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WO1994025476A1 - Proteines de recombinaison de type augmentant la permeabilite bactericide (bpi) et de type fixant le liposaccharide (lbd), molecules codant pour ces proteines, leurs procedes de production et leurs utilisations - Google Patents

Proteines de recombinaison de type augmentant la permeabilite bactericide (bpi) et de type fixant le liposaccharide (lbd), molecules codant pour ces proteines, leurs procedes de production et leurs utilisations Download PDF

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Publication number
WO1994025476A1
WO1994025476A1 PCT/US1994/004709 US9404709W WO9425476A1 WO 1994025476 A1 WO1994025476 A1 WO 1994025476A1 US 9404709 W US9404709 W US 9404709W WO 9425476 A1 WO9425476 A1 WO 9425476A1
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Prior art keywords
bpi
lbp
chimera
lps
variant
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PCT/US1994/004709
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English (en)
Inventor
Randal W. Scott
Marian N. Marra
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Incyte Pharmaceuticals, Inc.
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.)
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Application filed by Incyte Pharmaceuticals, Inc. filed Critical Incyte Pharmaceuticals, Inc.
Priority to AU69429/94A priority Critical patent/AU6942994A/en
Priority to CA002161971A priority patent/CA2161971A1/fr
Priority to JP6524554A priority patent/JPH08511682A/ja
Priority to EP94917901A priority patent/EP0760849A1/fr
Publication of WO1994025476A1 publication Critical patent/WO1994025476A1/fr
Priority to US08/431,517 priority patent/US6265187B1/en
Priority to US09/861,400 priority patent/US20020146761A1/en

