Attention Bluegreen......the holy grail is close......
>BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to proteins useful for the treatment of gram-negative bacterial infections and specifically to the neutralization of the effects of lipopolysaccharide (LPS) which is also known as endotoxin. LPS is a major component of the outer membrane of gram-negative bacteria and consists of serotype-specific O-side chain polysaccharides linked to a conserved region of core oligosaccharide and lipid A. LPS is an important mediator in the pathogenesis of septic shock and is one of the major causes of death in intensive-care units in the United States. It has been observed that exposure to LPS during sepsis stimulates an immune response in monocytes and macrophages that results in a toxic cascade resulting in the production of tumor necrosis factor (TNF) and other proinflammatory cytokines. Morrison and Ulevitch, Am. J. Pathol., 93:527 (1978). Endothelial damage in sepsis probably results from persistent and repetitive inflammatory insults. Bone, Annals Int. Med. 115:457 (1991).
[0003] LPS-binding proteins have been identified in various mammalian tissues. Among the most extensively studied of the LPS-binding proteins is bactericidal/permeability-increasing protein (BPI), a basic protein found in the azurophilic granules of polymorphonuclear leukocytes. Human BPI protein has been isolated from polymorphonuclear neutrophils (PMNs) by acid extraction combined with either ion exchange chromatography or E. coli affinity chromatography. Weiss et al., J. Biol. Chem., 253:2664 (1978); Mannion et al., J. Immunol. 142:2807 (1989).
[0004] The holo-BPI protein isolated from human PMNs has potent bactericidal activity against a broad spectrum of gram-negative bacteria. This antibacterial activity appears to be associated with the amino terminal region (i.e. amino acid residues 1-199) of the isolated human holo-BPI protein. In contrast, the C-terminal region (i.e. amino acid residues 200-456) of the isolated holo-BPI protein displays only slightly detectable anti-bacterial activity. Ooi et al., J. Exp. Med., 174:649 (1991). Human DNA encoding BPI has been cloned and the amino acid sequence of the encoded protein has been elucidated. Gray et al., J. Biol. Chem., 264:9505 (1989). Amino-terminal fragments of BPI include a natural 25 Kd fragment and a recombinant 23 Kd, 199 amino acid residue amino-terminal fragment of the human BPI holoprotein referred to as rBPI23. See, Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992). In that publication, an expression vector was used as a source of DNA encoding a recombinant expression product (rBPI23) having the 31-residue signal sequence and the first 199 amino acids of the N-terminus of the mature human BPI, as set out in SEQ ID NOS: 11 and 12 taken from Gray et al., supra, except that valine at position 151 is specified by GTG rather than GTC and residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG). Recombinant holoprotein referred to herein as rBPI has also been produced having the sequence set out in SEQ ID NOS: 11 and 12 taken from Gray et al., supra, with the exceptions noted for rBPI23. See also, Elsbach et al., U.S. Pat. No. 5,198,541 the disclosure of which is hereby incorporated by reference. In addition to its bactericidal effects, BPI has been shown to neutralize the toxic and cytokine-inducing effects of LPS to which it binds.
[0005] Lipopolysaccharide binding protein (LBP) is a 60 kD glycoprotein synthesized in the liver which shows significant structural homology with BPI. Schumann et al. disclose the amino acid sequences and encoding cDNA of both human and rabbit LBP. Like BPI, LBP has a binding site for lipid A and binds to the LPS from rough (R-) and smooth (S-) form bacteria. Unlike BPI, LBP does not possess significant bactericidal activity, and it enhances (rather than inhibits) LPS-induced TNF production. Schumann et al., Science, 249:1429 (1990). Thus, in contrast to BPI, LBP has been recognized as an immunostimulatory molecule. See, e.g., Seilhamer, PCT International Application WO 93/06228 which discloses a variant form of LBP which it terms LBP-[beta].
