CA2104217A1 - Treatment of endotoxemia - Google Patents

Abstract

Disclosed is a method for treating endotoxemia and other types of infection by gram-negative or similar pathogens. The method includes providing a therapeutic agent that is a non-immunoglobulin polypeptide, soluble in the circulation, and has the ability to bind endotoxin-related substances; and administering to the circulation of a subject a therapeutic amount of the agent. This conjugate has reduced toxicity and reduced pathogenicity relative to unconjugated endotoxin-related substances. In preferred embodiments, the therapeutic agent is at least a portion of the extracellular portion of the scavenger receptor protein.

Description

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2 1 ~ 1 '7 TREATMENT OF ENDOTOXEMIA

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,' ;; BACKGROUND OF THE INVENTION
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The technical field of this invention is the diagnosis and treatment of endotoxemia and related disease states and, in particular, relates to the inactivation of endoto~in-related substances by the systemic administration of genetically engineered or chemically modified native polypeptides of the scavenger receptor protein.

Endoto~ins are the lipopolysaccharides (LPS) uniquely found on the outer surface of gram-negative bacteria. They are responsible in large part for the pathophysiological phenomena associated with gram-negative infections.

The outer monolayer of the outer membrane of most gram-negative bacteria includes a unique hydrophobic component called lipid A which is the active moiety of lipopolysaccharide. The ~, .,:-, . . .

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:~ ~10~217 -1,2-aiacylglycerol moiety of classical membrane phospholipids is absent in lipid A, and the acyl chains linked to its glucosamine backbone differ from those of qlycerolphospholipids in that they are 2-6 carbon atoms shorter and contain an R-3-hydrosyl substituent. The unique structure of Lipid A
reflects its role in membrane assembly and function, including providing resistance to phospholipases.

Lipid A and its precursor lipid IvA are also potent activators of macrophages, resulting in the rapid production of a wide array of immune mediators such as interleukin-l, tumor necrosis factor, and platelet activating factor, among others (see, Raetz (1990) Ann. Rev. Biochem. 59:129-170). When tumor necrosis factor is rapidly secreted into the system, a shock syndrome (septic, endotosic, or to~ic shock or endotosemia) may occur (see e.g., Beutler et al.
(1986) Nature 320:584; and Old (1988) Sçientific American 258:59).

Incipient septic shock is characterized in part by body temperature estremes, altered mental status, a decrease in orthostatic blood pressure, decreased urine output, a decreased serum albumin concentration, and tachypnea with hyposemia. The high morbidity associated with endotosin-induced shock remains a major clinical problem, especially in debilitated and immunosuppressed patients.

Traditional anti-shock therapy includes replacement of plasma volume with plasma espanders containing, for esample, vasoactive compounds, . .. - . . . . : ~ - : -- -; . . , , .. . .: . . -, . . - ~ - :. :. : , : . ~. : . .-. :
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WO 92/14482 PCI`/US92tO1370 3-- j 21~2~7 anti-inflammatory drugs, and/or various anti-prostaglandins.

However, since such measures have been only partially successful in controlling the morbidity associated with endoto~emia, other types of therapy have been developed such as the systemic :
administration of transforming growth factor 8 (PCT/US89/03162), interleukin-l (PCT/US87~02065), agonists of platelet activating factor (EP
89104429.9), and the parenteral administration of taurolidine and/or taurultam (EP 87306297.0).

Immunotherapy methods have also been developed including the systemic administration of antibody to endoto~in (see, for example, U.S. Patent No. 4,120,950; PCT/US84/00688; EP 84308218.1) or to the TNF binding protein (Beutler et al. (1985) Science 229:869-871; EP 89104494.3). These immunotherapies may suffer from the disadvantages of unfavorable kinetics, short biological half-life, and the potential for anti-idiotype antibody generation that would in some cases neutralize the therapeutic antibody.
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: Accordingly, there exists a need for improved methods of treating endotoxemia, more effective techniques for inactivating gram-negative pathogens, and therapeutic agents useful in these methods.

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WO92/14482 PCTtUS92~01370 2 1 0 ~ 2 1 7 ., SUMMARY OF THE INVENTION
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It has been discovered that endoto~in-related substances can be inactivated by being bound to a non-immunoglobulin polypeptide therapeutic agent, such as a soluble form of the scavenger receptor protein. When an endotosin-related substance is bound to the therapeutic agent, a conjugate is formed that has reduced to~icity and reduced pathogenicity relative to the unconjugated endotoxin-related substance.

