EP0605653A1 - A new form of liposaccharide binding protein (lbp) - Google Patents

Abstract

The invention relates to a new form of lipposaccharide fixing protein, called LBP. This protein can be prepared by recombination and is used to stimulate the immune system.

Description

A NEW FORM OF LIPOSACCHARIDE BINDING PROTEIN (LBP)

Technical Field

The invention relates to medicaments useful in stimulating the immune system. More specifically, the invention concerns a new form of liposaccharide binding protein (LBP) .

Background Art

The lipopolysaccharides (LPS) are glycoprotein components of gram-negative bacterial cell walls and are an active part of the etiology of infection by these bacteria. LPS is apparently essential for immune response to the bacteria, and also is involved in the acute effects of such infection, such as septic shock. In order to exert its effects, however, LPS evidently binds to a number of indigenous proteins.

A number of proteins have been described in mammals that bind and mediate the effects of polar lipids such as lipopolysaccharides in circulation. Many of these molecules have been isolated and studied in detail and found to be similar in function and structure. It has been suggested that these proteins can be considered as encoded by a gene family with possible common evolutionary origin. One such protein, cholesterol ester transfer protein (CETP) binds cholesterol esters and other complex lipids associated with lipoprotein complexes and mediates their transfer between HDL and VLDL. Another protein, bacteriocidal permeability increasing protein (BPI) was originally isolated as a naturally-occurring host antibacterial agent. It has later been shown that BPI binds LPS released from bacterial cell walls and appears to neutralize many of its biological effects (Marra, J Immunol (1990) 144:662- 666) . A third protein, lipopolysaccharide binding protein (LBP) also binds LPS, but may mediate its biological effects in triggering cytokine release through a direct interaction of the molecular complex with a specific monocytic cell surface receptor. Thus, the complex of LBP and LPS has been proposed to mediate the activation of macrophages and neutrophils and the release of cytokines such as tumor necrosis factor (TNF) , interleukin-1 (IL-1) and interleukin-6 (IL-6) . See, for example, Schumann et al., Science (1990) 242:1429-1431; Wright, Science (1990) 249:1431-1433.

All of CETP, BPI and LBP show significant homology, and the homology between BPI and LBP is particularly striking. Both BPI and LBP contain a positively charged amino terminal domain of about 25 kd that appears to be responsible for lipid binding and a hydrophobic carboxy terminal domain of about 30 kd which^' may mediate interaction with membranes and/or receptors.

The known form of LBP (LBP-α) is synthesized in the liver and is present in normal human serum at a concentration of less than 0.5 μg/ l. The level rises to 50 μg/ml 24 hrs after induction of an immune response. The known LBP form has a molecular weight of about 60 kd of which about 10 kd represents added glycosyl residues. LBP has been shown to bind the lipid A moiety of LPS, a binding which presumably involves the amino terminal portion of LBP. The lipid A moiety of LPS is considered essential to induce shock. LBP also opsonizes LPS- bearing particles such as intact gram-negative bacteria, mediating attachment of the coated particles to macrophage. This binding appears to occur via a specific cellular receptor, CD14, which is free to move within the plane of the cell membrane (Wright et al. (supra)). Thus it is clear that LBP plays an important role in an inflammatory response to infection. Schumann et al. (supra) disclose the amino acid sequence and encoding cDNA of both human and rabbit LBP. The present invention provides a novel form of LBP (LBP-/3) not previously described.

Disclosure of the Invention

The present invention provides a novel form of LBP useful in activating the immune system. The novel LBP of the invention, designated herein LBP-β, adds to the repertoire of liposaccharide binding proteins known to stimulate the immune system. The availability of this additional form of LBP permits fine tuning of therapeutic protocols designed to enhance immune function.

Accordingly, in one aspect, the invention is directed to the immune-stimulating protein LBP-3 in purified and isolated form. In other aspects, the invention is directed to recombinant materials and methods useful in providing this protein. In still other aspects, the invention is directed to methods to stimulate the immune system using the LBP -β of the invention, to pharmaceutical compositions useful in this method, and to antibodies reactive with LBP-jS.

Brief Description of the Drawings

Figure 1 shows a comparison of the amino acid sequence and nucleic acids encoding LBP-α; and LBP-/3.

