanocytes (Rose et al. , 1986, Proc. Natl. Acad. Sci.
• Lactoferrin has a broad distribution, present in both external secretions that bathe the body surfaces (Masson et al , 1971, Comp. Biochem. Physiol. 22: 119-129; Hennart et al. , 1991 , Am. J. Clin. Nutr. 52:32-39; Masson et al., 1966, Clin. Chim. Acta. 14:735-739; Pentecost et al. , 1987, 7. Biol. Chem. 262: 10134-10139; Yu et al. , 1993, Biochem. J.
• lactoferrin include iron binding and delivery to the small intestine (Fransson et al. , 1980, J. Pediatrics 96_:380-384; Iyer et al. , 1993, Eur. J. Clin. Nutr. 47:232-241 ; Cox et al. , 1979, Biochem. Biophys. Acta. 558:129-141; Hu et al. , 1988, Biochem. J.
• Lactoferrin shares a high degree of structural homology with other members of the transferrin family. All of these proteins are monomeric glycoproteins with a molecular weight of - 80 kDa. Aisen et al. , 1980, Ann. Rev. Biochem. 49:357- 393; Metz-Boutique et al , 1984, Eur. J. Biochem. 145:659-676.
• lactoferrin (Anderson et al , 1989, J. Mol. Biol. 209:711- 734) and transferrin (Lindley et al. , 1988, Biochem. 27:5804-5812) have been precisely defined by X-ray crystallographic analysis.
• the proteins are folded into two globular lobes corresponding to the amino- and carboxy-terminal halves of the protein.
• This bilobal structure with — 40% conservation between the amino- and carboxy-terminal halves, is thought have evolved by intragenic duplication from a common ancestral gene. Williams et al. , 1982, Trends Biochem. Sci. 7:394-397.
• Each of these lobes can reversibly bind iron with high affinity and with the concomitant binding of an anion, usually carbonate.
• lactoferrin The amino acids required for iron binding by lactoferrin are highly conserved between members of the transferrin family. Baker et al. , 1992, J. Inorg. Biochem. 47: 147-160.
• lactoferrin an iron atom binds to Asp 396, Tyr 93, Tyr 193 and His 254 in the amino-terminal lobe, and the corresponding Asp 396, Tyr 436, Tyr 539 and His 598 in the carboxy-terminal lobe, respectively.
• lactoferrin displays much more avid iron binding properties than its serum counterpart, transferrin.
• the release of iron from lactoferrin displays greater pH stability than transferrin, the latter releasing iron in a pH range of about six (6) to about four (4) while the former releases iron a pH range of about four (4) to about two (2).
• Mazuier et al. 1989, Biochem. Biophys. Acta. 622:399-408. It has been suggested that the unique iron binding properties of lactoferrin contribute to some of the diverse functional activities proposed for this protein.
• lactoferrin variants with improved properties including, but not limited to, lactoferrin variants with higher affinity for iron for improved antimicrobial activities, lactoferrin variants with lower affinity for iron having improved iron releasing properties, or lactoferrin variants having modified pH dependent efficiencies for iron release.
• lactoferrin variants with higher affinity for iron for improved antimicrobial activities lactoferrin variants with lower affinity for iron having improved iron releasing properties
• lactoferrin variants having modified pH dependent efficiencies for iron release The Applicants of the present invention have previously reported the high level production and characterization of recombinant human lactoferrin in the filamentous fungi, including Aspergillus. Ward et al , 1995, Biotechnology 13:498-503; U.S. Patent Nos.
• the present invention relates to nucleic acid sequences encoding lactoferrin variants or portions thereof, wherein the portion comprises a sequence corresponding to at least one iron binding site of lactofer ⁇ n.
• the lactoferrin variant or portion thereof is further defined by having a modified iron binding capacity when compared to wild-type lactoferrin.
• the present invention elucidates the contribution of the two-lobe structure to the unique iron binding properties of lactoferrin.
• Human lactoferrin cDNA was selectively mutated in the two tyrosine residues involved in iron binding in either or both halves of the protein.
• the resulting three iron binding defective variants were expressed in and purified from Aspergillus awamori.