~ Endotoxin-related substances" as used herein refers to endotoxin, the lipopolysaccharides found on the surface of gram-negative pathogens, to endotoxin-like, lipid A-like molecules, to molecules which bind the scavenger receptor and have to~ic or otherwise pathogenic effects, and to organisms which express such endotoxin, endotoxin-like, tosic, or pathogenic molecules on their surfaces or at sites which are accessible to the soluble scavenger receptor protein.

In preferred embodiments of the invention, the therapeutic agent contains an extracellular fragment of the scavenger receptor protein which is soluble. Native scavenger receptor protein is not soluble because of the presence of transmembranous and cytoplasmic domains which anchor the protein firmly within the membrane of the macrophage. One preferred embodiment of the therapeutic agent includes the collagen-binding domain of the native scavenger receptor protein. Another embodiment includes the a-helical coiled coil domain and/or the , . : ~ , '': '' . ' . ' ' -4~2 PCT/US92/01370 : ~ .
2~ ~2~7 cysteine-rich domain. Yet another embodiment includes, in addition to the collagen binding domain, the spacer domain of the scavenger receptor protein.

The soluble fragment may be obtained from isolated native scavenger receptor protein that has been subjected to various biochemical cleavage techniques. Alternatively, the ~ragment can be an analog produced by recombinant DNA methodologies such as a secreted form of the scavenger receptor protein. This analog can be a truncated form of the scavenger receptor protein having at least its intracellular and transmembranous domains deleted, and can have an amino acid sequence sufficiently duplicative of the amino acid sequence of a portion of the estracellular region of the scavenger receptor protein such that it binds an~ thereby inactivates endotosin-related substances. In addition, the analog can be engineered to have a greater binding affinity for an endotoxi~-related substance than the native scavenger receptor protein or than the e~tracellular fragment thereof, or can be engineered to more effectively neutralize the to~ic or pathogenic effects of molecules which bind the scavenger receptor or of organsms which espress such molecules on their surfaces or at sites which are accessible to the soluble receptor protein. This neutralization is the consequence of direct inactivation, steric hindrance, more rapid clearance from the circulation, or some other mechanism.

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In some aspects of the invention, the therapeutic agent binds specifically to the lipid A
moiety of endotoxin. In other aspects, the therapeutic agent has an affinity for acetylated low density lipoprotein of about 0.5 to 5.0 microgram protein per milliliter. In addition, the therapeutic agent may be further characterized by the ability to bind chemically-modified low density lipoprotein (LDL), or negatively-charged macromolecules including polyvinylsulfate, maleyl-BSA, fucoidan, and purine polynucleotides, poly~I-C], poly[I] and poly[G], and/or gram-negative bacteria.
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It has also been discovered that the therapeutic agent with the ability to bind an endotoxin-related substance can be used for treating endoto~emia or other toxemias resulting from infection by an organism espressing the endotoxin-related substance, or from the ingestion or invasion of endoto~in-related substances. The terms nendotosemia,n nto~ic shock~, ~septic shock", and ~endotoxic shockU are used herein to describe the shock syndrome resulting in response to infection by gram-negative bacteria or in response to invasion by other endoto2in-related substances. The method includes providing the therapeutic agent and administering to the circulation of a subject an endoto~emia-inhibiting, effective amount of the agent in a pharmaceutically acceptible carrier. The term "subject" as used herein refers to humans and animals. The agent binds endoto~in-related substances it encounters, thereby forming a conjugate that has reduced to~icity and pathogenicity relative to unconjugated endotoxi~-related substances.

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~RIEF pES~RIPTIQN OF_rH~ DRAwI~Gs The foregoing and other objects of this invention, the various features thereof, as well as the invention itself may be more fully understood from the following description when read together with the accompanying drawings in which:

~; FIG. l is a schematic representation of the type I and type II scavenger receptor proteins and their protein domains; and FIGS. 2A-2D are diagrammatic representations of the construction of plasmids used to transfect host cells with a gene encoding a soluble scavenger receptor protein;
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; FIG. 2A shows the insertion of the myelin associated glycoprotein leader sequence into pCDNAl;
~' FIG. 2B shows the construction of the pCDNAl/common vector;

FIG. 2C shows the insertion of unique regions to create soluble scavenger receptor type I
and type II in the pCDNAl-MAG vector; and FIG. 2D shows the transfer of the secreted soluble scavenger receptor gene including the MAG
leader into a pRc/CMV vector.