Figure 2 shows the amino acid sequence and the cDNA encoding LBP-/S,

Figure 3 shows the structure of two aiπplimers used in PCR in the cloning of LBP -β . Figure 4 shows a comparison of the amino acid sequence at positions 240-250 of rabbit LBP, human BPI, a previously disclosed form of human LBP (LBP-o;) , and human LBP-β. The numbering shown refers to Figure 1 of Schumann, et al. (supra) .

Modes of Carrying- Out the Invention

The LBP-/3 of the invention shows enhanced homology to both human BPI and rabbit LBP as compared to human LBP-α previously disclosed. There are 7 amino acid substitutions in LBP-jδ relative to LBP-α (see Fig. 1) . In addition and, more significantly, the LBP-/3 of the invention includes an additional 4 amino acids in the carboxy terminal region which correspond to similar residues in human BPI and rabbit LBP. This sequence was not disclosed in Schumann's LBP-α_. Four additional amino acids could have a profound effect on the molecule's tertiary structure.

This additional quartet of amino acids, of the sequence Met-Ser-Leu-Pro, corresponds to the Met-Asn-Leu- Pro homologous sequence in rabbit LBP and the Met-Glu- Phe-Pro sequence in human BPI. Thus, the quartet found in LBP -β is unique to this protein. A sequence comparison of the residues at positions 240-250 of human BPI, the previously disclosed human LBP, rabbit LBP, and the LBP-/3 of the invention is shown in Figure 4.

The novel LBP-j3 of the invention can most conveniently be produced using recombinant techniques. The complete amino acid sequence and cDNA encoding this sequence is disclosed herein and is shown in Figure 2.

The amino acid sequence shown in Figure 2 shows the precursor protein which is cleaved to obtain the mature form. The first 25 amino acids (positions 1-25) effect the secretion of the mature form of LBP which has, as its N-terminus, the Ala residue shown at position 26. The numbering system shown in Figure 2 is thus appropriate to the precursor protein; the corresponding positions in the mature protein are obtained by subtracting 25 from the numbers shown. Thus, for example, the valine shown at position 266 in Figure 2 is at position 241 in the mature form.

While the invention has reference to LBP-3 corresponding to the amino acid sequence shown at positions 26-482 of Figure 2 herein, it is recognized that minor modifications can be made to the sequence while retaining activity. Specifically, one or several amino acids may be deleted from the N-terminus and/or C- terminus without affecting activity, and one or several amino acid substitutions with residues which are considered conservative with respect to the amino acid residue for which substitution is made may be included without affecting activity in a significant manner. Such minor and understood modifications provide molecules also included within the scope of the invention and are included within the designation "LBP-jδ." In addition, standard derivatization, by glycosylation, phosphorylation, or chemical modification of sidechains, such as by conversion of proline to hydroxyproline may also be included in proteins designated LBP-/3 and within the scope of the invention so long as these modifications do not destroy the immune system stimulating activity of the protein.

The cDNA shown in Figure 2 is identical to that retrieved as described in the illustrations below except for the deliberately engineered Clal site at numbered amino acid positions 222-223. This Clal site was added to enable portions of LBP-jS to be spliced into other constructs in cassette form. However, of course, any DNA encoding the amino acid sequence of LBP-3 can be included in suitable expression systems for production of this protein. The encoding DNA may be prepared by retrieving the native cDNA, optionally providing degenerate modifications thereof, or may be partially or completely synthesized by conventional solid-phase oligonucleotide synthesizing techniques. Construction of DNA of sufficient length to encode the 482 amino acid LBP-jS precursor or 457 amino acid mature protein can be effected by techniques readily available and known in the art. The DNA encoding the LBP-3 of the invention is ligated to control systems which effect the expression of the coding DNA in host cells with which the control sequences are compatible. The choice of control sequences will thus depend on the nature of the production host cell. Techniques for construction of expression systems, transformation of host cells, and culture of the transformed hosts are now well-established procedures. A variety of expression system/recombinant hosts can be used. For prokaryotic expression, conventionally, E_^ coli hosts are available and a variety of control sequences including regulatable promoters such as the trp promoter or P_L promoter are well established. Eukaryotic systems useful for production of recombinant proteins include yeast, insect cells, mammalian cells, plant cells, and more recently, intact plants and multicellular organisms. The expression systems may be constructed to provide the LBP-/3 of the invention as the mature protein, or the encoding DNA may be ligated to upstream DNA which encodes a peptide sequence that effects the secretion of the LBP -β from the recombinant host cell (including the native sequence) or provide LBP-j8 as a fusion protein. Such variations in the design of expression systems are also well known in the art.