• Iron binding analysis using 59 FeCl confirmed that mutation of the two tyrosine residues involved in iron binding in either lobe, resulted in selective loss of iron binding to the mutated lobe.
• the present invention provides guidance for the generation of lactoferrin variants having improved properties, such as modified iron binding characteristics, e.g. , increased or decreased affinity for iron, or modified pH or temperature requirements or ranges for iron binding. Furthermore, the present invention provides guidance for the modelling of lactoferrin variants having otherwise improved characteristics, e.g. , therapeutic tolerance, while retaining their iron binding activity.
• the present invention is further directed to vectors comprising nucleic acids encoding lactoferrin variants or portions thereof, suitable for expression of the lactoferrin variant or portion thereof in eukaryotic cells.
• the plasmid are suitable for expression in fungal cells, including Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans and Aspergillus awamori.
• the present invention is further directed to methods of producing such vectors, and to transfected cells harboring the same. Methods for the production of lactoferrin and variants thereof in various eukaryotic or prokaryotic cells are also provided.
• the invention is directed to lactoferrins and variants thereof produced by the processes of the invention.
• the invention provides an efficient and economical means for the production of recombinant lactoferrin protein and variants thereof, and lactoferrin related polypeptides.
• FIGURES 1A and IB depict a Western immunoblot and silver stain analysis of the purified iron binding defective lactoferrin variants.
• FIGURE 1A depicts a Western immunoblot analysis of 200 ng samples of purified recombinant human lactoferrin (Rec hLF), amino-terminal (MN-2Y), carboxy-terminal (MC-2Y) and amino- and carboxy-terminal (MNC-4Y) iron binding defective variants of lactoferrin, respectively.
• Rec hLF purified recombinant human lactoferrin
• MN-2Y amino-terminal
• MC-2Y carboxy-terminal
• MNC-4Y amino- and carboxy-terminal
• FIGURE IB depicts a Silver-stained SDS-polyacrylamide gel analysis of purified Rec hLF, MN-2Y, MC-2Y and MNC-4Y (1 ⁇ g each).
• FIGURE 2 A Iron-saturated biotinylated recombinant human lactoferrin (0.4 ⁇ M) was incubated with Caco-2 membranes (300 ng) in the presence or absence of increasing concentrations of unlabeled iron-saturated recombinant lactoferrin (Fe-RechLF) or apo-recombinant lactoferrin (Apo-RechLF). Inhibition of biotinlyated lactoferrin binding to Caco-2 membranes was determined using a biotin/avidin microtiter assay (Ward et al , 1995, Biotechnology 13:498-503).
• FIGURES 2B, 2C, and 2D Apobiotinylated recombinant human lactoferrin (0.4 ⁇ M) was incubated with Caco-2 membranes (300 ng) in the presence or absence of increasing concentrations of unlabeled Apo-RechLF or MN-2Y (FIGURE 2B), MC-2Y (FIGURE 2C) or MNC-4Y (FIGURE 2D). Inhibition of biotinylated lactoferrin binding to Caco-2 membranes was determined using a biotin/avidin microtiter assay. The data are represented as means +/- S.E.M.
• FIGURE 3 depicts iron-saturation analysis of the lactoferrin amino-terminal
• MN-2Y carboxy-terminal
• MNC-4Y amino- and carboxy-terminal iron binding defective variants.
• Iron-free recombinant human lactoferrin (Rec hLF) MN-2Y, MC-2Y, and MNC-4Y were saturated with iron as described in the experimental procedures.
• 59 Fe bound to the samples was quantified using liquid scintillation counting and the Fe/protein molar ratios were determined. The data are represented as means +/- S.E.M.
• FIGURE 4 depicts pH-dependent release of 59 Fe from the lactoferrin amino- terminal (MN-2Y) and carboxy-terminal (MC-2Y) iron binding defective variants.
• 59 Fe-saturated recombinant human lactoferrin (Rec hLF) MN-2Y and MC-2Y were dialyzed against buffers ranging in pH from 7-2.
• 59 Fe remaining bound to the lactoferrin samples after dialysis was quantified by liquid scintillation counting and the Fe/protein molar ratio was determined. The data are represented as means +/- S.E.M.
• lactoferrin variant refers to a polypeptide generated by mutation of a naturally occurring lactoferrin in at least one amino acid position.