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    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4742—Bactericidal/Permeability-increasing protein [BPI]
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • Gram-negative infections are a major cause of morbidity and mortality, especially in hospitalized and immunocompromised patients. [Duma, R.J., Am. J. of Med., 78 (Suppl. 6A):154-164 (1985); and Kreger, B.E., D.E. Craven and W.R. McCabe, Am. J. Med., 68:344-355 (1980)].
  • available antibiotics are generally effective in containing Gram-negative infections, they do not neutralize the patho-physiological effects associated with heat stable bacterial toxins (called endotoxins or lipopolysaccharides (LPS)) which are released from the outer membrane of Gram-negative bacteria upon lysis [Shenep, J.L. and K.A.
  • Endotoxin is a potent stimulator of the inflammatory response. Endotoxemia occurs when endotoxin enters the bloodstream resulting in a dramatic systemic inflammatory response.
  • TNF Tumor necrosis factor
  • TNF is also a potent stimulator of neutrophils.
  • Other cytokines such as IL-1, IL-6, and IL-8 also mediate many of the patho-physiologic effects of LPS, as well as other pathways involving endothelial cell activation by tissue factor, kininogen, nitric oxide and complement.
  • DIC disseminated intra- vascular coagulation
  • ARDS adult respiratory distress syndrome
  • cardiac dysfunction organ failure
  • liver failure hepatobiliary dysfunction
  • CNS dysfunction brain failure
  • renal failure multi-organ failure and shock.
  • SIRS systemic inflammatory response syndrome
  • sepsis syndrome septic shock
  • bacterial meningitis neonatal sepsis
  • cystic fibrosis inflammatory bowel disease and liver cirrhosis
  • gram-negative pneumonia gram-negative abdominal abscess
  • hemorrhagic shock disseminated intravascular coagulation.
  • Subjects that are leukopenic or neutropenic including subjects treated with chemotherapy or immunocompromised subjects (for example with AIDS), are particularly susceptible to bacterial infection and the subsequent effects of endotoxin. Endotoxin-associated disorders can be present whenever there is a gram-negative infection.
  • Endotoxin-associated disorders can also be present (a) when there is ischemia of the gastrointestinal tract, which ischemia may be present following hemorrhagic shock or during certain surgical procedures, or (b) when systemic or local inflammation causes increased permeability of the gut to endotoxin or gram-negative organisms.
  • Current methods for treating Gram-negative infections use antibiotics and supportive care. Despite successful antimicrobial therapy, morbidity and mortality associated with endotoxemia remain high. Antibiotics are not effective in neutralizing the toxic effects of LPS. Therefore, the need arises for a therapy with direct endotoxin-neutralizing activity.
  • Polymyxin B is a basic polypeptide antibiotic which has been shown to bind to, and structurally disrupt, the most toxic and biologically active component of endotoxin - - Lipid A. PMB has been shown to inhibit endotoxin activation of neutrophil granule release in vitro and is a potential therapeutic agent for Gram-negative infections. However, because of its systemic toxicity, this antibiotic has limited therapeutic use except as a topical agent.
  • MPSS methylprednisolone sodium succinate
  • cytokine blockers such as IL-1 receptor antagonist and anti-TNF antibodies
  • a cytokine blocker can only block the cytokine(s) for which it is specific, and cannot block other cytokines.
  • blocking cytokines may have other deleterious effects.
  • Two soluble endotoxin-binding proteins have now been identified which play a role in the physiological response to endotoxin.
  • lipopolysaccharide binding protein is a soluble receptor found in serum which mediates endotoxin activation of cells.
  • BPI bactericidal/ permeability-increasing protein
  • LBP was further shown to bind to the lipid A moiety of endotoxin, which binding accounts for much of the biological activity of endotoxin [Tobias, P.S., Soldau, K. and Ulevitch, R.J. (1989) J. Biol. Chem. 264:10867-10871].
  • BPI is a neutrophil granule protein first discovered in 1975 [Weiss, J., R.C. Eranson, S. Becherdite, K. Schmeidler, and
  • BPI was obtained in highly purified form from human neutrophils in
  • BPI retains its in vitro bactericidal activity after cleavage with neutrophil proteases, suggesting that fragments of the molecule retain activity [Ooi and Elsbach, Clinical Research 33(2):567A (1985)]. All of the in vitro bactericidal and permeability increasing activities of BPI are present in the N-terminal 25 kD fragment of the protein [Ooi, C.E., et al., J. Biol. Chem. 262:14891 (1987)]. BPI binding to Gram-negative bacteria was reported originally to disrupt LPS structure, alter microbial permeability to small hydrophobic molecules and cause cell death [Weiss, et al. (1978)].
  • BPI shares amino acid sequence homology and immuno-crossreactivity with LBP [Tobias et al., J. Biol. Chem. 263:13479-13481 (1988)], and the genes encoding both BPI and LBP have been cloned [Gray, P.W., Flaggs, G., Leong, S.R., Gumina, R.J., Weiss, Ooi, C.E. and Elsbach, P. (1989) J. Biol. Chem. 264:9505-9509]. Both genes code for hydrophobic leader sequences and polypeptides having 44% amino acid sequence identity. LBP was reported by Schumann et al.
  • LBP means a protein having the sequence shown for human LBP in Figure 5.
  • BPI can be described as having two distinct domains, an N-terminal domain, and a C-terminal domain, which domains are separated by a proline-rich hinge region.
  • the N-terminal domain of the LBP molecule has been shown to contain the bactericidal and LPS-binding domain of BPI [Ooi and Elsbach, Clinical Research 33(2):567A (1985) and Ooi, C.E., et al., J. Biol. Chem. 262:14891 (1987)].
  • the C-terminal domain of BPI has been reported to have modest LPS-binding activity.
  • the C-terminal domain of LBP is thought to be involved in the binding and activation of monocytes.
  • the N- and C-terminal domains of BPI have a striking charge asymmetry that is not shared by LBP.
  • the N-terminal domain of BPI is extremely rich in positively charged lysine residues, and this charge imparts a predicted pI > 10 to the full-length molecule, whereas the C-terminal domain is slightly negatively charged.
  • the bactericidal activity of BPI may result from its cationicity.
  • LBP is largely neutral, has no skewed charge distribution, and is not bactericidal [Tobias, P.S., Mathison, J.C. and Ulevitch, R.J. (1988) J. Biol. Chem. 263:13479-13481].
  • Table 1 provides a comparison of BPI and LBP structure and function.
  • the subject invention provides a recombinant nucleic acid molecule which encodes a BPI variant.
  • the subject invention also provides the BPI variant encoded by the recombinant nucleic acid molecule of the subject invention.
  • the subject invention further provides a vector comprising the recombinant cDNA molecule of the subject invention.
  • the subject invention further provides a host vector system for the production of a BPI variant, which comprises the vector of the subject invention in a suitable host.
  • the subject invention further provides a method for producing a BPI variant, which comprises growing. the host vector system of the subject invention under conditions permitting the production of the BPI variant and recovering the BPI variant produced thereby.
  • the subject invention provides a recombinant nucleic acid molecule which encodes an LBP variant.
  • the subject invention also provides the LBP variant encoded by the recombinant nucleic acid molecule of the subject invention.
  • the subject invention further provides a vector comprising the recombinant cDNA molecule of the subject invention.
  • the subject invention further provides a host vector system for the production of an LBP variant, which comprises the vector of the subject invention in a suitable host.
  • the subject invention further provides a method for producing an LBP variant, which comprises growing the host vector system of the subject invention under conditions permitting the production of the LBP variant and recovering the LBP variant produced thereby.
  • the subject invention provides a recombinant nucleic acid molecule which encodes an LBP-BPI chimera.
  • the subject invention also provides the LBP-BPI chimera encoded by the recombinant nucleic acid molecule of the subject invention.
  • the subject invention further provides a vector comprising the recombinant cDNA molecule of the subject invention.
  • the subject invention further provides a host vector system for the production of an LBP-BPI chimera, which comprises the vector of the subject invention in a suitable host.
  • the subject invention furt ⁇ er provides a method for producing an LBP-BPI chimera, which comprises growing the host vector system of the subject invention under conditions permitting the production of the LBP-BPI chimera and recovering the LBP-BPI chimera produced thereby.
  • the subject invention provides a recombinant nucleic acid molecule which encodes a BPI-IgG chimera.
  • the subject invention also provides the BPI-IgG chimera encoded by the recombinant nucleic acid molecule of the subject invention.
  • the subject invention further provides a vector comprising the recombinant cDNA molecule of the subject invention.
  • the subject invention further provides a host vector system for the production of a BPI-IgG chimera, which comprises the vector of the subject invention in a suitable host.
  • the subject invention further provides a method for producing a BPI-IgG chimera, which comprises growing the host vector system of the subject invention under conditions permitting the production of the BPI-IgG chimera and recovering the BPI-IgG chimera produced thereby.
  • the subject invention provides a recombinant nucleic acid molecule which encodes an LBP-IgG chimera.
  • the subject invention also provides the LBP-IgG chimera encoded by the recombinant nucleic acid molecule of the subject invention.
  • the subject invention further provides a vector comprising the recombinant cDNA molecule of the subject invention.
  • the subject invention further provides a host vector system for the production of an LBP-IgG chimera, which comprises the vector of the subject invention in a suitable host.
  • the subject invention further provides a method for producing an LBP-IgG chimera, which comprises growing the host vector system of the subject invention under conditions permitting the production of the LBP-IgG chimera and recovering the LBP-IgG chimera produced thereby.
  • the subject invention provides a recombinant nucleic acid molecule which encodes an LBP-BPI-IgG chimera.
  • the subject invention also provides the LBP-BPI-IgG chimera encoded by the recombinant nucleic acid molecule of the subject invention.
  • the subject invention further provides a vector comprising the recombinant nucleic acid molecule of the subject invention.
  • the subject invention further provides a host vector system for the production of an LBP-BPI-IgG chimera, which comprises the vector of the subject invention in a suitable host.
  • the subject invention further provides a method for producing an LBP-BPI-IgG chimera, which comprises growing the host vector system of the subject invention under conditions permitting the production of the LBP-BPI-IgG chimera and recovering the LBP-BPI-IgG chimera produced thereby.
  • the subject invention provides a pharmaceutical composition, which comprises a therapeutically effective amount of a BPI variant, an LBP variant, an LBP-BPI chimera, a BPI-IgG chimera, an LBP-IgG chimera, or an LBP-BPI-IgG chimera, and a pharmaceutically acceptable carrier.
  • the subject invention further provides a method of treating a subject suffering from an endotoxin-related disorder, which comprises administering to the subject a dose of the pharmaceutical composition of the subject invention effective to bind to LPS and thereby inhibit LPS-mediated stimulation of neutrophils and mononuclear cells, so as to thereby treat the subject.