The LBP-j8 of the invention may also be used to provide antibodies useful in immunoassay systems for the detection of LBP-/3 or related proteins. In particular, antibodies which recognize epitopes related to the novel portion of the LBP-β which distinguishes it from the previously-disclosed human LBP are thus useful. Antibodies may be prepared specifically with respect to this epitope by immunization with a peptide fragment including the portion including amino acids 241-245 of the mature form of LBP-j8 (shown as residues 266-270 of the precursor peptide in Figure 2) . Such fragments may be as short as the 5 amino acid sequence shown, or may include additional amino acid residues derived from LBP-0. Shorter peptides may require conjugation to carrier to enhance immunogenicity.

The antibodies of the invention are prepared by standard immunization protocols by repeated administra¬ tion to suitable mammalian subjects and monitoring antibody titers in the serum. The polyclonal antiserum may be harvested and used per se, or monoclonal antibodies may be prepared from the peripheral blood lymphocytes or spleens of the immunized animals using standard cell immortalization and screening techniques. As is well known, immunologically reactive fragments of the antibodies can be advantageously used in many assays and other applications in which the antibodies are useful.

The LBP-S of the invention is useful in stimulating the immune system, and thus can be formulated into pharmaceutical compositions for therapeutic use. Standard formulations for administration can be found, for example, in Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, PA (latest edition). For administration of proteins such as LBP-3, administration by injection is generally preferred; and suitable formulations for injection include, for example, Hank's solution, Ringer's solution, physiological saline, and the like. LBP-S may also be administered by transmucosal or transdermal formulations, generally transmucosal formulations include detergents or other surface active agents and bile salts or fusidic acids to enhance transition of the membrane. Dosage levels required are comparable to the previously disclosed form of LBP although identification of appropriate dosages for a particular subject and condition depend on the mode of administration, the nature of the subject, the severity of the condition, and the judgment of the attending practitione .

The following examples are included to illustrate but not to limit the invention.

Example 1

Retrieval of cDNA Encoding LBP-β Figure 1 shows the oligonucleotide sequence of amplimers designed to complement the regions upstream and downstream of the cDNA encoding LBP described by Schumann (1990) (supra) . As shown in Figure 3, the oligonucleotides also contained restriction sites Nhel, Clal, and Xhol, both external and internal to the LBP coding regions. The amplimers shown in Figure 3 were used to amplify LBP encoding DNA from a commercially available cDNA preparation derived from human liver RNA. The amplification utilized standard enzyme-mediated PCR techniques.

The amplified products were digested with the corresponding restriction enzymes Nhel, Clal and Xhol and isolated by polyacrylamide gel electrophoresis. The digested cDNA fragments were ligated into pMa Neo, a commercially available mammalian expression vector and cloned in EL. coli. DNA from the resulting clones was analyzed by gel electrophoresis and sequenced. The resulting sequence is shown in Figure 2. The sequence was similar to that reported by Schumann et al. (supra) but also contained an additional sequence of 12 nucleotides in the coding region beginning after base 826 of Genbank sequence 35533, and shown in Figure 2 as positions 824-835.

Claims

Clai s

1. A protein in purified and isolated form which protein comprises the amino acid sequence of mature LBP-/3 shown in positions 26-482 of Figure 2.

2. A recombinant DNA in purified and isolated form which encodes the protein of claim 1.

3. A recombinant expression system capable of expressing the DNA of claim 2, which expression system comprises a DNA encoding said protein operably linked to control sequences heterologous to said encoding DNA.

4. The expression system of claim -3 which is contained in a plasmid.

5. A recombinant host cell transformed with the expression system of claim 3.

6. A method to produce the protein of claim 1, which method comprises culturing the cells of claim 5 under conditions which permit expression of said encoding DNA; and recovering the LBP-J protein from the culture.

7. A pharmaceutical composition which composition contains, as active ingredient, the protein of claim 1 in admixture with a pharmaceutically acceptable excipient.

8. A method for stimulating the immune system in a subject which comprises administering to the subject in need of such stimulation an effective amount of a protein comprising the amino acid sequence of mature LBP- jS shown in positions 25-82 of Figure 2 or a pharmaceutical composition thereof.

9. Antibodies or immunologically reactive fragments thereof specifically reactive with LBP-/3.