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WO92t14482 PCT/US92/01370 ~1~4~17 DESCRIPTION QF_THE INVENTION
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It has been discovered that a soluble form of a scavenger receptor protein can act as a therapeutic agent by binding endotosin-related substances, thereby inactivating them and aiding in their clearance from the circulation. The therapeutic agent can be administered to patients who are at high risk, or symptomatic, of endoto~ic shock.

The native scavenger receptor is a membrane bound protein taking two forms, type I and type II, both found on the surface of macrophages. This protein has an apparent molecular weight on SDS-polyacrylamide gels of about 220,000 daltons (220 kD) in the case of the type I receptor, and a binding affinity for chemically modified low density -lipoprotein (LDL) such as acetylated or oxaloacetylated LDL. For example, it has a binding affinity for acetylated LDL (Ac-LDL) of about 0.5-5.0 micrograms protein per milliliter. This protein is functional when combined with two other scavenger receptor subunits forming a trimer of subunits, each of which having an apparent molecular weight on SDS-polyacrylamide gels of about 77 kD in the case of the type I receptor, and including an asparagine (Asn)-linked carbohydrate chain.

As shown in FIG. l, each native protein subunit includes an N-terminal cytoplasmic domain and a domain which spans the membrane (transmembrane domain), followed by a spacer region, an alpha-helical coiled coil domain, and an e~tracellular collagen domain. The term ~collagen .
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domain~ is used herein to encompass a region of a polypeptide which is substantially analogous to that of collagen or an anal~g or portion thereof. ~inked to the collagen domain of the type I receptor is another estracellular domain which is rich in cysteine residues (~Cys-rich~ domain).

TABLE I illustrates the location of the various domains within the amino acid sequence of the scavenger receptor protein.
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TAB~E I
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' amino acid # Domain Estimated_Mass 1 - 50 Cytoplasmic 5,740 51 - 76 Transmembrane2,766 , 77 - 109 Spacer 3,708 110 - 271 ~-Helical Coiled Coil 18,747 272 - 343 Collagen Binding 6,795 341 - 453 Cys-Rich 12,275 50,056 total ~based on the molecular weight of the individual amino acids Each subunit has at least one asparagine (Asn)-linked carbohydrate chain determined by treatment with various deglycosylation enzymes. By analysis of its amino acid sequence, these carbohydrate attachment sites are located in the spacer and alpha helical coiled coil domains.

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WO92/1~82 PCT/US92/01370 2~ 42~7 -lo- ~ i``

The assignment of residues 341-453 as a Cys-rich domain is based on the fact that it contains multiple Cys amino acids. In addition, because this domain is the most distal esternal domain, and because several other known cell surface receptors have Cys-rich ligand binding domains, this domain may also serve as a ligand binding site. However, since the collagen binding domain (amino acid numbers 272-343) is positively charged and since the ligands are often negatively charged, the collagen domain alternatively or additionally may participate in ligand binding. The same may be true for a portion or all of the alpha helical coiled coil in the Asn-linked sugar domain.
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The soluble therapeutic agent includes at least a portion of the e~tracellular region of the native scavenger receptor protein (amino acid nos.
77-453) responsible for binding endotoxin-related substances. This e~tracellular portion may include the spacer region, all or part of the a-helical coiled coil domain, all or part of the collagen binding domain, and/or all or part of the Cys-rich domain. Alternatively, the soluble protein is a ` recombinantly produced analog having an amino acid sequence sufficiently duplicative of the amino acid sequences of at least a portion of the e~tracellular region such that the protein binds endoto~in-related substances with a similar, qreater, or slightly lesser affinity as the native, insoluble, membrane-bound protein or as a soluble fragment thereof.

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The therapeutic agent can be prepared by isolating native scavenger receptor protein from macrophages or a related cell line e~pressing the scavenger receptor protein on its surface, as disclosed in PCT~US~9/05115, herein incorporated by reference, and then subjecting the purified protein to proteolytic cleavage to remove the intracellular and transmembranous portions of the protein from the estracellular domains. A number of proteolytic enzymes are known in the art that recognize and cleave at a particular amino acid or amino acid sequence. Such commercially available enzymes include trypsin, chymotrypsin, pepsin, Endo Lys C, and Endo Arg C. After digestion, the fragments of the protein can be isolated by any number of chromatographic methods including, differential centrifugation, affinity and column chromatography, among many others.

