Classifications

• • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders

Definitions

• the invention relates to medicaments useful in stimulating the immune system. More specifically, the invention concerns a new form of liposaccharide binding protein (LBP) .
• LBP liposaccharide binding protein
• LPS lipopolysaccharides
• a number of proteins have been described in mammals that bind and mediate the effects of polar lipids such as lipopolysaccharides in circulation. Many of these molecules have been isolated and studied in detail and found to be similar in function and structure. It has been suggested that these proteins can be considered as encoded by a gene family with possible common evolutionary origin.
• One such protein cholesterol ester transfer protein (CETP) binds cholesterol esters and other complex lipids associated with lipoprotein complexes and mediates their transfer between HDL and VLDL.
• CETP cholesterol ester transfer protein
• BPI bacteriocidal permeability increasing protein
• LBP lipopolysaccharide binding protein
• BPI and LBP show significant homology, and the homology between BPI and LBP is particularly striking. Both BPI and LBP contain a positively charged amino terminal domain of about 25 kd that appears to be responsible for lipid binding and a hydrophobic carboxy terminal domain of about 30 kd which ' may mediate interaction with membranes and/or receptors.
• LBP- ⁇ The known form of LBP (LBP- ⁇ ) is synthesized in the liver and is present in normal human serum at a concentration of less than 0.5 ⁇ g/ l. The level rises to 50 ⁇ g/ml 24 hrs after induction of an immune response.
• the known LBP form has a molecular weight of about 60 kd of which about 10 kd represents added glycosyl residues.
• LBP has been shown to bind the lipid A moiety of LPS, a binding which presumably involves the amino terminal portion of LBP. The lipid A moiety of LPS is considered essential to induce shock.
• LBP also opsonizes LPS- bearing particles such as intact gram-negative bacteria, mediating attachment of the coated particles to macrophage.
• LBP plays an important role in an inflammatory response to infection.
• Schumann et al. disclose the amino acid sequence and encoding cDNA of both human and rabbit LBP.
• the present invention provides a novel form of LBP (LBP-/3) not previously described.
• the present invention provides a novel form of LBP useful in activating the immune system.
• the novel LBP of the invention designated herein LBP- ⁇ , adds to the repertoire of liposaccharide binding proteins known to stimulate the immune system.
• LBP- ⁇ adds to the repertoire of liposaccharide binding proteins known to stimulate the immune system.
• the availability of this additional form of LBP permits fine tuning of therapeutic protocols designed to enhance immune function.
• the invention is directed to the immune-stimulating protein LBP-3 in purified and isolated form.
• the invention is directed to recombinant materials and methods useful in providing this protein.
• the invention is directed to methods to stimulate the immune system using the LBP - ⁇ of the invention, to pharmaceutical compositions useful in this method, and to antibodies reactive with LBP-jS.
• Figure 1 shows a comparison of the amino acid sequence and nucleic acids encoding LBP- ⁇ ; and LBP-/3.
• Figure 2 shows the amino acid sequence and the cDNA encoding LBP-/S
• Figure 3 shows the structure of two ai ⁇ plimers used in PCR in the cloning of LBP - ⁇ .
• Figure 4 shows a comparison of the amino acid sequence at positions 240-250 of rabbit LBP, human BPI, a previously disclosed form of human LBP (LBP-o;) , and human LBP- ⁇ . The numbering shown refers to Figure 1 of Schumann, et al. (supra) .
• the LBP-/3 of the invention shows enhanced homology to both human BPI and rabbit LBP as compared to human LBP- ⁇ previously disclosed. There are 7 amino acid substitutions in LBP-j ⁇ relative to LBP- ⁇ (see Fig. 1) . In addition and, more significantly, the LBP-/3 of the invention includes an additional 4 amino acids in the carboxy terminal region which correspond to similar residues in human BPI and rabbit LBP. This sequence was not disclosed in Schumann's LBP- ⁇ _. Four additional amino acids could have a profound effect on the molecule's tertiary structure.
• novel LBP-j3 of the invention can most conveniently be produced using recombinant techniques.
• the complete amino acid sequence and cDNA encoding this sequence is disclosed herein and is shown in Figure 2.
• the amino acid sequence shown in Figure 2 shows the precursor protein which is cleaved to obtain the mature form.
• the first 25 amino acids effect the secretion of the mature form of LBP which has, as its N-terminus, the Ala residue shown at position 26.
• the numbering system shown in Figure 2 is thus appropriate to the precursor protein; the corresponding positions in the mature protein are obtained by subtracting 25 from the numbers shown.
• the valine shown at position 266 in Figure 2 is at position 241 in the mature form.
• LBP-j ⁇ Such minor and understood modifications provide molecules also included within the scope of the invention and are included within the designation "LBP-j ⁇ .”
• standard derivatization, by glycosylation, phosphorylation, or chemical modification of sidechains, such as by conversion of proline to hydroxyproline may also be included in proteins designated LBP-/3 and within the scope of the invention so long as these modifications do not destroy the immune system stimulating activity of the protein.
• the cDNA shown in Figure 2 is identical to that retrieved as described in the illustrations below except for the deliberately engineered Clal site at numbered amino acid positions 222-223. This Clal site was added to enable portions of LBP-jS to be spliced into other constructs in cassette form.