  • the subject invention provides a method of preventing an endotoxin-related disorder in a subject, which comprises administering to the subject a prophylactically effective amount of a BPI variant, an LBP variant, an LBP-BPI chimera, a BPI-IgG chimera, an LBP-IgG chimera, or an LBP-BPI-IgG chimera, so as to thereby prevent the endotoxin-related disorder in the subject.
  • LBP-b Differences between LBP sequence as used herein (LBP-b) and LBP sequence as published by Schumann, et al. (LBP-a).
  • LBP binds to LPS to form the LPB-LPS complex which then binds CD14 and activates monocytes to produce inflammatory cytokines.
  • BPI binds to LPS but the BPI-LPS complex does not bind CD14 or activate monocytes.
  • Figure 6 Amino acid sequence of NCY118 protein.
  • Figure 8 Effects of BPI, NCY102, NCY103 and NCY104 on b iotinylated BPI binding to LPS .
  • Figure 9 Effects of BPI, NCY102, NCY103, NCY104 and NCY105 protein on LPS activity in the chromogenic LAL assay.
  • Figure 10 FITC-LPS binding to monocytes in the presence of BPI and NCY103.
  • FIG. 12 Clearance of BPI, NCY102, NCY103 and NCY104 from mouse serum after intravenous injection.
  • NCY115, and NCY117 on biotinylated BPI binding to LPS.
  • NCY104, NCY117 and PLL poly-L-lysine
  • FIG. 17 LPS-mediated TNF production in THP-l cells cultured without serum.
  • Figures 18A, 18B, 18C, 18D, 18E and 18F LPS-mediated TNF production in THP-l cells cultured without serum.
  • NCY118 panel A; BPI, NCY114, NCY115 and
  • NCY139 panel B
  • BPI LBP, NCY117 and NCY118 (panel C); BPI, LBP and NCY144 (assayed for both Fc and BPI) in CD-1 mice (panel D); LBP, NCY116, NCY117, NCY118 (panel E); NCY102, NCY103, NCY115, NCY135, and NCY134 (panel F); NCY102, NCY141, NCY142, NCY143, and BPI (panel G); and BPI, NCY115, and NCY114 (panel H).
  • Pichia pastoris Pichia pastoris.
  • Figure 20 Effects of BPI and NCY103 on endotoxin activation of monocytes.
  • the subject invention Toward the goal of ameliorating the morbidity and mortality associated with endotoxemia and septic shock, the subject invention provides BPI and LBP variants, BPI-LBP chimeras, and BPI-IgG and LBP-IgG chimeras having biological properties distinct from and advantageous to either native BPI or native LBP.
  • the subject invention also provides therapeutic and prophylactic uses for these molecules.
  • the subject invention provides a recombinant nucleic acid molecule which encodes a BPI variant.
  • the recombinant nucleic acid molecule is a DNA molecule.
  • the DNA molecule is a cDNA molecule.
  • BPI or bactericidal permeability increasing protein means a protein having the amino acid sequence shown for human BPI in Figure 5.
  • the BPI nucleotide and amino acid sequences are shown in Figure 3.
  • a BPI variant means a protein comprising a portion of BPI, which protein is capable of (a) binding to LPS, (b) competing with BPI or LBP for binding to LPS, and (c) inhibiting the LPS-mediated production of TNF ⁇ . by human monocytes.
  • a BPI variant may comprise a fragment of BPI, a point mutant of BPI, a deletion mutant of BPI, or both a point and deletion mutant of BPI.
  • LPS means lipopolysaccharide, which is used synonymously with the word "endotoxin.”
  • TNF ⁇ means tumor necrosis factor alpha.
  • the BPI variant has the structure BPI (S351- >X) , serine residue 351 being substituted for X, an ammo acid residue other than serine.
  • X is alanine.
  • the portion of BPI in BPI variants and chimeras is designated by the letter B, followed by amino acid sequence numbers which correspond to those shown in Figure 5 for human BPI.
  • Figure 5 designates the mature N-terminal amino acid as residue 1.
  • the portion of LBP in LBP variants and chimeras is designated by the letter L, followed by amino acid sequence numbers which correspond to those shown in Figure 5 for human LBP.
  • Figure 5 designates the mature N-terminal amino acid as residue 1.
  • L 1-197 B 200-456 contains amino acid residues 1-197 of LBP fused at its C-terminus to the N-terminus of BPI amino acid residues 200-456.
  • L 1-197 B 200-456 is shown in Figure 6.
  • L 1-197 B 200-456 has the N-terminal domain of LBP (having an endotoxin-binding domain) fused to the C-terminal domain of BPI (having a putative LPS-clearing domain).
  • Suitable amino acid substitutions include but are not limited to substitutions of a particular amino acid residue in one protein with the residue which resides at the corresponding position in a different protein.
  • BPI Xn->Y
  • X amino acid residue X at position n in BPI is substituted with residue Y which is found at position n in LBP (or rabbit or bovine LBP).
  • X and Y denote amino acid positions in a primary amino acid sequence.
  • "Y” as used in this context is not to be confused with the symbol “Y” denoting the amino acid residue tyrosine.
  • LBP (Xn->Y) is another example of such a substitution, wherein amino acid residue X at position n in LBP is substituted with residue Y which is found at position n in BPI (or rabbit or bovine BPI).
  • Amino acid residue insertions are also indicated in parentheses. First, the amino acid residue after which the insertion occurs and its number are given. After an arrow the amino acid residue before the insertion and the inserted amino acid are given. For example, in B (DS200 . >DP) , a proline residue is substituted for the serine residue at position 200.
  • the subject invention also provides the BPI variant encoded by the recombinant nucleic acid molecule of the subject invention.
  • the subject invention further provides a vector comprising the recombinant cDNA molecule of the subject invention.
  • Vectors not comprising the recombinant cDNA molecule of the subject invention are readily available to those skilled in the art, and can readily be used to form the vector of the subject invention.
  • vectors for expressing the inventive proteins may be employed.
  • Such vectors including plasmid vectors, cosmid vectors, bacteriophage vectors and other viruses, are well known in the art.
  • one class of vectors utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MoMLV), Semliki Forest virus or SV40 virus.
  • cells which have stably integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow for the selection of transfected host cells.
  • the markers may provide, for example, prototrophy to an auxotrophic host, biocide resistance or resistance to heavy metals such as copper.
  • the selectable marker gene can be either directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation.
  • Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. Additional elements may also be needed for optimal synthesis of mRNA. These additional elements may include splice signals, as well as enhancers and termination signals.
  • a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG.
  • a eukaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome.
  • Such vectors may be obtained commercially or assembled from the sequences described by methods well known in the art, for example the methods described above for constructing vectors in general.
  • the subject invention further provides a host vector system for the production of a BPI variant, which comprises the vector of the subject invention in a suitable host. Methods of producing host vector systems are well known to those skilled in the art.
  • Suitable host cells include, but are not limited to, bacterial cells (including gram positive cells), yeast cells, fungal cells, insect cells and animal cells.
  • Suitable animal cells include, but are not limited to, HeLa cells, COS cells (including COS-7 cells), CV1 cells, NIH-3T3 cells, CHO cells, and Ltk- cells.
  • Certain animal cells, i.e., mammalian cells may be transfected by methods well known in the art such as calcium phosphate precipitation, electroporation and microinjection.
  • the suitable host is a bacterial cell.
  • Bacterial cells include, for example, gram negative cells (e.g. E. coli cells).
  • the suitable host is an eucaryotic cell.
  • the eucaryotic cell may be a mammalian cell. Mammalian cells include, for example, Chinese Hamster Ovary cells (CHO).
  • the eucaryotic cell may also be a yeast cell.
  • Yeast cells include, for example, Pichia cells.
  • the subject invention further provides a method for producing a BPI variant, which comprises growing the host vector system of the subject invention under conditions permitting the production of the BPI variant and recovering the BPI variant produced thereby.
  • Protein recovery is accomplished by methods well known to those skilled in the art. Such methods include, but are not limited to, gel electrophoresis, ion exchange chromatography, affinity chromatography or combinations thereof.
  • the subject invention provides a recombinant nucleic acid molecule which encodes an LBP variant.
  • the recombinant nucleic acid molecule is a DNA molecule.
  • the DNA molecule is a cDNA molecule.
  • LBP or lipopolysaccharide binding protein means a protein having the amino acid sequence shown for human LBP in Figure 5.
  • the amino acid sequence shown for human LBP in Figure 5 is distinct from the amino acid sequence reported by Schumann et al. (Science 249:1429-1431 (1990)). Therefore, the amino acid sequence shown for human LBP in Figure 5 should not be confused with the sequence reported by Schumann et al.
  • Figure 1 shows differences between LBP sequence as used herein and LBP sequence as published by Schumann, et al.
  • the LBP nucleotide and amino acid sequences are shown in Figure 4.
  • an LBP variant means a protein comprising a portion of LBP, which protein is capable of (a) binding to
  • LBP variant may comprise, by way of example, a fragment of
  • LBP LBP
  • a point mutant of LBP a deletion mutant of LBP
  • a point and deletion mutant of LBP a point and deletion mutant of LBP.
  • the subject invention provides a recombinant nucleic acid molecule which encodes an LBP-BPI chimera.
  • the recombinant nucleic acid molecule is a DNA molecule.
  • the DNA molecule is a cDNA molecule.
  • a chimera means a protein comprising all or a portion of a first protein fused to all or a portion of a second protein, which resulting fusion protein may in turn be fused to all or a portion of a third protein.
  • Chimeras include but are not limited to (a) a protein comprising a portion of LBP fused to a portion of BPI, (b) a protein comprising an LBP portion fused to a BPI portion which in turn is fused to a portion of an immunoglobulin, and (c) a protein comprising an LBP portion fused to a BPI portion, which in turn is fused to ah LBP portion.
  • Each protein portion of the chimera may comprise a fragment of the protein, a point mutant of the protein, a deletion mutant of the protein, or both a point and deletion mutant of the protein.
  • an LBP-BPI chimera means a protein which (i) comprises an LBP portion fused to a BPI portion, and (ii) is capable of (a) binding to LPS, (b) competing with BPI or LBP for binding to LPS, and (c) inhibiting the production of TNF ⁇ by human monocytes.
  • Chimeras of LBP and BPI may share properties of both BPI and LBP.
  • LBP-BPI chimera e.g., NCY103 or NCY118.
  • the resulting LBP-BPI chimera differs from LBP in that the chimera neutralizes endotoxin in whole blood and differs from BPI in that the chimera has a longer half-life in vivo.
  • Such chimeras can be used to clear endotoxin from the blood of a patient with endotoxemia.
  • a BPI-LBP chimera is a protein wherein all or a part of the N-terminal domain of BPI is fused to all or a part of the C-terminal domain of LBP (e.g., NCY104). This chimera competes effectively with BPI binding to endotoxin but activates monocytes in the presence of endotoxin as does LBP.
  • one or more of the nonconserved positively-charged residues in BPI may be substituted with the corresponding residue or residues in LBP (as in, e.g., NCY139). Such substitutions would render BPI less cationic.