[0006] One of the normal host effector mechanisms for clearance of bacteria involves the binding to and subsequent phagocytosis by neutrophils and monocytes. As part of this process, bacteria are exposed to bactericidal and bacteriostatic factors, including oxygen radicals, lysosomal enzymes, lactoferrin and various cationic proteins. LBP opsonizes LPS-bearing particles and intact Gram-negative bacteria, mediating attachment of these LBP-coated particles to macrophages. Wright et al., J. Exp. Med. 170:1231 (1989). The attachment appears to be through the CD14 receptor of monocytes which binds complexes of LPS and LBP. Wright et al., Science 249:1431 (1990). Anti-CD14 mAbs have been shown to block the synthesis of TNF by whole blood incubated with LPS. Wright et al. Science 249:1431 (1990). Interaction of CD14, which is present on the surface of polymorphonuclear leukocytes as well as monocytes, with LPS in the presence of LBP has been shown to increase the adhesive activity of neutrophils. Wright et al., J. Exp. Med. 173:1281 (1991), Worthen et al., J. Clin. Invest. 90:2526 (1992). Thus, while BPI has been shown to be cytotoxic to bacteria and to inhibit proflammatory cytokine production stimulated by bacteria, LBP promotes bacterial binding to and activation of monocytes through a CD14-dependent mechanism.
[0007] LPS, either directly or by inducing proinflammatory cytokines such as IL-1 and TNF, induces the expresion of adhesion molecules including CD54 (intercellular adhesion molecule-1, ICAM-1) and E-selectin (endothelial-leukocyte adhesion molecule-1, ELAM-1) on endothelial cells, and thereby increases binding of leukocytes in vitro. Schleimer and Rutledge, J. Immunol. 136:649 (1986); Pohlman et al., J. Immunol. 136:4548 (1986); Bevilacqua et al., J. Clin. Invest. 76:2003 (1985); Gamble et al., Proc. Natl. Acad. Sci. USA. 82:8667 (1985); Smith et al., J. Clin. Invest. 82:1746 (1988); and Bevilacqua et al., Proc. Natl. Sci. USA 84:9238 (1987). However, as CD14 has not been detected on the surface of endothelial cells (Beekhuizen et al., J. Immunol. 147:3761 (1990)) and no other receptor for LPS on endothelial cells has been identified, a different mechanism may exist whereby LPS can affect the endothelium.
[0008] Soluble CD14, found in serum (Bazil et al., Eur. J. Immunol. 16:1583 (1986)), has been hypothesized to be responsible for transmitting the LPS signal to endothelial cells. Specifically, soluble CD14 has been shown to mediate a number of LPS-dependent effects on endothelial cells, including E-selectin and VCAM expression, IL-1, IL-6 and IL-8 secretion, and cell death, Frey et al., J. Exp. Med. 176:1665 (1992); Pugin et al., Proc. Natl. Acad. Sci. USA. 90:2744 (1993).
[0009] Recent studies have shown that soluble CD14 is involved in the LPS-mediated adhesion of neutrophils to endothelial cells. Anti-CD14 mAbs were able to completely inhibit the adhesion induced by LPS, indicating that the contribution of other CD14-independent LPS receptors to these effects is minimal. The protein(s) on the endothelial cells that soluble CD14 might associate with to transduce the LPS signal remains to be identified. LBP has been shown to be involved in the signal transduction of LPS through soluble CD14; however, at high concentrations of LPS or soluble CD14, LBP does not further enhance the response of endothelial cells to LPS (Pugin et al., Proc. Natl. Acad. Sci. USA. 90:2744 (1993).
[0010] Larrick et al., Biochem. and Biophysical Res. Commun., 179:170 (1991) relates to a cationic protein obtained from rabbit granulocytes which is identified as CAP18. CAP18 is identified as bearing no sequence homology with either BPI or LBP. In the course of their disclosure, Larrick et al. characterize other publications which discuss the structure of proteins including LBP and incorrectly attribute to the Wright et al., supra disclosure the speculation that "LBP is believed to be composed of two regions: an amino-terminal domain that binds to LPS and a carboxy-terminal domain that may (emphasis supplied) mediate binding of the LBP-LPS complex to the CD14 receptor on leukocytes."