• any DNA encoding the amino acid sequence of LBP-3 can be included in suitable expression systems for production of this protein.
• the encoding DNA may be prepared by retrieving the native cDNA, optionally providing degenerate modifications thereof, or may be partially or completely synthesized by conventional solid-phase oligonucleotide synthesizing techniques.
• Construction of DNA of sufficient length to encode the 482 amino acid LBP-jS precursor or 457 amino acid mature protein can be effected by techniques readily available and known in the art.
• the DNA encoding the LBP-3 of the invention is ligated to control systems which effect the expression of the coding DNA in host cells with which the control sequences are compatible.
• the choice of control sequences will thus depend on the nature of the production host cell.
• Techniques for construction of expression systems, transformation of host cells, and culture of the transformed hosts are now well-established procedures.
• a variety of expression system/recombinant hosts can be used.
• E ⁇ coli hosts are available and a variety of control sequences including regulatable promoters such as the trp promoter or P L promoter are well established.
• Eukaryotic systems useful for production of recombinant proteins include yeast, insect cells, mammalian cells, plant cells, and more recently, intact plants and multicellular organisms.
• the expression systems may be constructed to provide the LBP-/3 of the invention as the mature protein, or the encoding DNA may be ligated to upstream DNA which encodes a peptide sequence that effects the secretion of the LBP - ⁇ from the recombinant host cell (including the native sequence) or provide LBP-j8 as a fusion protein.
• Such variations in the design of expression systems are also well known in the art.
• the LBP-j8 of the invention may also be used to provide antibodies useful in immunoassay systems for the detection of LBP-/3 or related proteins.
• antibodies which recognize epitopes related to the novel portion of the LBP- ⁇ which distinguishes it from the previously-disclosed human LBP are thus useful.
• Antibodies may be prepared specifically with respect to this epitope by immunization with a peptide fragment including the portion including amino acids 241-245 of the mature form of LBP-j8 (shown as residues 266-270 of the precursor peptide in Figure 2) .
• Such fragments may be as short as the 5 amino acid sequence shown, or may include additional amino acid residues derived from LBP-0. Shorter peptides may require conjugation to carrier to enhance immunogenicity.
• the antibodies of the invention are prepared by standard immunization protocols by repeated administra ⁇ tion to suitable mammalian subjects and monitoring antibody titers in the serum.
• the polyclonal antiserum may be harvested and used per se, or monoclonal antibodies may be prepared from the peripheral blood lymphocytes or spleens of the immunized animals using standard cell immortalization and screening techniques.
• immunologically reactive fragments of the antibodies can be advantageously used in many assays and other applications in which the antibodies are useful.
• the LBP-S of the invention is useful in stimulating the immune system, and thus can be formulated into pharmaceutical compositions for therapeutic use.
• Standard formulations for administration can be found, for example, in Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, PA (latest edition).
• suitable formulations for injection include, for example, Hank's solution, Ringer's solution, physiological saline, and the like.
• LBP-S may also be administered by transmucosal or transdermal formulations, generally transmucosal formulations include detergents or other surface active agents and bile salts or fusidic acids to enhance transition of the membrane. Dosage levels required are comparable to the previously disclosed form of LBP although identification of appropriate dosages for a particular subject and condition depend on the mode of administration, the nature of the subject, the severity of the condition, and the judgment of the attending practitione .
• Figure 1 shows the oligonucleotide sequence of amplimers designed to complement the regions upstream and downstream of the cDNA encoding LBP described by Schumann (1990) (supra) .
• the oligonucleotides also contained restriction sites Nhel, Clal, and Xhol, both external and internal to the LBP coding regions.
• the amplimers shown in Figure 3 were used to amplify LBP encoding DNA from a commercially available cDNA preparation derived from human liver RNA. The amplification utilized standard enzyme-mediated PCR techniques.
• the amplified products were digested with the corresponding restriction enzymes Nhel, Clal and Xhol and isolated by polyacrylamide gel electrophoresis.
• the digested cDNA fragments were ligated into pMa Neo, a commercially available mammalian expression vector and cloned in EL. coli.
• DNA from the resulting clones was analyzed by gel electrophoresis and sequenced. The resulting sequence is shown in Figure 2.
• the sequence was similar to that reported by Schumann et al. (supra) but also contained an additional sequence of 12 nucleotides in the coding region beginning after base 826 of Genbank sequence 35533, and shown in Figure 2 as positions 824-835.

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• 1992
  • 1992-09-28 JP JP5506414A patent/JPH07502642A/ja active Pending
  • 1992-09-28 WO PCT/US1992/008298 patent/WO1993006228A1/en not_active Application Discontinuation
  • 1992-09-28 AU AU28727/92A patent/AU2872792A/en not_active Abandoned
  • 1992-09-28 EP EP92922186A patent/EP0605653A4/de not_active Withdrawn

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