Alternatively, the therapeutic agent can be obtained from cell lines genetically engineered to espress the scavenger receptor protein or fragments thereof. For esample, a nucleic acid sequence encoding the estracellular domain of the scavenger receptor protein, or a particular fragment thereof, may be used to produce a protein in an appropriate microbial, yeast, insect, or mammalian host cell. To accomplish this, the sequence is inserted into an e~pression system such as a vector which is suitable for transforming or transfecting a prokaryotic ~bacterial) or eucaryotic (yeast, insect, or mammalian) host cell. Some useful mammalian host cells include Chinese Hamster Ovary (CHO) cells, COS
M6 cells, and THP-l cells. These standard .~ .
procedures have been followed to produce well-known proteins such as insulin, interferons, human growth ; hormone, and the like.
:
Similar procedures, or obvious modifications thereof, can also be employed to prepare the .
scavenger receptor protein and fragments or analogs thereof in accord with the subject invention, using the nucleic acid sequence for the scavenger receptor protein set forth below in the sequence listing as SEQ ID NO: 1 and SEQ ID NO: 3.

A major portion of the amino acid sequence of the protein has been derived from the nucleic acid sequence of a gene encodiny the protein. However, because more than one nucleotide triplet (codon) can encode a single amino acid, a number of different nucleotide sequences can encode a single protein.
Hence, the peptide fragment disclosed herein may be encoded by nucleic acid sequences which are func~ionally equivalent to the one shown above, and which may also be prepared by known synthetic procedures. Accordingly, the invention includes such functionally equivalent nucleotide sequences. In addition, one skilled in the art, knowing the amino acid ~equence of the receptor protein, could synthetically or biosynthetically prepare a functionally equivalent analog of the receptor protein of the invention having substantially the same biological activity. In particular, fragments of the protein, especially portions of the estracellular domain, can be obtained for the disclosures herein without undue esperimentation.

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WO92/14~2 PCT/US92/01370 . .
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Thus, the scope of the invention includes the amino acid sequences and corresponding nucleic acid sequences depicted herein, as well as all functionally equivalent amino acid sequences (and corresponding nucleic acid ~e~uences) for molecules with substantially the same biological activities as the soluble scavenger receptor protein. For e~ample, one embodiment of the invention includes a polypeptide having an amino acid sequence sufficiently duplicative of the amino acid sequence of a subunit of the soluble scavenger receptor protein such that the polypeptide, when trimerized with two other like polypeptides or with two subunits of the scavenqer receptor protein, bind chemically modified forms of LDL and endoto~in-related substances with the same or greater affinity than the native protein.

The scavenger receptor protein can be used for a variety of diagnostic ana therapeutic purposes. In a simple embodiment, insoluble or soluble receptor proteins are harvested and purified from eucaryotic cells which are preferably mammalian, or from eucaryotic or prokaryotic cells engineered by recombinant means to produce such proteins, and used in both radiolabelled and unlabelled states in competitive binding assays to test for the presence of the receptor. The receptor protein, or fragments or analogs thereof, can also be fi~ed to inert supports for purification and assay purposes. ~or e~ample, the collagen binding domain of the receptor protein can be linked to an inert support material for uses in affinity chromatographic methods to ,: ~ : . - .. .
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.' ; isolated lipids and lipid-containing substance such as endoto~in, or to purify inhibitors which may be useful diagnostic, analytic, or therapeutic agents.

Other uses include various assay techniques which can be practiced employing the reagents disclosed hérein, including radioimmunoassays, enzyme immunoassays, heterogeneous and homogeneous assays, enzyme linked immunoabsorbent assays (~ELISA~), and the like.

An exemplary assay for endotosin-related substances can be carried out as follows. The sample (having an unknown concentration of an endotosin-; related substance) is first contacted with a known quantity of insoluble receptor protein (or analog or a portion thereof containing the epitope and ability to bind an endotosin-related substance), during which time the endotosin-related substance in the sample becomes bound to the receptor protein. The mi~ture is then treated with a known quantity of radiolabeled analyte which binds to those sites on the fised support which were unoccupied. Escess label is then washed off, and the quantity of label remaining on the support is inversely proportional to the amount of analyte originally present in the sample.