[0011] Ulevitch, PCT International Application WO 91/01639 discloses methods and compositions for treatment of sepsis comprising administering anti-CD14 antibodies. The published application also describes "LBP peptide analogs" at page 17 which are stated to be polypeptides capable of competitively inhibiting the binding of LPS-LBP complexes to CD14 expressed on the surface of monocyte derived macrophages. The sequences of the three disclosed "LBP peptide analogs" show 90 to 100% homology with CD14 polypeptide sequences and no homology with LBP sequences.
[0012] Ulevitch et al., U.S. Pat. No. 5,245,013 discloses a lipopolysaccharide binding protein which binds to Gram-negative bacterially secreted LPS and retards in vitro binding of LPS to high density lipoprotein.
[0013] Marra, PCT International Application WO 92/03535 discloses various chimeric BPI molecules including an rLBP/BPI chimeric molecule designated LBP25K/BPI30K [LBP(1-197)/BPI(200-456)] and comprising the first 197 amino acid residues of LBP and amino acid residues 200-456 of BPI wherein the coding sequence for the amino-terminal 25 kD portion of LBP was linked to the coding sequence for the carboxy-terminal portion of the BPI protein by virtue of an engineered ClaI site within the coding sequence. The resulting molecule reacted positively in an ELISA assay utilizing anti-BPI protein antibodies and also reacted positively in an endotoxin binding assay. Rogy et al., J. Clin. Immunol., 14: 120-133 (1994) describes experiments utilizing the LBP(1-197)/BPI(200-456) molecule wherein animals treated with the molecule in a primate bacteremia model demonstrated decreased LPS levels compared to controls, but still developed the sequalae of septic shock. For example, no significant reduction in endotoxin mediated cytokine synthesis was observed in endotoxin-treated baboons to whom the compound was administered.
[0014] There exists a need in the art for LPS binding and neutralizing proteins which lack CD14-mediated immunostimulatory properties, including the ability to mediate LPS activity through the CD14 receptor.
SUMMARY OF THE INVENTION
[0015] The present invention provides novel biologically active polypeptide derivatives of Lipopolysaccharide Binding Protein (LBP), including LBP derivative hybrid proteins, which are characterized by the ability to bind to LPS and which lack CD14-mediated immunostimulatory properties, including the ability of LBP holoprotein to mediate LPS activity via the CD14 receptor. More particularly, LBP protein derivatives including LBP derivative hybrid proteins according to the invention lack those carboxy terminal-associated elements characteristic of the LBP holoprotein which enable LBP to bind to and interact with the CD14 receptor on monocytes and macrophages so as to provide an immunostimulatory signal to monocytes and macrophages.
[0016] Presently preferred LBP protein derivatives are characterized by a molecular weight less than or equal to about 25 kD. Particularly preferred LBP protein derivatives of the invention are LBP fragments comprising an amino-terminal region of LBP (e.g., amino acid residues 1-197). A molecule comprising the first 197 amino terminal residues of LBP and designated rLBP25 exemplifies the derivatives of the invention. This particular derivative includes amino acid regions comprising LBP residues 17 through 45, 65 through 99 and 141 through 167 which correspond to respective LPS binding domains (e.g., residues 17 through 45, 65 through 99 and 142 through 169) of Bactericidal/Permeability-Increasing protein (BPI).
[0017] LBP derivative hybrid proteins of the invention comprise hybrids of LBP protein sequences with the amino acid sequences of other polypeptides and are also characterized by the ability to bind to LPS and the absence of CD14-mediated immunostimulatory properties. Such hybrid proteins can comprise fusions of LBP amino-terminal fragments with polypeptide sequences of other proteins such as BPI, immunoglobulins and the like. Preferred LBP/BPI hybrids of the invention comprise at least a portion (i.e., at least five consecutive amino acids and preferably ten or more amino acids) of an LPS binding domain of BPI. One preferred LBP derivative hybrid protein of the LBP/BPI type comprises an amino-terminal LBP amino acid sequence selected from within the amino terminal half of LBP (e.g. within amino acid residues 1-197 of LBP) in which one or more portions of that sequence is replaced by the corresponding sequence of BPI selected from within the amino terminal half of BPI (e.g., within amino acid residues 1-199 of BPI). Another preferred LBP derivative hybrid protein comprises a fusion of amino terminal portions of LBP and heavy chain regions of IgG. Other LBP derivative hybrid proteins comprise LBP amino acid sequences into which all or portions of LPS binding domains of e.g., BPI or other LPS binding protein have been inserted or substituted for all or part of an LPS binding region of LBP. Preferred LBP derivative hybrid proteins include those in which all or portions of the previously-noted amino terminal LPS binding domains of BPI replace the corresponding region within LBP.