• vector means plasmid, cosmid, phage, virus, retrovirus or other vehicle to allow insertion, propagation and/or expression of a nucleic acid encoding a lactoferrin variant or portion thereof.
• host cell means any cell that will allow lactoferrin expression.
• promoter means regulatory DNA sequences that controls transcription of the lactoferrin cDNA.
• transformation means incorporation permitting expression of heterologous nucleic acid sequences by a cell.
• iron binding capacity means ability to bind Fe. Fully functional human lactoferrin can bind two atoms of iron per molecule of lactoferrin.
• biological activity/biological active means biological activity of lactoferrin as measured by its ability to bind iron, or kill microorganisms, or retard the growth of microorganisms, or to function as an iron transfer protein.
• the present invention is based, in part, on the identification those domains, sequences, and structures in the lactoferrin polypeptide which contribute to the iron binding capacity/properties of the protein.
• the present invention provides guidance for the generation of lactoferrin variants having improved characteristics, such as modified iron binding characteristics, e.g. , increased or decreased iron binding capacity, or modified pH or temperature requirements or ranges for the binding of iron.
• the present invention provides guidance for the modelling of lactoferrin va ⁇ ants having otherwise improved characteristics, e.g. , therapeutic tolerance, stability against degradation, or immunoreactivity, while retaining their iron binding activity.
• the present invention is directed to recombinant nucleic acids encoding lactoferrins variants and portions thereof, and to vectors comprising same recombinant nucleic acids.
• the present invention is further directed to methods of producing such vectors, and to transfected cells harboring the same. Methods for the production of lactoferrin variants and portions thereof in various eukaryotic or prokaryotic cells are also provided.
• the invention is directed to lactoferrins variants and portions thereof encoded by the nucleic acids of the invention and/or produced by the processes of the invention.
• the invention provides guidance for the design of lactoferrin va ⁇ ants or portions thereof having improved characte ⁇ stics, and an efficient and economical means for the production of such lactoferrin va ⁇ ants and portions thereof.
• lactoferrin variants as determined by silverstain, western immunoblotting and enteric receptor binding analysis were similar to wild-type recombinant human lactofemn indicating that the amino-acid substitutions had no adverse effect on the protein.
• Iron- saturation analysis using 59 FeCl 3 showed that while the lactoferrin va ⁇ ant with an intact carboxy-terminal iron binding lobe saturate at the expected 1 : 1 ratio of iron to protein, the variant with an intact amino-terminal iron binding function consistently saturated at less than 1 : 1 suggesting a reduced stability of iron binding of this lactoferrin variant at pH 7.0.
• the present invention extends these studies and show that cooperative interactions, driven primarily by a functional carboxy-terminal lobe are necessary for iron binding stabilization.
• the bias for selection of a bilobal structure in the evolution of the transferrin family is unknown.
• the instant disclosure provides a functional rationale for this selection in the case of lactoferrin.
• the present invention shows that the evolution of a two lobe structure has endowed lactoferrin with unique iron binding properties which are likely to impinge on the unique functional activity of this protein.
• transferrin differs from lactoferrin in that it has been shown that the pH dependent iron release properties of this protein and a proteolytically derived amino-terminal fragment are similar. Mazuier et al , 1989, Biochem.
• Lactoferrin Variants Based on its identification of those domains, sequences and structures in the lactoferrin polypeptide contributing to the iron binding properties of the protein, the present invention provides guidance for the design and generation of novel lactoferrin variants or portions thereof having a modified iron binding capacity.
• the lactoferrin variants of the invention have improved properties, including, but not limited to, lactoferrin variants with higher affinity for iron for improved antimicrobial activities, lactoferrin variants with lower affinity for iron having improved iron-releasing properties, or lactoferrin variants having modified pH or temperature requirements or ranges for the binding and/or release of iron.
• the invention allows for the design of lactoferrin variants having otherwise improved characteristics, e.g.
• the lactoferrin variants of the invention may be derived from wild-type lactoferrin of a variety of mammalian species, including, but not limited to, human, murine, rat, bovine, and porcine lactoferrin.