The soluble form of the receptor protein can be useful as a therapeutic sequestering agent which would render less to~ic and pathoqenic endotosin-related substances which bind to the scavenger receptor.
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Effective dosages of the therapeutic agents and modes of their administration in the treatment of endoto~emia can be determined by routine e~perimentation. ~he pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the estemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the estent that easy syringability e~ists. It must be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacterial and fungi. The carrier can be a solvent or dispersion medium containing, for esample, water, polyol (for e~ample, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mistures thereof, and vegetable oils. ~he proper fluidity can be maintained, for esample, by the use of a coating, such as lecithin, by the maintenance of the reguired particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for esample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some cases, it may be preferable to include isotonic agents, for esample, sugars or sodium chloride. Prolonged absorption of the injectable therapeutic agents can be brought about by the use in the compositions of agents delaying absorption.

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i WO92/14482 PCT/US92/01370 l 2~0 ~2~7 -16-, Sterile injectable solutions are prepared by incorporating the therapeutic agent in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

The therapeutic agent may be administered parenterally or intraperitoneally. Solutions of the therapeutic agent as pharmacologically acceptable salts can be prepared in water suitably mised with a surfactant, such as hydrosypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mistures thereof and in oils. Under ordinary conditions of stora~e and use, these preparations contain a preservative to prevent the growth of microorganisms.

The therapeutic agent also may be orally administered, for esample, with an inert dilutent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For ,. , , . ~ . .
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oral administratioD, the therapeutic agent may be incorporated with escipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, eli~irs, suspension syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of therapeutic agent. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60~ of the weight of the unit. The amount of therapeutic agent in such useful compositions is ~uch that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: escipients, such as dicalcium phosphate; a disintegrating agent;
and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. A syrup or eli2ir may contain the active compounds sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparations and formulations.

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As used herein, ~pharmaceutically acceptable -carrier~ includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents an~ the like. The use of such media and agents for pharmaceutically active substances is well known in the art. E~cept insofar a~ any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
Supplementary active ingredients can also be incorporated into the compositions.
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The invention will ne~t be described in connection with certain illustrated embodiments.
~; However, it should be clear that various ~, modifications, additions and subtractions can be made without departing from the spirit or ~cope of the ~i invention.
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Preparation of the Soluble Scaven~er ReceptQr from Native Pro~eins The soluble form of the scavenger receptor protein may be obtained by enzymatic cleavage of isolated native protein as follows. Membrane proteins from 500 g of li~er are prepared essentially by the method of Schneider et al. tvol- 225, ~ Biol.
Chem., pp. 11442-11447 (1980)), herein incorporated as reference. The proteins are resuspended in 500 ml of 10 mM Tris-HCl, pH 8, 1 mM CaC12, 0.15 M NaCl and 1 mM PMSF (Buffer A), sonicated twice, and then dissolved by the addition of 55 ml of 20% Triton X-100 with stirring for 30 min. Insoluble material .
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is removed by centrifugation (33,000 rpm, 1 hr, Beckman Type 35 rotor). The supernatant (500 ml) is applied at 75 ml/hr to an M-BSA-coupled Sepharose 4B
column (Pharmacia, 9.8 s 12 cm, containing about 10 mg of M-BSA/ml of gel) which had been equilibrated with Buffer A con~aining 1~ Triton X-100. The column is washed overnight with the same buffer and then washed with two column volumes of Buffer A containing 40 mM octyglucoside. The receptor protein is eluted with Buffer B (1 M NaCl, 20 mM Tris HCl, pH 8, 1 mM
CaC12, 1 mM PMSF, and 40 mM octyglucoside).

The fractions obtained are tested for their ability to bind Ac-LDL and endoto~inas described below; those containing Ac-LDL and endotosin-related substances binding activity are pooled and concentrated using ultrafiltration (Diaflo membrane PM30, Amicon). The sample buffer is changed to 25 mM
potassium phosphate, 40 mM octyglucoside, 1 mN PMSF, pH 6.8 using PD10 desalting columns (Pharmacia). The M-~SA affinity purified fraction (50 ml) is then applied to an Ultrogel-Ha (LKB) column (2.5 ~ 13 cm) at a flow rate of 75 ml~hr, and the proteins eluted with a gradient of phosphate buffer (25 mM to 3S0 mM) containing 40 mM octyglucoside.