[0018] LBP protein derivatives and LBP derivative hybrid proteins of the invention are expected to display one or more advantageous properties in terms of pharmacokinetics, LPS binding, LPS neutralization and the like.
[0019] The present invention further provides novel pharmaceutical compositions comprising the LBP protein derivatives and LBP derivative hybrid proteins along with pharmaceutically acceptable diluents, adjuvants, and carriers and correpsondingly addresses the use of LBP protein derivatives and LBP derivative hybrid proteins in the manufacture of medicaments for treating gram negative bacterial infections and the sequelae thereof.
[0020] Polypeptides of the invention may be synthesized by assembly of amino acids. In addition, the invention provides DNA sequences, plasmid vectors, and transformed cells for producing the LBP protein derivatives and LBP hybrid derivative proteins of the invention.
[0021] Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention which describes presently preferred embodiments thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 depicts the DNA sequence and translated amino acid sequence (1-197) of rLBP25 [SEQ ID NOS: 1 and 2];
[0023] FIG. 2 depicts the DNA sequences and translated amino acid sequence (1-456) of rLBP [SEQ ID NOS: 3 and 4];
[0024] FIG. 3 depicts the construction of an rLBP25 mammalian expression vector pING4505;
[0025] FIG. 4 depicts construction of a vector pIC111 encoding an LBP (1-43)/BPI (44-199) hybrid protein;
[0026] FIG. 5 depicts constriction of mammalian expression vectors pING4525 and pING4526 for LBP/BPI hybrid proteins;
[0027] FIG. 6 depicts construction of plasmids pML105 and pML103 for in vitro transcription/translation of LBP/BPI hybrid proteins;
[0028] FIG. 7 depicts construction of a vector pIC112 encoding a BPI (1-159)/LBP (158-197) hybrid protein;
[0029] FIG. 8 depicts a graph illustrating the pharmacokinetics of I labeled rLBP25 and rBPI23;
[0030] FIG. 9 depicts binding of rLBP25 to E. coli J5 lipid A;
[0031] FIG. 10 depicts competition by rBPI23 and by rLBP25 for the binding of I-rLBP25 to immobilized lipid A;
[0032] FIG. 11 depicts competition by rBPI23 and by recombinant LBP holoprotein (rLBP) for the binding of I-rLBP to immobilized lipid A;
[0033] FIG. 12 depicts the ability of rLBP25 and rLBP to inhibit the LAL assay;
[0034] FIG. 13 depicts the effect of rLBP25 and rLBP on binding uptake of I-labeled LPS by THP-1 cells;
[0035] FIG. 14 depicts the effect of rLBP25 and rLBP molecules on TNF production by THP-1 cells;
[0036] FIGS. 15A and 15B depict the effect of rLBP on Tissue Factor (TF) and TNF production, respectively, in PBMCs;
[0037] FIG. 16 depicts the effect of rLBP on TNF production in PBMCs;
[0038] FIG. 17 depicts the effect of rLBP25 on TNF production in PBMCs;
[0039] FIG. 18 depicts the effect of LBP molecules on Tissue Factor production in PBMCs; and
[0040] FIG. 19 depicts the effect of rLBP25 on LPS induced endothelial adhesiveness for neutrophils:
[0041] FIG. 20 depicts the effect of rLBP on bacterial binding to monocytes;
[0042] FIG. 21 depicts the effect of rLBP25 on bacterial binding to monocytes;
[0043] FIG. 22 depicts the binding of rLBP and rLBP25 in an LBP sandwich ELISA assay; and
[0044] FIG. 23 depicts a homology comparison of the amino-terminal amino acid residues of human LBP and human BPI.