• the wild-type lactoferrin may be mutated by a variety of methods generally known in the art. See, among other places, Sambrook et al , 1990, Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press, New York; Kunkel et al , 1987, Meth. Enzymol. 154:367-382; Kunkel, 1985, Proc. Natl. Acad. Sci. USA 82:488-492.
• the lactoferrin variant comprises at least one mutation affecting at least one iron binding site.
• Such iron binding site may have modified iron binding characteristics when compared to wild-type lactoferrin. For example, affinity for iron may increased or decreased. In addition, affinity for iron may exhibit modified pH range characteristics. Alternatively, the iron binding site may be modified such that the lactoferrin variant or portion thereof does not bind iron at all. In another embodiment, the lactoferrin variant does comprise at least one mutation effecting modification of the therapeutic tolerance, biological stability or immunotolerance of lactoferrin, while retaining its biological activity.
• nucleic acid sequences of the invention encoding lactoferrin variants may be inserted in a vector suitable for its expression in a eukaryotic cell in such way that allows expression of the lactoferrin variant.
• nucleic acid sequences encoding portions of the lactoferrin variants of the invention may be inserted in vectors allowing their expression in eukaryotic cells.
• the portion of the lactoferrin variant comprises at least one iron binding site, which may be modified.
• lactoferrin is produced in recombinant expression systems. See, among other places, Ward et al , 1992, Biotechnology 10:784-789; Ward et al , 1995, Biotechnology 12:498-503.
• nucleic acids coding for the desired form of lactoferrin (see, e.g. , U.S. Patent 5,571 ,691 , incorporated by reference in its entity) is incorporated expressibly in a cellular host, which is then cultured under conditions appropriate for expression of that particular peptide or protein.
• a variety of gene expression systems have been adapted for this purpose, and typically drive expression of the desired gene from expression controls used naturally by the chosen host.
• lactoferrin variants of the invention typically, as natural occurring lactoferrin, requires post-translational modifications, such as glycosylation at several amino acid residues, many of the lactoferrins variants or portions thereof need to be produced in an eukaryotic host.
• the lactoferrin product is produced by an Aspergillus expression system, as described in Ward et al , 1992, Gene 122:219-223; and U.S. Patent Nos. 5,571,896 and 5,571,697, incorporated by reference in its entirety. If unglycosylated forms of lactoferrin variants or portions thereof are produced, however, their production may conveniently be achieved in bacterial hosts such as E.
• nucleic acid coding for the selected lactoferrin variant or portion thereof may usefully be placed under an expression control, e.g. , of the lac, tip or PL genes of E. coli.
• the host can be adapted to express the lactoferrin product as a fusion protein in which the lactoferrin product is linked releasably to a carrier protein that facilitates isolation and stability of the expression product.
• the lactoferrin variant or portion thereof may be generated by organic synthesis. In particular where production of a portion of a lactoferrin variant, e.g.
• a peptide of about twenty (20) through about fifty (50) amino acids in length is the objective preferably the well established techniques of automated peptide synthesis are employed, general descriptions of which appear, for example, in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis. 2nd Edition, 1984, Pierce Chemical Company, Rockford, Illinois; and in M.
• Bodanszky and A. Bodanszky The Practice of Peptide Synthesis. 1984, Springer- Verlag, New York; Applied Biosystems 430A Users Manual, 1987, ABI Inc. , Foster City, California; and Solid Phase Peptide Synthesis - A Practical Approach. by: E. Atherton & R.C. Sheppard, IRL Press, Oxford (1989). In these techniques, the lactoferrin variant portion is grown from its C-terminal, resin-conjugated residue by the sequential addition of appropriately protected amino acids, using either the Fmoc or tBoc protocols. E. Applications
• lactofemn variants of the present invention may be used for a broad variety of applications.
• a lactoferrin variant or portion thereof having improved iron binding capacity may be used for antimicrobial applications, including the treatment of infections, as additive to human or animal foods, as additive for eyedrops, eardrops, contact lense and other eye care solutions, topical skin care products, mouthwashes, chewing gum and toothpaste.
• Lactoferrin variants or portions thereof having modified pH or temperature requirements may also be useful for the above applications. They further may be used as therapeutic additives for enhanced or modified iron transport and delivery.