The 220 kD scavenger receptor protein is recovered at phosphate concentrations between 100 and 200 mM and is further purified by non-reducing SDS-PAGE on a 3-10~ acrylamide gradient gel as described by Laemmli (Vol. 227, Nature pp. 680-685 (1970)), herein incorporated as reference). A 220 kD
protein with Ac-LDL binding activity was electroeluted from the gel in 0.1~ SDS, 10 mM

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Tris-HCl, pH 8 using an ISCO 1750 electrophoretic concentrator.
i The scavenger receptor protein was also purified by a combination of M-BSA affinity chromatography, and IgG-Dl immunoaffinity chromatography, using an antibody to the scavenger receptor protein. All procedures are performed at 4C. 100 ml of Buffer C (0.1% SDS, 0.1~ sodium deo~ycholate, 1% Nonidet P40, 50 mM Tris-HCl, pH 8, 150 mM NaCl, and 1 mM PMSF) are added to M-BSA
affinity purified proteins from 500 9 of liver or lung (or a smaller amount of THP-l cells) in 100 ml Buffer B. The sample is applied to Sepharose 4B
~Pharmacia) coupled with IgG-Dl (4 mg antibody/ml gel), prepared as described below, at a flow rate of 50 ml/hr, and recycled overnight. The column is washed consecutively with 50 ml of Buffer C, 50 ml of Buffer D (0.2~ Triton X-I00, 10 mM Tris-HCl, pH 8), 50 ml of Buffer D containing 2 M NaCl, and 20 ml of suffer E (40 mM octylglucoside containing 10 mM
Tris-HCl, pH 8). The bound proteins are then eluted with 20 ml of Buffer E containing 2 M guanidine thiocyanate. After elution, the buffer is changed to Buffer A containing 40 mM octyglucoside using PD10 columns (Pharmacia).

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Isolated scavenger receptor protein is then subjected to proteolytic cleavage using serine-, sulfhydryl-, metallo-, or aspartyl proteases to '~ cleave the receptor to remove the membrane spanning domain from the estracellular domain containing the ligand binding site.

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Alternatively, a soluble form of the scavenger receptor protein can be generated by cleaving at the sulfhydryl group in the spacer region using cyanogen bromide ~see, ç.q,, Cross in Meth.
En2_ (Heis, ed.) Academic Press, N.Y. (1967) ~:238).
;

EXAMPL~ 2 Prep~ration of the Soluble Scaven~er Receptor Protein by Recombinant DNA Technology A soluble form of the scavenger receptor protein can also be obtained by using recombinant DNA
technoloqy. Methodology for the production of recombinant soluble scavenger receptor, unless otherwise noted, were standard procedures such as those described in Maniatis et al. (Mole~ll~E
Cls~nin~. A La~oratorY Model (1982) Cold Spring Harbor Laboratory), and Davis et al. (~asic Methods In Molecular Bioloqy (1988) Elsevier Scientific Publishing Co., Inc., NY).
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The nucleic acid seguence of the scavenger receptor protein is determined as follows. Isolated scavenger receptor protein is further purified using i RP-300 (Brownlee Laboratory, Emeryville, CA) reverse phase HPLC. ~he receptor is solubilized in 70~
formic acid. It is then cleaved by cyanogen bromide (CN~r) (25 mg/100 ml solution) at room temperature, overnight, resulting in the formation of a number of cleavage fragments. The fragments are separated chromotographically on the RP-300 column.

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'2~7 One of the fragments is isolated and sub~ected to automated amino acid analysis using an Applied Biosystems Amino Acid Sequencer (Foster City, CA). A second sequence is obtained from a similar CNBr digestion using the gel electrophoresis/
immunoblotting method of Matsudaira (J. Biol. Chem.
(1987) ~2:10035-10038).
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A size fractionated (less than 800 base pairs) cDNA library in lambda ZAPII (Stratogene) is prepared as follows. Poly(Z)+mRNA is isolated by acid guanidium ' thiocyanate/phenol/chloroform extraction as described by Chomczynski. (Anal. Biochem. (1987) 162:156-159) from bovine lung, and is used to construct a random primed cDNA library. The library is screened with pools of a 32p end-labelled 41mer oligonucleotide probes that include 5-fluorodeoxyuridine (F) as described by Habener et al. (Proc. Natl. Acad. Sci.
(USA) (1988) 85:1735-1739), ANTCAGTANlNl TN2TCN2GANlAA
NlGAGGCN2AANl NlTXN1TN2GANG C. For each pool, 5 x 105 plaques are screened by hybridization at 37C and washing at 50C in 6 x SSC (Maniatis et al. ibid. pp.
t~ 447) with 0.1% SDS. Putative positive clones are purified and ln vivo excised into picoblue (pBluscript)-derived plasmids (Short, ibid.). The inserts are screened by Southern blot hybridization (Southern (1975) J. Mol. Biol. 98:503) using the 17mer oligonucleotide probe ARRTTNGCYT CRTTRTC, and the 26mer oligonucleotide probes ATN2CARTANlNl TNlTCNlGANlAA
NlGARGC and ATN2CARTANlNl TNlAGNlGANlAA NlGARGC.