DETAILED DESCRIPTION
[0045] The present invention encompasses LBP protein derivatives and LBP derivative hybrid proteins which are characterized by the ability to bind to LPS but which lack the carboxy terminal-associated immunostimulatory element(s) characteristic of the LBP holoprotein and thus lack the CD14-mediated immunostimulatory activity characteristic of LBP holoprotein. Preferred LBP protein derivatives are characterized as including N-terminal LBP fragments having a molecular weight of about 25 kD. Most preferred are LBP N-terminal fragments characterized by the amino acid sequence of the first 197 amino acids of the amino-terminus of LBP set out in FIG. 1 and SEQ ID NOS: 1 and 2. It is also contemplated that LBP protein derivatives containing N-terminal fragments considerably smaller than 25 kD and comprising substantially fewer than the first 197 amino acids of the N-terminus of the LBP holoprotein molecule are suitable for use according to the invention provided they retain the ability to bind to LPS. Thus, specifically contemplated are LBP derivatives comprising part or all of one or more of three regions (defined by LBP amino acid sequences 17-45, 65-99 and 141-167) corresponding (by reason of amino acid homology) to LPS binding regions (comprising amino acid sequences 17-45, 65-99 and 142-169) of BPI. Moreover, it is contemplated that LBP protein derivatives comprising greater than the first 197 amino acid residues of the holo-LBP molecule, i.e., including amino acids on the carboxy-terminal side of residue 197 of rLBP as disclosed in FIG. 2 and SEQ ID NOS: 3 and 4 will likewise prove useful according to the methods of the invention provided they lack CD14-mediated immunostimulatory activity. It is further contemplated that those of skill in the art are capable of making additions, deletions and substitutions of the amino-acid residues of SEQ ID NOS: 1-4 without loss of the desired biological activities of the molecules. Such, LBP protein derivatives may be obtained by deletion, substitution, addition or mutation, including mutation by site-directed mutagenesis of the DNA sequence encoding the LBP holoprotein, wherein the LBP protein derivative maintains LPS-binding activity and lacks CD14-mediated immunostimulatory activity. One preferred LBP derivative is that wherein the alanine residue at position 131 of the illustrative LBP (1-197) polypeptide fragment is substituted with a cysteine residue. The resulting LBP (1-197) (Cys 131) polypeptide may have the ability to dimerize via interchain disulfide bond formation through cysteine 131 and the resulting dimer may be characterized by improved biological activity.
[0046] Also contemplated are LBP derivative hybrid proteins including LBP/BPI hybrid proteins [but excluding the hybrid designated LBP(1-197)/BPI(200-456) noted above] and LBP-Ig fusion proteins which are characterized by the ability to bind LPS but which lack CD-14 immunostimulatory activity. A preferred LBP/BPI hybrid protein of the invention is a protein comprising one or more portion of the amino-terminal half of LBP, e.g., selected from within amino acid residues 1-197 of LBP, and a one or more portions of the amino-terminal half of BPI, e.g., selected from within amino acid residues 1-199 of BPI.
[0047] Other LBP hybrid proteins comprise LBP amino acid sequences into which all or portions of LPS binding domains of other LPS binding proteins (such as BPI) have been inserted or substituted. Preferred LBP hybrid proteins include those in which all or portions of the LPS binding domains of BPI (comprising BPI residues 17-45, 65-99 and 142-169) are substituted into the corresponding region of LBP. Portions of such BPI domains substituted into the hybrid proteins may comprise as few as five continuous amino acids but preferably include ten or more continuous amino acids.