  • one or more of the nonconserved amino acid residues in LBP may be substituted with the corresponding positively-charged residue in BPI (as in, e.g., NCY141), and thus result in an LBP variant having an increased positive charge which enhances binding to the negatively charged phosphate groups in LPS, or facilitates interaction with the negatively charged surfaces of Gram-negative bacteria.
  • BPI positively-charged residues
  • positively-charged residues are lysine, arginine, and histidine.
  • Other BPI and LBP variants and chimeras have one or more cysteine residues deleted or substituted with serine or another amino acid.
  • Such variants and chimeras help prevent the aggregation of BPI or LBP variants or chimeras during their production or use.
  • cysteine residue 132 in BPI which is not conserved in LBP
  • alanine the corresponding residue in LBP
  • BPI and LBP variants and chimera have one or more nonconserved glycosylation sites deleted (as in, e.g., NCY105) by amino acid substitution or deletion. Alternatively, a glycosylation site is added to other BPI and LBP variants and chimera by amino acid insertion or substitution. Other BPI and LBP- variants and chimera have one or more secondary structure-altering amino acid residues deleted or added. For example, one or more of the nonconserved proline residues in BPI may be substituted with the corresponding non-proline residue in LBP.
  • the LBP-BPI chimera has the structure LBP 1-197 BPI 200-456 .
  • the LBP-BPI chimera has the structure LBP 1-197(I43->V) BPI 200-456(N206->D) .
  • the LBP-BPI chimera comprises all or a portion of the amino acid sequence of BPI from residue 199 to residue 359.
  • the amino acid sequence of BPI from residue 199 to residue 359 contains a region required for neutralizing LPS, i.e., preventing LPS from stimulating an inflammatory response.
  • the subject invention also provides the recombinant nucleic acid molecule encoding the LBP-BPI chimera, vector and host vector system.
  • the subject invention provides a BPI-IgG chimera and a recombinant nucleic acid molecule which encodes a BPI-IgG chimera.
  • the recombinant nucleic acid molecule is a DNA molecule.
  • the DNA molecule is a cDNA molecule.
  • a BPI-IgG chimera means a protein which (i) comprises a BPI portion (at least 10 amino acid residues in length) fused at its C-terminus to the N-terminus of a portion of an IgG molecule; and (ii) is capable of (a) binding to LPS, (b) competing with BPI or LBP for binding to LPS, and (c) inhibiting the production of TNF ⁇ by human monocytes.
  • the portion of the IgG molecule is an IgG heavy chain Fc domain.
  • the IgG heavy chain Fc domain may be the IgG heavy chain Fc domain whose sequence is shown in Figure 7.
  • An example of a BPI-IgG chimera is B 1-199 Fc.
  • the subject invention provides an LBP-IgG chimera and a recombinant nucleic acid molecule which encodes an LBP-IgG chimera.
  • the recombinant nucleic acid molecule is a DNA molecule.
  • the DNA molecule is a cDNA molecule.
  • an LBP-IgG chimera means a protein which (i) comprises an LBP portion (at least 10 amino acid residues in length) fused at its C-terminus to the N-terminus of a portion of an IgG molecule; and (ii) is capable of (a) binding to LPS, (b) competing with BPI or LBP for binding to LPS, and (c) inhibiting the production of TNF ⁇ by human monocytes.
  • the subject invention also provides the LBP-IgG chimera encoded by the recombinant nucleic acid molecule of the subject invention.
  • the subject invention further provides a vector comprising the recombinant cDNA molecule of the subject invention.
  • the subject invention provides an LBP-BPI-IgG chimera and a recombinant nucleic acid molecule which encodes an LBP-BPI-IgG chimera.
  • the recombinant nucleic acid molecule is a DNA molecule.
  • the DNA molecule is a cDNA molecule.
  • an LBP-BPI-IgG chimera means a protein which (i) comprises an LBP-BPI chimera fused at its C-terminus to the N-terminus of a portion of an IgG molecule; and (ii) is capable of (a) binding to LPS, (b) competing with BPI or LBP for binding to LPS, and (c) inhibiting the production of TNF ⁇ by human monocytes.
  • the BPI variant, LBP variant, LBP-BPI chimera, BPI-IgG chimera, LBP-IgG chimera, and LBP-BPI-IgG chimera of the subject invention may be modified with polyethylene glycol to increase the circulating half-life and/or bioavailability of the molecules.
  • the subject invention provides a pharmaceutical composition, which comprises a therapeutically effective amount of a BPI variant, an LBP variant, an LBP-BPI chimera, a BPI-IgG chimera, an LBP-IgG chimera, or an LBP-BPI-IgG chimera; and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01-0.1M and preferably 0.05M succinate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Further, pharmaceutically acceptable carriers may include detergents, phospholipids, fatty acids, or other lipid carriers.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • a lipid carrier is any lipid-soluble substance which inhibits protein precipitation and in which the proteins of the subject invention are soluble.
  • Lipid carriers may be in the form of sterile solutions or gels. Lipid carriers may be detergents or detergent-containing biological surfactants.
  • nonionic detergents examples include polysorbate 80 (also known as TWEEN 80 or polyoxyethylenesorbitan monooleate). Examples of ionic detergents include, but are not limited to, alykltrimethyl-ammonium bromide.
  • the lipid carrier may be a liposome.
  • a liposome is any phospholipid membrane-bound vesicle capable of containing a desired substance, such as BPI or BPI variant, in its hydrophilic interior.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
  • the subject invention further provides a method of treating a subject suffering from an endotoxin-related disorder, which comprises administering to the subject a dose of the pharmaceutical composition of the subject invention effective to bind to LPS and thereby inhibit LPS biological activity.
  • an endotoxin-related disorder includes, but is not limited to endotoxin-related shock, endotoxin-related disseminated intravascular coagulation, endotoxin-related anemia, endotoxin-related thrombocytopenia, endotoxin-related adult respiratory distress syndrome, endotoxin-related renal failure, endotoxin-related liver disease or hepatitis, SIRS (systemic immune response syndrome) resulting from Gram-negative infection, Gram-negative neonatal sepsis, Gram-negative meningitis, Gram-negative pneumonia, neutropenia and/or leucopenia resulting from Gram-negative infection, hemodynamic shock and endotoxin-related pyresis.
  • SIRS systemic immune response syndrome
  • Endotoxin-related pyresis is associated with certain surgical procedures, such as trans-urethral resection of the prostate and gingival surgery.
  • the presence of endotoxin may result from infection at any site with a Gram-negative organism, or conditions which may cause ischemia of the gastrointestinal tract, such as hemorrhage, or surgical procedures requiring extracorporeal circulation.
  • the admmisteration may be performed by methods known to those skilled in the art.
  • the administerati ⁇ n comprises delivery to the lungs via an aerosol delivery system or via direct instillation.
  • the aerosol may be nebulized.
  • Other admmisteration modes include but are not limited to intravenous, intramuscular, and subcutaneous administration as well as direct delivery into an infected body cavity.
  • the dose of the pharmaceutical composition of the subject invention effective to bind to LPS and thereby inhibit LPS-mediated stimulation of neutrophils and mononuclear cells is an amount sufficient to deliver to the subject an inventive protein at a concentration of between about O.lmg/kg of body weight and about lOOmg/kg of body weight. In one embodiment, the dose is an amount sufficient to deliver to the subject an inventive protein at a concentration of between about lmg/kg of body weight and about lOmg/kg of body weight.
  • the therapeutically effective amounts of inventive proteins in the pharmaceutical composition may be determined according to known methods based on the effective dosages discussed above.
  • inhibit means to inhibit at a level which is statistically significant and dose dependent.
  • the terms “statistically significant” and “dose dependent” are well known to those skilled in the art.
  • the subject invention further provides a method of preventing an endotoxin- related disorder in a subject, which comprises administering to the subject a prophylactically effective amount of a BPI variant, an LBP variant, an LBP-BPI chimera, a BPI-IgG chimera, an LBP-IgG chimera, or an LBP-BPI-IgG chimera, so as to thereby prevent the endotoxin- related disorder in the subject.
  • a prophylactically effective amount is an amount between about 0.1mg/kg of body weight and about 100mg/kg of body weight. In the preferred embodiment, a prophylactically effective amount is an amount between about lmg/kg of body weight and about 10mg/kg of body weight.
  • the term “inventive proteins” is used throughout the subject application.
  • the term “inventive proteins” means a BPI variant, an LBP variant, an LBP-BPI chimera, a BPI-LBP chimera, a BPI-IgG chimera, an LBP-IgG chimera, an LBP-BPI-IgG chimera, a recombinant protein comprising a portion of LBP or BPI, or any combination thereof.
  • the subject invention provides recombinant nucleic acid molecules which encode L 1-199 (NCY109), L 1-357 B 360-456 (NCY117), LBP (NCY102), L 1-199 Fc (NCY111), L 200-458 (NCY113), LBP (A132->c) (NCY126), LBP (C61->F) (NCY127), LBP (C61->S) (NCY128), LBP (C135->S) (NCY129), LBP (A175->S) (NCY130), LBP C61->F)(C135->S)(A175->S) (NCY131), or LBP (C61->S)(C135- >S)(A175->S) (NCY132).
  • the recombinant nucleic acid molecules are DNA molecules.
  • the DNA molecules are cDNA molecules.
  • the subject invention also provides the proteins encoded by these recombinant nucleic acid molecules.
  • the subject invention further provides vectors comprising these recombinant cDNA molecules.
  • the subject invention further provides host vector systems for the production of these proteins, which comprise these vectors in suitable hosts.
  • the suitable hosts are bacterial cells.
  • the suitable hosts are eucaryotic cells.
  • the eucaryotic cells may be mammalian cells.
  • the eucaryotic cells may also be yeast cells.
  • the subject invention further provides methods for producing these proteins, which comprise growing these host vector systems under conditions permitting the production of these proteins and recovering the proteins produced thereby.
  • the proteins L 1-199 (NCY109), L 1-357 B 360-456 (NCY117), LBP (NCY102), L 1-199 Fc (NCY111), L 200-458 (NCY113), LBP (A132->C) (NCY126), LBP (C61->F) (NCY127), LBP (C61->S) (NCY128), LBP (C135->S) (NCY129), LBP (A175->S) (NCY130), LBP C61->F)(C135->S)(A175->S) (NCY131), or LBP (C61->S)(C135->S)(A175->S) (NCY132) are useful for inhibiting the LPS-mediated cellular response both in vitro and in vivo.
  • the subject invention provides an article of manufacture comprising packaging material and a pharmaceutical composition contained within said packaging material wherein (a) the packaging material comprises a label which indicates that the pharmaceutical composition can be used for treating a subject suffering from an endotoxin-related disorder and for preventing endotoxin-related inflammation in a subject, and (b) said pharmaceutical composition comprises a therapeutically effective amount of a BPI variant, an LBP variant, an LBP-BPI chimera, a BPI-IgG chimera, an LBP-IgG chimera, or an LBP-BPI-IgG chimera, and a pharmaceutically acceptable carrier.
  • the packaging material comprises a label which indicates that the pharmaceutical composition can be used for treating a subject suffering from an endotoxin-related disorder and for preventing endotoxin-related inflammation in a subject