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WO92/1~82 PCT/US92/01370 ~ _~3_ 2 3 0 !~ 21 7 Five positive clones (including pBSR7 an pBSR31~) were sequenced by the dideoxy-chain termination method, all contained sequences encoding peptides I and II. Additional overlapping clones were isolated from an oligo (dT)-primed bovine lung cDNA
library. SEQ. ID NOS: 3 and 4 show the nuclei~ acid sequence and correspondingly deduced amino acid sequence, respectively, of soluble scavenger receptor derived from bovine clones. The Human sequence can also be derived, for example, by the method of Matsumopo et al. (Proc. Natl. Acad. Sci. (l990) 87:9133-9137). The human nucleic acid sequence and amino acid sequence are shown in SEQ. ID NOS: l and 2, respectively.
Vectors for the e~pression of soluble, secreted scavenger receptor type I (bSRI) and type II
(bSRII) are generated as follows. A DNA fragment containing the myelin associated glycoprotein (MAG) leader sequence and a portion of the fibronectin gene is obtained by digesting the vector pMIT (gift from Dr. Richard Hynes, MIT) with BamHl and Xbal. This is ligated with a pCDNAl backbone generated by digestion of pXbSR3 (pCDNAl/Type II) with BamHl/Xbal. The pCDNAl vector is commercially available (Invitrogen);
however the pCDNAl/Type II (pXbSR3) vector shown in FIG. 2A has additional features (see Rohrer et al, Nature). The resulting vector is called pCDNAl/MAG
; (see FIG. 2A).
.: `
The pCDNAl~MAG vector is digested with Xhol, Klenow blunted, and digested with Xbal to yield a linear fragment with a 5' blunt end and a 3' sticky Xbal end. This construction is then ligated to a Smal and Xbal digested polymerase chain reaction (PCR) product described below, to form the construct , . . . . . . . . .......................... . . . .

:

WO9~/l4482 PCT/U592/0l370 2~0~2~7 ", ~.
called pCDNAl/common (see FIG. 2B). The term ~common~ refers to the fact that the PCR product contains sequence common to both the type I and the type II bovine ~cavenger receptor cDNAs.

A region common to secreted bSRI and bSRII
is obtained by utilizing PCR technology as described in E~LTechnology: Principles and Applications for ~A Amplification (Henry R. Erlich, ed.) Stockton Press, 1989, and in Freeman et al (Proc. Natl. Acad.
` Sci. (USA) 87:8B10-8814). The oligonucleotides MKSec5' and MXTK8 are used to generate a 620 base pair fragment by PCR using native pCDNAl-bSRI
(pXbSR7) as a template. This fragment is digested with Smal (site in the primer MkSec5') and Xbal (site in the common scavenger receptor cDNA sequence), and ligated to the pCDNAl-MAG construct as described above to generate pCDNAl/common. The pCDNAl/common encodes at its 5' end the MAG leader sequence which is attached directly to the cDNA which encodes scavenger receptor amino acids 77-227. The construction results in the conversion of the lysine at position 77 of the receptor to the two amino acids, argenine-glycine. This site is e~pected to be the N-terminal amino acid after the MAG-receptor primary translation product is cleaved during translocation into the endoplasmic reticulum.

The remainder of the secreted bSRl is added by obtaining an Xbal-Xbal fragment from the full length pCDNAl/Type I (pXbSR7, see Kodama et al., Nature (1990) 343:531-535) encoding the 3' portion of the gene, and ligating it with the Xbal digested pCDNAl/common vector, resulting in the creation of - . . . .
. -.:
.. -.- ~ ~ , -W092/14482 PCT/US92/0137~ j .

, -the construction called pCDNAl/bSR-I-sec (see FIG.
2C). The vector pCDNAl/bSR-II-sec is generated identically using an Xbal-Xbal fragment from pCDNAl/Type II (psbSR3) as described by Rohrer et al.
(Natu~e (1990) ~ 531-535).
i The region encoding the secreted bSRI
including the MAG leader is escised from pCDNAl/bSR-I-sec with HindIII and ligated with a HindIII digested pRc/CMV (Invitrogen) backbone as shown in FIG. 2D. Secreted bSRII is transferred to PRc/CMV using an identical strategy.