[0048] LBP derivative hybrid proteins in which all or portions of the LPS binding regions of BPI are substituted into the corresponding region of LBP thus include those comprising at least a part of an LPS binding domain of BPI selected from the group of amino acid sequences consisting of:
[0049] ASQQGTAALQKELKRIKPDYSDSFKIKH (SEQ ID NO: 17) designated Domain I comprising the amino acid sequence of human BPI from about position 17 to about position 45;
[0050] SSQISMVPNVGLKFSISNANIKISGKWKAQKRFLK (SEQ ID NO: 18) designated Domain II comprising the amino acid sequence of human BPI from about position 65 to about 99; and
[0051] VHVHISKSKVGWLIQLFHKKIESALRNK (SEQ ID NO: 19) designated Domain III comprising the amino acid sequence of human BPI from about position 142 to about position 169.
[0052] These LPS binding domains of BPI correspond to LBP regions consisting of:
[0053] AAQEGLLALQSELLRITLPDFTGDLRIPH (SEQ IS NO: 20) comprising the amino acid sequence of human LBP from about position 17 to about position 45;
[0054] HSALRPVPGQGLSLSISDSSIRVQGRWKVRKSFFK (SEQ ID NO: 21) comprising the amino acid sequence of human LBP from about position 65 to about 99; and
[0055] VEVDMSGDLGWLLNLFHNQIESKFQKV (SEQ ID NO: 22) comprising the amino acid sequence of human LBP from about position 141 to about position 167.
[0056] According to another aspect of the invention, DNA sequences are provided which encode the above-described LBP protein derivatives and LBP derivative hybrid proteins. Also provided are autonomously replicating DNA plasmid vectors including such DNA sequences and host cells stably transformed or transfected with such DNA sequences in a manner allowing their expression. Transformed host cells of the invention are of manifest utility in procedures for the large-scale production of the LBP protein derivatives and LBP derivative hybrid proteins of the invention involving the cultured growth of the hosts in a suitable medium and the isolation of the proteins from the cells or their growth medium.
[0057] The invention further provides novel pharmaceutical compositions comprising an LBP protein derivative or an LBP derivative hybrid protein which retains LPS-binding activity and lacks CD14-mediated immunostimulatory activity together with pharmaceutically acceptable diluents, adjuvants, and carriers. The compositions are useful in methods for treating a gram-negative bacterial infection, including the sequelae thereof such as endotoxin related shock, and one or more of conditions associated with gram-negative bacterial infection and resulting endotoxic shock such as disseminated intravascular coagulation, anemia, thrombocytopenia, leukopenia, adult respiratory distress syndrome, renal failure, hypotension, fever and metabolic acidosis. Such methods comprise administering an LBP protein derivative or LBP derivative hybrid protein to a subject suffering from a gram-negative bacterial infection, including the sequelae thereof.
[0058] When employed for treatment of a gram-negative bacterial infection, including the sequelae thereof, LBP protein derivatives and LBP derivative hybrid proteins of the invention are preferably administered parenterally and most preferably intravenously in amounts broadly ranging from about 0.1 milligram and about 100 milligrams per kilogram of body weight of the treated subject with preferred treatments ranging from about 1 milligrams and 25 milligrams per kilogram of body weight. It is contemplated that administration of LBP derivative protein derivatives, such as rLBP25, and LBP hybrid proteins may be useful as one aspect of a combination therapy in which BPI or other antibiotics are administered to a subject.
[0059] According to a further aspect of the invention, LBP protein derivatives and LBP derivative hybrid proteins may be administered in combination with other therapeutic compositions which are not strictly antibiotics but rather which neutralize the endotoxic effects of LPS such as anti-LPS antibodies and antibodies to constituents of the LPS mediated toxic cascade such as anti-TNF antibodies.