• lactoferrin variants and portions thereof include the treatment of inflammatory skin diseases, as described in the co-pending provisional application, identified as Attorney Docket Number 8206-041 -888, filed April 10, 1997, hereby incorporated by reference in its entirety.
• lactoferrin variants and portions thereof having improved therapeutic tolerance, modified biological stability, or immunotolerance may be particularly preferred.
• the lactoferrin variants or portions thereof chosen can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents. Each can be administered alone but is generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
• the pharmaceutical compositions of the invention may be adapted for oral, parenteral, topical or rectal administration, or as inhalant, and may be in unit dosage form, in a manner well known to those skilled in the pharmaceutical art.
• Parenteral administration includes but is not limited to, injection subcutaneously, intravenously, intraperitoneally or intramuscularly. Treatment with the active ingredient may begin at any time after the indication to be treated, e.g.
• infections, or skin disorders e.g. , psoriasis, contact dermatitis, UV-induced inflammation, infant diaper rash, asthma, arthritis, and the like.
• treatment is commenced as a prophylactic or at early stages of the disease, in order to prevent massive infection or inflammation in the first place.
• treatment will continue until the disorder is cured.
• chronic diseases such as psoriasis, asthma or arthritis, or in cases of continued exposure to an allergen, the treatment may have to be extended beyond the cure of the symptoms.
• the dose administered will, of course, vary depending upon known factors, such as (1) the pharmacodynamic characteristics of the particular lactoferrin product and its mode and route of administration, (2) the age, health, height and weight of the recipient, (3) the nature and extent of the symptoms, (4) the kind of concurrent treatment(s), (5) the frequency of treatment(s), and (6) the effect desired.
• a daily dose of active ingredient can be determined by those skilled in the art depending on the above factors.
• the active ingredient may be administered topically, as inhalant, or as injection, e.g. , in inflamed joints or cartilage.
• the lactoferrin variant may be administered orally in solid or semi-solid dosage forms, such as hard or soft-gelatin capsules, tablets, or powders, or in liquid dosage forms, such as elixirs, syrups, or suspensions. It can also be administered parenterally, in ste ⁇ le liquid dosage forms. Other dosage forms are potentially possible such as patches or ointment or transdermal administration.
• the lactoferrin variants of the invention may also be formulated as a slow release implantation device for extended and sustained administration of the lactoferrin product.
• sustained release formulations include composites of biocompatible polymers, such as poly(lactic acid), poly(lactic-co- glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like.
• biocompatible polymers such as poly(lactic acid), poly(lactic-co- glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like.
• degradable polymers in drug delivery vehicles have been reviewed in several publications, including, A. Domb et al , Polymers for Advanced Technologies 2:279-292 (1992). Additional guidance in selecting and using polymers in pharmaceutical formulations can be found in the text by M. Chasin and R. Langer (eds.), "Biodegradable Polymers as Drug Delivery Systems, " Vol.
• Liposomes may also be used to provide for the sustained release of a lactoferrin variant or portion thereof. Details concerning how to use and make liposomal formulations of drugs of interest can be found in, among other places, U.S. Pat. No 4,944,948; U.S. Pat. No. 5,008,050; U.S. Pat. No. 4,921,706; U.S. Pat. No. 4,927,637; U.S. Pat. No. 4,452,747; U.S. Pat. No. 4,016,100; U.S. Pat. No. 4,311 ,712; U.S. Pat. No. 4,370,349; U.S.
• Sustained release formulations are of particular interest when it is desirable to provide a high local concentration of a lactoferrin variant, e.g. , in arthritic joints, or at local skin inflammations, etc.
• Gelatin capsules or liquid-filled soft gelatin capsules may contain the active ingredient and powdered or liquid carriers, such as lactose, lecithin starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and to protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and/or flavoring to increase patient acceptance.
• Suitable pharmaceutical carriers are further described in Remington's Pharmaceutical Sciences. 17th ed., Mack Publishing Company, Easton, PA (1990) a standard reference text in this field, which is incorporated herein by reference in its entirety.
• pPLF-26 A Universal Aspergillus Awamori Expression Vector.