  • said pharmaceutical composition comprises a therapeutically effective amount of a BPI variant, an LBP variant, an LBP-BPI chimera, a BPI-IgG chimera,
  • vectors may be introduced into a suitable host cell to form a host vector system for producing the inventive proteins.
  • Methods of making host vector systems are well known, to those skilled in the art. This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the subject inventions which follow thereafter.
  • BPI and LBP variants and chimeras are described in Tables 2 and 3.
  • Table 2 describes some general classes of BPI and LBP variants and chimeras which are given by way of example. Specific. examples of BPI and LBP variants and chimeras are described in Table 3 and are additionally designated by a product name (e.g., NCY103).
  • the Fc, or constant domain, or a human immunoglobulin heavy chain can be linked to the BPI variant protein.
  • n represents an amino acid residue position in the mature sequence of BPI or LBP
  • x represents an amino acid residue in a position which is C-terminal to n in the sequence of BPI or LBP
  • y represents an amino acid residue in a position which is C-terminal to x in the sequence of BPI or LBP.
  • the symbols n, x and y denote the amino acid residue positions as they occur in the mature sequence of the native protein, and not necessarily the positions as they occur in the variant protein.
  • cDNA sequences of BPI and LBP are shown in Figures 3 and 4, respectively, with each nucleotide designated numerically.
  • DNA encoding the inventive proteins was prepared by site-directed mutagenesis using standard techniques well known in the art [Zoller, M.J., et al., Methods Enzymol. 154:329 (1977)].
  • sequences "ATAGAT 723 " and “ATTGAC 700 " were chosen as a convenient site to insert a Clal restriction site (ATCGAT) by which to recombine portions of BPI and LBP, respectively.
  • Oligonucleotide primers were designed to overlap this region and to add the Clal sequence, and were synthesized on an ABI 380B synthesizer (Applied Biosystems Inc., Foster City, CA). Additional primers were designed to bind to the 5' and 3'-ends of both molecules and to provide Nhel (5') and Xhol
  • LBP-BPI useful LBP-BPI, BPI-IgG, LBP-IgG, and LBP-BPI-IgG chimeras
  • DNA molecules encoding these chimeras may be constructed using methods well known to those skilled in the art.
  • the cDNA sequences were inserted into a suitable plasmid vector.
  • a suitable vector for such an application is pSE, which contains early and late promoters of SV40, followed by multiple insert cloning sites, followed by the termination sequences from the hepatitis B surface antigen gene.
  • pSE which contains early and late promoters of SV40, followed by multiple insert cloning sites, followed by the termination sequences from the hepatitis B surface antigen gene.
  • Also contained within the plasmid are an origin of bacterial DNA replication, and the genes encoding ampicillin resistance and dihydrofolate reductase. Similar vectors have been used to express other foreign genes (McGrogan, et al. Biotechnology 6, 172-177).
  • pCEP4 Another suitable vector, particularly for rapidly obtaining small quantities of inventive proteins was pCEP4 (Invitrogen Corp., San Diego, California).
  • pCEP4 contains a CMV promoter, followed by multiple insert cloning sites, followed by SV40 termination sequences. Also contained within the plasmid are an origin of bacterial DNA replication, and the genes encoding resistance to ampicillin and hygromycin B. With pCEP4 and pSE, the same insert cloning sites as pSE for easy insert shuttling between the vectors were used.
  • this specialized plasmid replicates as an episome, allowing semistable amplification of introduced DNA sequences. The high gene copy number is maintained through the selective pressure of culture in the presence of hygromycin B.
  • vector DNA was prepared for acceptance of cDNA by digestion with Nhe I and Xho I, and was subsequently dephosphorylated by treatment with alkaline phosphatase.
  • the prepared cDNA fragments encoding BPI, LBP, or other inventive proteins were ligated into pSE or pCEP4, and the resulting recombinant colonies were screened by agarose gel electrophoresis. Subsequently, the DNA sequences were confirmed by standard enzymatic sequencing methods (e.g., Sanger, 1974).
  • BPI and NCY118 were successfully expressed in the methylotrophic yeast Pichia pastoris.
  • Pichia was chosen as a suitable expression system for BPI and BPI variants due to its lack of LPS (endotoxin to which BPI binds) and its ability to produce high levels of mammalian proteins.
  • P. pastoris strain GS115 (Invitrogen, San Diego, California) was transformed with plasmids encoding BPI and NCY118, and transformed colonies were selected for following the procedures outlined by Invitrogen (A Manual of Methods for Expression of Recombinant Proteins in Pichia pastoris, Version 1.5, Invitrogen, San Diego, California).
  • Invitrogen A Manual of Methods for Expression of Recombinant Proteins in Pichia pastoris, Version 1.5, Invitrogen, San Diego, California.
  • protein was secreted into the medium in a small-scale batch fermentation run. 116 ng/ml were secreted for the one BPI construct assayed, and 14, 11, and 10 ng/ml were secreted for the three NCY118 constructs assayed. Secretion was assayed by enzyme-linked immunosorbant analysis (ELISA).
  • ELISA enzyme-linked immunosorbant analysis
  • ⁇ Purified BPI from Chinese Hamster ovary cells (CHOs) was used as a control (lane 12).
  • lane 1 was a sample from untransformed GS115 cells. The antibodies did not recognize any proteins from such cells subject to the detection limits of the assay.
  • the next three lanes (2-4) were samples from colonies transformed with the construct for BPI and the last 6 lanes (5-10) were samples from colonies transformed with the construct for NCY118.
  • BPI (NCY101) was purified from conditioned media using the following four-step purification.
  • BPI was captured on CM Sepharose (Pharmacia LKB Biotechnology). The column was washed in 50mM Tris pH 7.4, and protein was eluted with 5OmM Tris buffer pH 7.4 + 1M NaCl. The eluate was diluted 10X with 50mM Tris pH 8.5, run over Fast Q Sepharose, and the flow through collected. BPI was re-captured on CM Sepharose, and again eluted as before. Buffer exchange into lOmM Succinate + 110mM NaCl pH 6 was performed using Sepharose CL6B (Pharmacia LKB Biotechnology).
  • TWEEN 20 was added to the formulated material to a final concentration of 0.05%.
  • LBP (NCY102) was captured from cell culture medium on Fast S Sepharose (Pharmacia). The column was washed with 50mM Tris pH 7.4, and protein was eluted using 50mM Tris pH 7.4 + 1M NaCl. The eluate was diluted 10X in 50mM Tris pH 8.5, and run over HiLoad Q Sepharose (Pharmacia). Protein was eluted with a 0-1M NaCl gradient in 50m0 Tris pH 8.5. Appropriate fractions were pooled according to migration on SDS PAGE electrophoresis. NCY102 concentration was diluted to 4.0 mg/ml, and the pH adjusted to 7.0 with 100mM HCl. NCY103 was purified from cell culture medium using the same method described for NCY102.
  • NCY104 and NCY105 were purified using the same protocol as for BPI, except that the size exclusion step was omitted.
  • NCY114, NCY115 and NCY138 were captured on a Poros II HS cation exchange column (PerSeptive Biosystems, Cambridge, MA) at pH 7.4.
  • the column was washed with 20mM HEPES buffer at pH 7.5, and eluted with 20mM HEPES pH 7.5 with 1M NaCl.
  • the eluate was diluted 5X in 20mM HEPES pH 7.5 and applied to a Poros HQ anion exchange column (PerSeptive) with the flow through applied directly to a POROS II HS column.
  • the POROS II HS column was eluted with 3.3mM acetate, 3.3mM MES and 3.3mM HEPES, pH 6.5 with a 0-1M NaCl gradient.
  • NCY117 and NCY144 were captured from conditioned medium at pH 7.4 on a Poros II HS column.
  • the column was washed with 20mM HEPES buffer at pH 7.5, and eluted with 20mM HEPES pH 7.5 + 1M NaCl.
  • the eluate was diluted 10X with 20mM HEPES pH 7.5, loaded on a second, smaller Poros II HS column, and eluted with 3.3mM acetate, 3.3mM MES and 3.3mM HEPES, pH 6 with a 0-1M NaCl gradient.
  • In vitro and in vivo tests were performed on representative compounds disclosed herein.
  • In vitro tests included LPS binding competition assay, Limulus amebocyte lysate (LAL) inhibition test, TNF release inhibition test, FITC-labeled LPS binding inhibition, THP-l cell TNF production and BPI activity against Neisseria.
  • In vivo tests included mouse LPS half-lives, mouse endotoxin challenges and LPS-induced cytokine function and mortality in rats, and LPS activation in bronchial fluids.
  • E. coli 055 :B5 LPS were obtained from List Biological Laboratories (Campbell, CA).
  • E. coli 0111:B4 LPS was obtained from Whitaker Biologicals (Walkersville, MD) .
  • S. abortus equi LPS was obtained from Sigma Chemical Co. (St. Louis, MO).
  • HBSS without calcium and magnesium and Roswell Park Memorial Institute (RPMI solution) 1640 was obtained from Gibco BRL (Grand Island, MD).
  • Fluorescent-activated cell sorting (FACS) analysis was performed on a FACStar, Beeton Dickinson Immunocytometry Systems (Mountain View, CA).
  • Binding to LPS immobilized on microtiter plates was performed using a modified procedure described by Ulevitch et al. (15). Briefly, Immulon 3 microtiter plates (96-well, Dynatech Biotechnology Products, Chantilly, VA) were coated with 1 or 4 ⁇ g of S. minnesota R595 Re LPS (LIST Biological Labs, Inc., #304) in 50mM borate, pH 9.5-9.8 + 20-25 mM EDTA overnight at 37°C. Blank, non-LPS coated wells were included on each plate and binding to these walls was used to determine non-specific binding. Absorbance values from wells which were not pre-coated with LPS consistently gave optical density readings of less than 0.05.
  • biotinylated BPI was incubated in the presence or absence of unlabeled BPI or inventive protein (pyrogen-free TBS + lmg/ml low endotoxin BSA, and 0.05% Tween-20) was incubated in the LPS coated and uncoated wells for 2-3 hours at 37°C in a total volume of 100 ⁇ l/well. After four washes in assay buffer, plates were developed with streptavidin conjugated to alkaline phosphatase (BioRad, Burlingame, California) followed by 100 ⁇ l of PNP substrate solution (Sigma) freshly prepared from two 5 mg tablets dissolved in 10ml substrate buffer.
  • unlabeled BPI or inventive protein pyrogen-free TBS + lmg/ml low endotoxin BSA, and 0.05% Tween-20
  • Substrate buffer is prepared with 24.5 mg MgCl 2 , 48 ml diethanolamine, brought up to 400 ml, pH adjusted to 9.8 and volume brought up to 500 ml. Absorbances were read at 405 nm on a Vmax kinetic microplate reader (Molecular Devices Inc., Menlo Park, CA).
  • BPI and inventive proteins 25 ⁇ l of 0-200 ⁇ g/ml were preincubated for 1 hour at 37°C with lEU/ml of E. coli 0111 :B4 LPS (25 ⁇ l of 2 EU/ml solution) (Whitaker Biologicals, Walkersville, Maryland). Then the mixtures were tested for LAL activity using the chromogenic LAL assay kit (Whitaker Biologicals, Walkersville, MD).
  • HBSS Hank's balanced salt solution
  • Mononuclear cells were isolated using Ficol-Paque (Pharmacia Inc., Piscataway, NJ). Cells were washed three times in HBSS, then brought up to an appropriate volume of RPMI 1640 with glutamine and antibiotics to give approximately 1 X 10 6 cells/ml. To one ml aliquots of cells, FITC-LPS was added to a final concentration of 500 ng/ml. Tubes were closed and incubated at 37°C on a rocking platform.
  • HSA Human Serum Albumin
  • TNF levels were determined by ELISA using human recombinant TNF (Genzyme, Cambridge, MA or Genentech Inc., South San Francisco, CA) as a standard.
  • THP-1 cells were obtained from the American Tissue Culture
  • THP-l cells Collection (Rockville, MD) and were maintained in REM medium containing 10% fetal bovine serum, 2mM L-glutamine, 100 units penicillin and 100 ⁇ g/ml streptomycin. Cells were passed at 2 ⁇ 10 5 cells/ml every 3 days. Responsiveness of THP-l cells to LPS was induced by culturing the cells for 48 hours in REM medium containing 10% fetal calf serum, 2mM L-glutamine, 100 units penicillin, 100 ⁇ g/ml of streptomycin and 100 nM PMA at 37°C in a humidified atmosphere with 5% CO 2 .