[0060] The following detailed description relates to the manufacture and properties of LBP protein derivatives and LBP hybrid proteins of the invention. More specifically, Example 1 relates to the construction of vectors for expression of an exemplary LBP protein derivative, rLBP25. Example 2 relates to the construction of vectors for expression of rLBP. Examples 3 and 4 relate to the incorporation of the vectors of Examples 1 and 2 into appropriate host cells and further describes the expression and purification of rLBP25 and rLBP. Examples 5 and 6 relate to construction of vectors encoding LBP/BPI hybrid proteins. Example 7 relates to in vitro transcription translation of the LBP/BPI hybrid protein, LBP(1-43)/BPI(44-199) and BPI(1-159)/LBP(158-197). Example 8 relates to the pharmacokinetics of rLBP25, rLBP and rBPI23 in vivo. Example 9 relates to the binding of rLBP25 and rLBP to Lipid A. Example 10 relates to competition by rLBP25 and rBPI23 for the binding of I-rLBP25 to immobilized lipid A. Example 11 relates to competition by rLBP and rBPI23 for the binding of I-rLBP to immobilized lipid A. Example 12 relates to the effect of rLBP25 and rLBP on an LAL assay. Example 13 relates to the effect of rLBP25 and rLBP on the binding/uptake I-labeled LPS on TNF production by a human monocyte cell line THP-1. Example 14 relates to the effect of rLBP25 and rLBP on TF and TNF production by isolated PBMCs. Example 15 relates to the effect of rLBP25 on LPS-induced adhesiveness of endothelial cells for neutrophils. Example 16 relates to the effect of rLBP and rLBP25 on bacterial binding to monocytes and polymorphonuclear cells. Example 17 relates to a sandwich ELISA assay for rLBP and rLBP25. Example 18 relates to construction of vectors for production of LBP (1-197) (Cys 131). Example 19 relates to construction of vectors for production of LBP-IgG hybrid fusion proteins. Example 20 relates to in vitro transcription/translations of truncated LBP fragments and determination of their ability to mediate LPS stimulation of TNF activity. Example 21 relates to construction of vectors for production of LBP/BPI hybrid proteins. Example 22 relates to construction of vectors for production of LBP/BPI hybrid proteins comprising BPI/LBP active domain replacement and partial replacement mutants. Example 23 relates to properties of synthetic LBP peptides.
EXAMPLE 1
[0061] Construction of Vectors for Expression of rLBP25 Protein
[0062] A. Cloning and Sequencing of Human rLBP25
[0063] The DNA encoding amino acids 1-197 of human LBP without the signal sequence (designated "rLBP25") was obtained by PCR using human liver poly (A) RNA (Clontech Laboratories, Palo Alto, Calif.) as the source of material for amplification. Reverse transcription of the RNA to cDNA and PCR amplification were carried out using the GeneAmp RNA PCR Kit (Perkin Elmer Cetus, Norwalk, Conn.) according to the manufacturer's protocols. The sequence of human LBP was obtained from GenBank, accession number M35533, as published by Schumann et al., Science, 249:1429-1431 (1990). The 5' PCR primer corresponded to the amino terminal sequence of the coding region of mature LBP and included a BsmI recognition site at its 5' end. The sequence of this primer, LBP-Bsm, was: 5'-GAATGCAGCCAACCCCGGCT TGGTCGCCA-3' (SEQ ID NO: 5). The 3' PCR primer was designed to place a stop codon and an XhoI site following the isoleucine at amino acid position No. 197. The sequence of this primer, LBP-2, was: 5'-CTCGAGCTAAATC TCTGTTGTAACTGGC-3' (SEQ ID NO: 6). This amino acid was chosen as the endpoint of rLBP25 based on sequence homology with an amino-terminal active fragment of BPI (designated rBPI23).
[0064] The amplified rLBP25 DNA was blunt-end cloned into SmaI cut pT7T318U (Pharmacia, LKB Biotechnology, Piscataway, N.J.) to generate plasmid pIC106. The LBP insert in pIC 106 was sequenced using Sequenase (USB). The rLBP25 DNA sequence obtained and the derived amino acid sequence is set out in SEQ ID NOS: 1 and 2. The sequence differs in two areas from the sequence of the corresponding region of LBP as published by Schumann et al. (1990) Science, 249:1429-1431, which involve changes in amino acids. These sequence differences are detailed in Table 1.
[0065] B. Construction of Vectors for Expression of rLBP,5 in Mammalian Cells
[0066] A vector for the expression of rLBP25 using the BPI signal sequence was constructed using the rLBP25 coding region |