• the construction of an expression vector, pPLF-19, for production of lactoferrin in A. awamori has previously been described. Ward et al , 1995, Biotechnology 12:498-503; U.S. Patent No. 5,571 ,896.
• pPLF-26 was generated. Briefly, pPLF-18 (Ward et al , 1995, Biotechnology 13:498-503) was digested with Sphl generating two fragments of 3.3 kb and 4.4 kb.
• the 3.3 kb Sphl fragment containing the lactoferrin cDNA was subcloned into Sphl digested pALTER (Promega, Madison, WI) generating pLF18sp.Alt.
• the 4.4 kb Sphl fragment was relegated generating PR18.2.
• In vitro mutagenesis using the commercially available pALTER kit (Promega, Madison, WT) was used to introduce a Notl restriction enzyme site at the start of mature lactoferrin cDNA in pLF18Sp.Alt generating pNot.9.
• the 5' phosphorylated oligonucleotide used for the mutagenesis was as follows: 5'
• PR18.2 was digested with EcoRI and the resulting two fragments were repaired using the Klenow fragment of DNA polymerase I and religated in the correct orientation.
• the resulting plasmid, P-.E12 was digested with Sphl and ligated with a 3.3 kb Sphl fragment from pNot.9 generating pPLF-25.
• PL03 encoding the phleomycin resistance gene under the control of the /3-tubulin promoter (Gatignol et al , 1987, Mol. Gen. Genet.
• pPLF-26 was digested with Notl/EcoRI and the resulting 2.1 kb fragment containing the human lactoferrin cDNA was subcloned into Notl/EcoRI digested pALTLINK. The resulting plasmid, pALThLF was used for subsequent mutagenesis experiments.
• the tyrosine residues involved in iron binding by lactoferrin in the amino-terminal lobe (Tyr 93, Tyr 193), carboxy-terminal lobe (Tyr 436, Tyr 529 and amino- and carboxy-terminal lobes (Tyr 93, Tyr 193, Tyr 436 and Tyr 529) were converted to corresponding alanine residues using in vitro mutagenesis using the pALTER kit.
• the 5' phosphorylated oligonucleotides used for the mutagenesis were as follows.
• TYR 193 ⁇ Ala 193 5' CTTGAAGGCACCAGAGGCGCTGAAGTACGGTTC 3' [SEQ ID NO:5]; Tyr 436 ⁇ Ala 436: 5' CACCGCCACAGCAAGGGCTCCTTCCACAGGTCT 3' [SEQ ID NO: 6];
• plasmids pALTMN-2Y, pALTMC-2Y, and pALTMNC-4Y, were digested with Notl/EcoRI and subcloned into Notl/EcoRI digested pPLF-26 generating expression plasmids suitable for expression of lactoferrin in Aspergillus fungal cells, i.e. , p26MN-2Y, p26MC-2Y and p26MNC-4Y, respectively. All oligonucleotides sequences and construction junction were sequenced using the commercially available Sequenase Version 2.0 kit (United States Biochemical Corporation, Cleveland OH).
• the A.awamori expression vectors, p26MN-2Y, p26MC-2Y and p26MNC-4Y were transformed into A. awamori and transformants obtained were cultured for 7 days as previously described. Ward e/ al , 1995, Biotechnology 12:498-503.
• the culture medium was screened for the iron binding variants using an ELISA assay. Vilja et al , 1985, J. Immunol. Meth. 76:73-83. Positive cultures ( > 50 mg/1) were cultured in 2 liter flasks for 7 days and the lactoferrin variant wa purified using ion-exchange chromatography using CM-sephadex. Ward et al , 1995, Biotechnology 13:498-503. The proteins were dialyzed against 0.1 M citric acid followed by extensive dialysis against H 2 0 and 5 mM sodium phosphate, pH 7.5
• Receptor Binding Assays were performed using a biotin/avidin microtiter plate assay (Rejman et al , 1994, Int. J. Biochem. 26:201- 206) using 8 day old Caco-2 solubilized membranes (300 ng) essentially as described. Ward et al , 1995, Biotechnology 13:498-503.