  • BPI, LBP, or inventive protein at 1 mg/ml
  • blood was collected from the retroorbital plexus from three animals at each time point tested.
  • a typical blood collection schedule was 5, 10, 15, 30, 45, 60, 90, 120, 240, and 360 minutes.
  • the blood was centrifuged for about 10 min at 1000 x g and the supernatant plasma frozen on dry ice until tested.
  • Levels of BPI, LBP, or inventive protein in the plasma samples were determined by ELISA using the appropriate protein as the standard.
  • mice Female CD-1 mice were injected in the lateral tail vein with a LD 100 dose (25-35 mg/kg) of Salmonella abortus equi endotoxin, which was followed by an injection of BPI, inventive protein, or vehicle control into the opposite lateral tail vein at the indicated time. Protein injection concentrations varied and provided doses of 0.5, 1 and 5 mg/kg. Use of vehicle control illustrated the effectiveness of the endotoxin challenge in the test animal. Animals were observed for mortality at 24, 48 and 72 hours. BPI Reduction of LPS-Induced Cytokine Function- and Mortality in Rats
  • NCY101 BPI
  • LPS-related cytokine formation and mortality The potential effect of NCY101 (BPI) against LPS-related cytokine formation and mortality was investigated in rats with either (a) hemorrhagic shock (bled to lower pressure to 30-35 mmHg mean arterial pressure for 90 minutes, followed by reinfusion of shed blood and an equal volume of Ringer's solution over 30 minutes), or (b) endotoxin shock (caused by 100 ⁇ g LPS and 500mg D-galactosamine/kg). Treatment comprised 5mg BPI/kg i.v. for the BPI group, or 1ml saline i.v. for the control group.
  • LPS lipopolysaccharide
  • LOS Gram-negative lipooligosaccharide
  • non-viable bacteria were pre -treated with recombinant BPI and incubated with human whole blood for 4 hours at 37°C. Without BPI, N.
  • BPI may be useful as a therapeutic agent against LOS -mediated tissue damage associated with these pathogenic Neisseria species.
  • NCY118->NCY103 (I43->V) and (N206->D)] had no effect on affinity for immobilized LPS.
  • NCY144 an IgG chimera consisting of NCY118 linked to human IgGl Fc constant region of the immunoglobulin molecule
  • Figure 14, panel A does not have an altered ability to compete with biotinylated BPI
  • NCY114 and NCY115 showed LPS affinity very similar to that observed for BPI, suggesting that the region between amino acid residues 1-59 (or 1-134) probably plays a minimal role in LPS binding (Figure 14, panel B). Together with data showing the NCY104 competes effectively with BPI
  • Amino acid residues 359 to 456 of BPI are not involved in LPS binding as demonstrated by the relative inability of NCY117 to displace biotinylated BPI from LPS ( Figure 14, panel C).
  • the apparent binding affinity of NCY117 for LPS is similar to that of LBP and NCY139, which affinity is approximately two orders of magnitude lower than the apparent affinity of BPI for LPS.
  • the domain of BPI which participates in binding to immobilized LPS is localized in the N-terminal half of the BPI molecule, since NCY104 has the greatest ability to displace native BPI from LPS coated onto microtiter plates. This domain of BPI has been more specifically localized to a region between amino acid residues 134-199.
  • NCY103 28.0 ⁇ 20 .0 (n 4)
  • NCY104 which contains the N-terminal domain of BPI, is a relatively poor inhibitor of LPS in the LAL assay.
  • NCY103 was a more potent inhibitor than NCY102 (LBP) or NCY104.
  • NCY139 which contains the entire BPI sequence except for nine cationic residues between positions 148 and 197, showed very poor LPS-neutralizing activity, suggesting that these residues are important in LPS-neutralizing activity. Similarly, this compound was relatively ineffective at LPS binding. These cationic residues may permit correct structural conformation of the molecule, since both NCY103 and NCY139 contain the C-terminal domain of BPI, yet NCY103 has potent neutralizing activity while NCY139 does not.
  • Normal human serum contains about l-10 ⁇ g/ml LBP.
  • BPI and NCY103 potently inhibited FITC LPS binding to monocytes, with BPI showing slightly greater potency.
  • NCY104 had marginal activity, and LBP had no effect ( Figure 15, panel A). These data indicate that the C-terminal half of the BPI molecule was neutralizing LPS in this test.
  • NCY104 which does not contain the C-terminal domain of BPI, is approximately two orders of magnitude less potent at blocking LPS binding in the presence of serum. LBP, as expected, had no effect. This demonstrated that BPI can intervene in the sepsis cascade by preventing LPS from binding to monocytes and causing release of TNF ⁇ .
  • the domain of LBP responsible for facilitating LPS-induced TNF release is within amino acid residues 199-357.
  • NCY104 did not mediate TNF release in a serum-free system. This may indicate that the N-terminal domain of BPI binds too tightly to LPS to allow transfer of LPS to CD14 on the macrophage surface.
  • Figure 17 shows an additional comparison of TNF production. NCY135, containing LBP domain 274-456, shows great activity, narrowing the active domain to 274-357.
  • NCY103 was the most potent at blocking TNF release, followed by BPI as the next most potent blocker.
  • NCY104 and LBP had essentially no effect.
  • NCY103 proved to be the most potent inhibitor of LPS-mediated cytokine stimulation.
  • a potent anti-endotoxin therapeutic should not only neutralize endotoxin, but should also have the capacity to clear endotoxin from the circulation.
  • the circulating levels of radioactively labeled 125 I-BPI were measured in the mouse in the presence and absence of endotoxin (Table 7). In the absence of endotoxin, the elimination (alpha) phase for 125 I-BPI was less than two minutes. In the presence of LPS, the alpha phase was extended to 6.2 minutes. 125 I-LPS alone has a single phase distribution (beta) with a half-life of about 101 minutes. When 125 I-LPS and unlabeled BPI were administered, a 6.2 minute elimination (alpha) phase was observed, indicating that elimination was remarkably facilitated by BPI. Table 7
  • Figure 12 shows that NCY103 indeed has a longer half-life than BPI.
  • NCY104 with the N-terminal domain of BPI, had an even shorter half-life than that of BPI.
  • the N-terminal domain of BPI appears to play a major role in its short circulating half-life.
  • NCY104 was similar to that of BPI.
  • NCY103 and NCY118 had overlapping elimination curves and persisted in the circulation significantly longer than BPI or NCY104, but not as long as the serum protein LBP.
  • Comparison of the elimination curves of NCY114, NCY115 and NCY139 revealed that the N-terminus of LBP plays a role in extending circulating half-life.
  • NCY114 circulates slightly longer than BPI and contains the least LBP sequence of any of the recombinant proteins tested (amino acid residues 1-59).
  • NCY115 was cleared somewhat more slowly, indicating that LBP amino acid residues 60-134 impart a longer circulating half-life.
  • the cationic residues of BPI between amino acid residues 134-199 shorten the half-life, since in NCY139, where the cationic residues in this region were replaced with the corresponding residues of LBP, the half-life was similar to that of NCY115.
  • Including more LBP residues in the N-terminal domain further extends the half life. If amino acid residues 199-357 of LBP are added (NCY117) the half-life is longer, but not quite as long as that of LBP.
  • NCY135 (with LBP domain 1-199 and 274-456 has a relatively long T 1 ⁇ 2 .
  • NCY103 was markedly more effective than BPI when given more than an hour before or after LPS (Figure 13). These results indicate that the longer circulating half-life of NCY103, or perhaps the increased ability of NCY103 to inhibit endotoxin in whole blood, has a dramatic effect on NCY103 efficacy in vivo.
  • NCY101 (BPI) against LPS related cytokine formation and mortality was investigated in rats with either (a) hemorrhagic shock or (b) endotoxin shock.
  • endotoxin shock The important role of endotoxin in hemorrhage (with endogenous LPS translocation from the gut), trauma and sepsis is well known.
  • BPI binds LPS and inhibits LPS- mediated neutrophil and monocyte stimulation.
  • recombinant BPI binds LPS and inhibits TNF formation in vitro.
  • mice Anesthetized male CD-1 mice were treated intra-nasally with 1 or 10 ⁇ g of either BPI or NCY103 in 50 ⁇ l. Control animals received 50 ⁇ l of saline for injection. After 20 minutes, animals were re-anesthetized, and challenged with 10ng of E. coli 055:B5 LPS. One hour after endotoxin challenge, mice were re-anesthetized, and 0.7ml of saline containing 1% human serum albumin was added to the lungs via the trachea. The lungs were gently kneaded.
  • BAL bronchoalveolar lavage
  • cells were pelleted by centrifugation, and the BAL sample was stored at -70°C.
  • the TNF-alpha level in the BAL fluid was determined by ELISA (results shown in Figure 20).
  • Figure 20 shows that endotoxin-neutralizing proteins such as BPI and NCY103 can also neutralize endotoxin-mediated TNF release in the lung. These results indicate that these proteins are effective when delivered directly into the lung. This supports use in the treatment of pneumonias and other endotoxin-related disorders of the lung, such as ARDS.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des molécules d'acide nucléique recombinées codant pour un variant de BPI, un variant de LBP, une chimère LBP-BPI, une chimère BPI-IgG, et une chimère LBP-IgG, ou une chimère LBP-BPI-IgG, ainsi que les protéines pour lesquelles codent ces dernières. L'invention porte également sur des systèmes de vecteurs hôtes utilisés pour la production d'un variant de BPI, d'un variant de LBP, d'une chimère LBP-BPI, d'une chimère BPI-IgG, d'une chimère LBP-IgG, ou d'une chimère LBP-BPI-IgG, et sur leurs procédés d'utilisation pour produire lesdites protéines. L'invention se rapporte également à une composition pharmaceutique contenant un variant de BPI, un variant de LBP, une chimère LBP-BPI, une chimère BPI-IgG, une chimère LBP-IgG ou une chimère LBP-BPI-IgG en doses pharmaceutiquement efficaces, ainsi qu'un excipient pharmaceutiquement acceptable, et à son procédé d'utilisation pour traiter un sujet souffrant d'une affection liée à l'endotoxine. Un procédé permettant de prévenir toute affection liée à l'endotoxine chez un sujet consistant à administrer à ce dernier une dose prophylactique efficace d'un variant de BPI, d'un variant de LBP, d'une chimère LBP-BPI, d'une chimère BPI-IgG et d'une chimère LBP-IgG est également décrit.
PCT/US1994/004709 1989-02-14 1994-04-29 Proteines de recombinaison de type augmentant la permeabilite bactericide (bpi) et de type fixant le liposaccharide (lbd), molecules codant pour ces proteines, leurs procedes de production et leurs utilisations WO1994025476A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
AU69429/94A AU6942994A (en) 1993-04-30 1994-04-29 Recombinant bpi-based and lbp-based proteins, nucleic acid molecules encoding same, methods of producing same, and uses thereof
CA002161971A CA2161971A1 (fr) 1993-04-30 1994-04-29 Proteines recombinantes a base de bpi et de lbp; molecules d'acide nucleique codant ces proteines; methodes de preparation et utilisations
JP6524554A JPH08511682A (ja) 1993-04-30 1994-04-29 組み換えｂｐｉ蛋白およびｌｂｐ蛋白、それをコードする核酸分子、その生産方法並びにそれらの使用
EP94917901A EP0760849A1 (fr) 1993-04-30 1994-04-29 Proteines de recombinaison de type augmentant la permeabilite bactericide (bpi) et de type fixant le liposaccharide (lbd), molecules codant pour ces proteines, leurs procedes de production et leurs utilisations
US08/431,517 US6265187B1 (en) 1989-02-14 1995-05-01 Recombinant endotoxin-neutralizing proteins
US09/861,400 US20020146761A1 (en) 1989-02-14 2001-05-18 Recombinant endotoxin-neutralizing proteins