• MN-2Y, MC-2Y and MNC-4Y (5 mg) were incubated with a four-fold excess of FeCl 3 : 59 FeCl 3 :NTA (400: 1:8). Ward et al , 1995, Biotechnology L3:498- 503. The samples were incubated at room temperature for 30 minutes. The samples were purified through NAP- 10 column (Pharmacia, Piscataway, NJ) followed by dialysis against 0.05 M Tris/HCl, 0.2 M NaCl, pH 7.0 for 12 hours to remove any non-specific iron bound to the lactoferrin variants.
• Iron bound to the lactoferrin variants after dialysis was quantified by liquid scintillation counting.
• the pH stability of iron binding for each of the lactoferrin variants was carried out as described previously. Ward et al , 1995, Biotechnology 13:498-503.
• lactoferrin variants were expressed and purified from A. awamori as previously described for recombinant human lactoferrin. Ward et al , 1995,
• FIGURE 1 Western immunoblot analysis using a specific polyclonal IgG directed against human lactoferrin detected an immunoreactive band co ⁇ esponding to the size of wild-type recombinant lactoferrin for each of the three lactoferrin variants, as depicted in FIGURE 1A, lane 1-4.
• lactoferrin variants were > 95 % pure and a single band at the expected molecular weight of ⁇ 80 kDa was observed for each of the proteins. See, FIGURE IB, lane 1-4. Hence, the size and immunoreactivity of the lactoferrin variants were indistinguishable from wild-type recombinant lactoferrin.
• both apo- and iron-saturated lactoferrin showed comparable capacity to displace binding of iron- saturated biotinylated lactoferrin to the human enteric Caco-2 cell membranes indicating that both forms of lactoferrin have similar affinity for the lactoferrin enterocyte receptor. While lactoferrin has been proposed to deliver iron to enterocyte cells through these receptors, the similar relative receptor binding affinities or iron-free and iron-saturated lactoferrin suggest that the ability of lactoferrin to deliver iron via these receptors depends primarily on the relative amounts of iron-saturated versus iron-free lactoferrin present in the intestinal lumen.
• Example 3 Mutation Of The Tyrosine Residues Involved In Iron Binding In The Amino- And Carboxy-Terminal Lobes Of Lactoferrin Selectively Disrupts The Iron Binding Capacity Of The Mutated Lobes Of The Protein To confirm that mutation of the two tyrosine residues in either or both lobes of lactoferrin was sufficient to disrupt the iron binding ability of the mutated lobe, iron-saturation analysis using 59 FeCl 3 was performed.
• the lactoferrin variants were saturated with 59 FeCl 3 and dialyzed against buffers ranging in pH from 7-2 for 48 hours at 4°C.
• the amount of iron remaining bound to the lactoferrin variants was quantified by liquid scintillation counting.
• the results of this analysis are shown in FIGURE 4.
• the iron release profile from MN-2Y, containing an intact carboxy-terminal iron binding lobe, was similar to that of recombinant lactoferrin, iron release beginning at a pH of 5.0 and completed at pH 2.0.
• the pH dependent release of iron from MC-2Y containing an intact amino-terminal iron binding lobe, was markedly different.

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• 1997
  • 1997-05-30 US US08/866,544 patent/US6111081A/en not_active Expired - Lifetime
  • 1997-06-02 NZ NZ333215A patent/NZ333215A/xx not_active IP Right Cessation
  • 1997-06-02 DE DE0959898T patent/DE959898T1/de active Pending
  • 1997-06-02 IL IL12731997A patent/IL127319A/en not_active IP Right Cessation
  • 1997-06-02 WO PCT/US1997/009531 patent/WO1997045136A1/en not_active Ceased
  • 1997-06-02 AU AU32271/97A patent/AU707476B2/en not_active Ceased
  • 1997-06-02 CA CA002256453A patent/CA2256453A1/en not_active Abandoned
  • 1997-06-02 GR GR990300043T patent/GR990300043T1/el unknown
  • 1997-06-02 JP JP09543045A patent/JP2000511424A/ja not_active Ceased
  • 1997-06-02 ES ES97927932T patent/ES2140362T1/es active Pending
  • 1997-06-02 EP EP97927932A patent/EP0959898A4/en not_active Withdrawn
  • 1997-06-02 KR KR1019980709727A patent/KR20000016159A/ko not_active Ceased

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