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US5629293A 1993-04-30 1993-04-30
US08/056,292 1993-04-30
US16571793A 1993-12-10 1993-12-10
US08/165,717 1993-12-10

Related Parent Applications (3)

Application Number Title Priority Date Filing Date
PCT/US1991/005758 Continuation-In-Part WO1992003535A1 (fr) 1989-02-14 1991-08-13 Proteines bpi recombinees, utilisation et procedes de preparation de ces proteines
US07/915,720 Continuation-In-Part US5770694A (en) 1989-02-14 1992-07-22 Genetically engineered BPI variant proteins
US16571793A Continuation-In-Part 1989-02-14 1993-12-10

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US08/431,517 Continuation-In-Part US6265187B1 (en) 1989-02-14 1995-05-01 Recombinant endotoxin-neutralizing proteins
US09/861,400 Continuation-In-Part US20020146761A1 (en) 1989-02-14 2001-05-18 Recombinant endotoxin-neutralizing proteins

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WO1994025476A1 true WO1994025476A1 (fr) 1994-11-10

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PCT/US1994/004709 WO1994025476A1 (fr) 1989-02-14 1994-04-29 Proteines de recombinaison de type augmentant la permeabilite bactericide (bpi) et de type fixant le liposaccharide (lbd), molecules codant pour ces proteines, leurs procedes de production et leurs utilisations

Country Status (5)

Country Link
EP (1) EP0760849A1 (fr)
JP (1) JPH08511682A (fr)
AU (1) AU6942994A (fr)
CA (1) CA2161971A1 (fr)
WO (1) WO1994025476A1 (fr)

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034873A1 (fr) * 1995-05-01 1996-11-07 Incyte Pharmaceuticals, Inc. Proteines de recombinaison neutralisant une endotoxine
WO1997042966A1 (fr) * 1996-05-10 1997-11-20 Xoma Corporation Applications therapeutiques de produits proteiques bactericides/augmentant la permeabilite (bpi) dans le cas de meningococcemies chez l'homme
US5741779A (en) * 1996-05-10 1998-04-21 Xoma Corporation Antithrombotic materials and methods
US5756464A (en) * 1996-05-23 1998-05-26 Xoma Corporation Therapeutic uses of BPI protein products in humans with hemmorhage due to trauma
US5780429A (en) * 1995-12-22 1998-07-14 Marine Biological Laboratory Anti-LPS factor from horseshoe crabs and methods of use
US5786324A (en) * 1994-03-24 1998-07-28 Regents Of The University Of Minnesota Synthetic peptides with bactericidal activity and endotoxin neutralizing activity for gram negative bacteria and methods for their use
US5804367A (en) * 1994-01-24 1998-09-08 Xoma Corporation Method for quantifying LBP in body fluids
US5830860A (en) * 1994-03-24 1998-11-03 Regents Of The University Of Minnesota Peptides with bactericidal activity and endotoxin neutralizing activity for gram negative bacteria and methods for their use
US5856438A (en) * 1993-03-12 1999-01-05 Xoma Corporation Biologically active peptides from functional domains of bactericidal/permeability-increasing protein and uses thereof
US5858974A (en) * 1995-07-20 1999-01-12 Xoma Corporation Anti-fungal peptides
EP0890648A1 (fr) * 1996-12-10 1999-01-13 Council of Scientific & Industrial Research Lipopolysaccharide non toxique et sa préparation
DE19729810A1 (de) * 1997-07-11 1999-01-14 Max Delbrueck Centrum Mittel zur Sepsistherapie, seine Herstellung und seine Verwendung
US5888973A (en) * 1996-08-09 1999-03-30 Xoma Corporation Anti-chlamydial uses of BPI protein products
US5891618A (en) * 1994-01-24 1999-04-06 Xoma Corporation Method for quantifying LBP in body fluids
US5990082A (en) * 1997-10-22 1999-11-23 Xoma Corporation Uses of lipopolysaccharide binding protein
US6013631A (en) * 1998-06-19 2000-01-11 Xoma Corporation Bactericidal/permeability-increasing protein (BPI) deletion analogs
US6093573A (en) * 1997-06-20 2000-07-25 Xoma Three-dimensional structure of bactericidal/permeability-increasing protein (BPI)
US6482796B2 (en) 1996-11-01 2002-11-19 Xoma Corporation Therapeutic uses of N-terminal BPI protein products in ANCA-positive patients
US6486125B1 (en) 1996-05-24 2002-11-26 Regents Of The University Of Minnesota Synthesis of soluble β-sheet forming peptides
EP1310558A3 (fr) * 1993-02-02 2003-08-13 XOMA Technology Ltd. Produits protéiniques stables bactéricides ou renforcant la perméabilité et compositions pharmaceutiques les contenant
US7056514B2 (en) 2002-02-20 2006-06-06 Regents Of The University Of Minnesota Partial peptide mimetics and methods
US8394379B2 (en) 2003-05-15 2013-03-12 Iogenetics, Llc Targeted cryptosporidium biocides
US8703134B2 (en) 2003-05-15 2014-04-22 Iogenetics, Llc Targeted cryptosporidium biocides

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5171739A (en) * 1989-02-14 1992-12-15 Incyte Pharmaceuticals, Inc. Treatment of endotoxin-associated shock and preventation thereof using a BPI protein
US5234912A (en) * 1989-02-14 1993-08-10 Incyte Pharmaceuticals, Inc. Pharmaceutical compositions comprising recombinant BPI proteins and a lipid carrier and uses thereof
US5089274A (en) * 1989-02-14 1992-02-18 Incyte Pharmaceuticals, Inc. Use of bactericidal/permeability increasing protein or biologically active analogs thereof to treat endotoxin-related disorders
ATE169030T1 (de) * 1990-06-28 1998-08-15 Hoechst Ag Fusionsproteine mit immunglobulinanteilen, ihre herstellung und verwendung
AU2872792A (en) * 1991-09-26 1993-04-27 Incyte Pharmaceuticals, Inc. A new form of liposaccharide binding protein (lbp)
AU4382193A (en) * 1992-05-19 1993-12-13 Xoma Corporation BPI-immunoglobulin fusion proteins

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 179, No. 1, issued 30 August 1991, LARRICK et al., "Complementary DNA Sequence of Rabbit CAP18--A Unique Lipopolysaccharide Binding Protein", pages 170-175. *
JOURNAL OF EXPERIMENTAL MEDICINE, Volume 174, issued September 1991, OOI et al., "Endotoxin-Neutralizing Properties of the 25 kD N-Terminal Fragment and a Newly Isolated 30 kD C-Terminal Fragment of the 55-60 kD Bactericidal/Permeability-Increasing Protein of Human Neutrophils", pages 649-655. *
See also references of EP0760849A4 *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 264, No. 16, 05 June 1989, GRAY et al., "Cloning of the cDNA of a Human Neutrophil Bactericidal Protein", pages 9505-9509. *

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US6828418B2 (en) 1993-02-02 2004-12-07 Xoma Corporation Stable bactericidal/permeability-increasing protein products and pharmaceutical compositions containing the same
EP1310558A3 (fr) * 1993-02-02 2003-08-13 XOMA Technology Ltd. Produits protéiniques stables bactéricides ou renforcant la perméabilité et compositions pharmaceutiques les contenant
US5856438A (en) * 1993-03-12 1999-01-05 Xoma Corporation Biologically active peptides from functional domains of bactericidal/permeability-increasing protein and uses thereof
US6274328B1 (en) 1994-01-24 2001-08-14 Xoma Corporation Method for quantifying LBP in body fluids
US5891618A (en) * 1994-01-24 1999-04-06 Xoma Corporation Method for quantifying LBP in body fluids
US5804367A (en) * 1994-01-24 1998-09-08 Xoma Corporation Method for quantifying LBP in body fluids
US7081334B2 (en) 1994-01-24 2006-07-25 Xoma Corporation Method for quantifying LBP in body fluids
US5990086A (en) * 1994-01-24 1999-11-23 Xoma Corporation Therapeutic uses of BPI protein products for human meningococcemia
US5786324A (en) * 1994-03-24 1998-07-28 Regents Of The University Of Minnesota Synthetic peptides with bactericidal activity and endotoxin neutralizing activity for gram negative bacteria and methods for their use
US5830860A (en) * 1994-03-24 1998-11-03 Regents Of The University Of Minnesota Peptides with bactericidal activity and endotoxin neutralizing activity for gram negative bacteria and methods for their use
WO1996034873A1 (fr) * 1995-05-01 1996-11-07 Incyte Pharmaceuticals, Inc. Proteines de recombinaison neutralisant une endotoxine
US5858974A (en) * 1995-07-20 1999-01-12 Xoma Corporation Anti-fungal peptides
US5780429A (en) * 1995-12-22 1998-07-14 Marine Biological Laboratory Anti-LPS factor from horseshoe crabs and methods of use
US5935930A (en) * 1996-05-10 1999-08-10 Xoma Corporation Antithrombotic materials and methods
US6242418B1 (en) 1996-05-10 2001-06-05 Xoma Corporation Therapeutic uses of BPI protein products for human meningococcemia
US6599881B1 (en) 1996-05-10 2003-07-29 Xoma Corporation Antithrombotic materials and methods
US6596691B2 (en) 1996-05-10 2003-07-22 Xoma Corporation Therapeutic uses of BPI protein products for human meningococcemia
US5888977A (en) * 1996-05-10 1999-03-30 Giroir; Brett P. Therapeutic uses of BPI protein products for human meningococcemia
US5741779A (en) * 1996-05-10 1998-04-21 Xoma Corporation Antithrombotic materials and methods
WO1997042966A1 (fr) * 1996-05-10 1997-11-20 Xoma Corporation Applications therapeutiques de produits proteiques bactericides/augmentant la permeabilite (bpi) dans le cas de meningococcemies chez l'homme
US6107280A (en) * 1996-05-10 2000-08-22 Xoma Corporation Antithrombotic materials and methods
US5756464A (en) * 1996-05-23 1998-05-26 Xoma Corporation Therapeutic uses of BPI protein products in humans with hemmorhage due to trauma
US5945399A (en) * 1996-05-23 1999-08-31 Xoma Corporation Therapeutic uses of BPI protein products in humans with hemorrhage due to trauma
US7030087B2 (en) 1996-05-24 2006-04-18 Regents Of The University Of Minnesota Synthetic approach to designed chemical structures
US6486125B1 (en) 1996-05-24 2002-11-26 Regents Of The University Of Minnesota Synthesis of soluble β-sheet forming peptides
US5888973A (en) * 1996-08-09 1999-03-30 Xoma Corporation Anti-chlamydial uses of BPI protein products
US6162788A (en) * 1996-08-09 2000-12-19 Xoma Corporation Uses of BPI protein products
US6583116B1 (en) 1996-08-09 2003-06-24 Xoma Corporation Anti-chlamydial methods and materials
US6482796B2 (en) 1996-11-01 2002-11-19 Xoma Corporation Therapeutic uses of N-terminal BPI protein products in ANCA-positive patients
EP0890648A1 (fr) * 1996-12-10 1999-01-13 Council of Scientific & Industrial Research Lipopolysaccharide non toxique et sa préparation
US6093573A (en) * 1997-06-20 2000-07-25 Xoma Three-dimensional structure of bactericidal/permeability-increasing protein (BPI)
WO1999002178A1 (fr) * 1997-07-11 1999-01-21 Max-Delbrück-Centrum für Molekulare Medizin Agent therapeutique pour le traitement de la septicemie, sa preparation et son utilisation
US6949512B1 (en) * 1997-07-11 2005-09-27 Max-Delbruck-Zentrum Fur Molekulare Medizin Therapeutic agent for the treatment of septicaemia its preparation and use
DE19729810C2 (de) * 1997-07-11 2000-01-13 Max Delbrueck Centrum Mittel zur Sepsistherapie, seine Herstellung und seine Verwendung
DE19729810A1 (de) * 1997-07-11 1999-01-14 Max Delbrueck Centrum Mittel zur Sepsistherapie, seine Herstellung und seine Verwendung
US6306824B1 (en) 1997-10-22 2001-10-23 Xoma Corporation Uses of lipopolysaccharide binding protein
US5990082A (en) * 1997-10-22 1999-11-23 Xoma Corporation Uses of lipopolysaccharide binding protein
US6087126A (en) * 1998-06-19 2000-07-11 Xoma Corporation Bactericidal/permeability-increasing protein (BPI) deletion analogs
US6599880B1 (en) 1998-06-19 2003-07-29 Xoma Corporation Bactericidal/permeability-increasing protein (BPI) deletion analogs
US6013631A (en) * 1998-06-19 2000-01-11 Xoma Corporation Bactericidal/permeability-increasing protein (BPI) deletion analogs
US7056514B2 (en) 2002-02-20 2006-06-06 Regents Of The University Of Minnesota Partial peptide mimetics and methods
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US8703134B2 (en) 2003-05-15 2014-04-22 Iogenetics, Llc Targeted cryptosporidium biocides

Also Published As

Publication number Publication date
EP0760849A1 (fr) 1997-03-12
AU6942994A (en) 1994-11-21
JPH08511682A (ja) 1996-12-10
CA2161971A1 (fr) 1994-11-10
EP0760849A4 (fr) 1996-12-02

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