CH0 host cells are transfected with the pRc/CMV/bSR-I-sec vector using the polybrene method described in Maniatis et al. (Molecular Cloning. A
Laboratory Manual, Cold Spring Harbor Laboratory, 1982, pp. 16-47~. Neomycin-resistant cells are selected using G418 (Gibco), a neomycin analog.
G418-resistant colonies are picked at random and screened for e~pression of the protein product using a 30 minute pulse with 400 ~Ci/ml 35S-methionine followed by lysis and immunoprecipitation with an anti-peptide antibody which was raised against a peptide in the Cys rich domain.
. :
Media from a positive colony are esamined for the presence of secreted bSR-I as follows. The cells are grown in the presence of 80 ~Ci/ml 35S-methionine for 5 hours, at which time the media is harvested. PMSF was added to 1 mM and leupeptin to O.l mM. ~he media is then centrifuged at 1500 s g ifor 15 minutes to remove cellular debris. The ~ -' : .:

.- " .' ' '-'. ': , ~ ''' ' ' '' ',' - : '.'- ''' .
. .: " .

WO92/1~82 PCT/US92/01370 ;,; ~'' i `~ 2194~7 labelled media is diluted 3:1 with buffer A (20 nM
Tris, pH 8.0, 150 mM NaCl, l mM CaCl2) containing 2 mg/ml bovine serum albumin. To this is added 25 ~I
AG-PolyG beads ~Pharmacia) which had been washed in buffer A. This mi~ture is vortesed and placed on a rotator at 4C overnight. The beads are then washed twice with buffer A and protein eluted by the addition of 30 ~l sample buffer and boiling. The eluate is run on an 8% Laemmli gel, which was dried and esposed to pre-flashed Koda~ XR7 film. A band of 72 kD is seen in transfected cells but not in untransfected CHO cells.

Dem~nstration of Utility The utility of the soluble scavenger receptor protein is determined by measuring its ability to block scavenger receptor-mediated cellular metabolism of known scavenger receptor ligands (such as radiolabelled endoto~in or l251-AcLDL). That the soluble receptor protein is inhibited by the same ligands as the membrane-bound form is demonstrated by the ability of such ligands to interfere with the binding of radioactively or otherwise labelled soluble forms of the receptor to PolyG beads (Pharmacia). For e~ample, the inhibitors poly G, poly I, malelyated BSA, and AcLDL are successful competitors at 400 ~g/ml, while poly C, LDL, and ~SA
fail to compete. This demonstrates that the soluble receptor protein has similar binding specificity and and hence utility as the full-len~th, membrane-bound , "

,' ~

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~ 27- 2iO~2l~

form. This bead-binding assay can be used to measure the association of endotosin, lipid IYA, and similar molecules, to soluble forms of the cavenger receptor.

The ability of the scavenger receptor to bind lipid IVA ls determinod by the methods of Raetz et al. (Cold Spri~g Harbor S~mp. Ouant. Biol. (1988) 53:973-982) and Hampton et al. (~. ~iQl~_Chem~ (1988) 263:1480~-14807).

The binding activity of the soluble receptor protein is also measured by filter binding and ligand blotting assays performed with minor modification, according to the methods of Schneider et al. (ibid.) and Daniel et al. (Vol. 25B, ~ Biol. Chem. pp.
4606-4611 (1983)), respectively, herein incorporated as reference. Ligand binding specificity is also determined by polynucleic acid affinity chromatoqraphy. M-BSA-purified soluble proteins having endoto~in-related-sustance binding activity in 4 ml of buffer containing 40 mM octyl glucoside, are applied to polynucleic acid coupled agarose columns (AG-POLY series, prepacked column, Pharmacia). After washing with the same buffer, the bound protein is removed with 5 ml of elution buffer. It is this protein that is useful as a therapeutic agent in the treatment of endotosemia.
. . .
It can be seen from the foregoing description and esamples that soluble forms of the scavenqer receptor protein can be prepared by purification from various appropriate mammalian tissues followed by proteolytic cleavage, or by recombinant DNA methodologies. Because these : . . : -.: . . , ~ : ., . ~

~ I
. -2~-21042~7 proteins can bind endoto~in-related substances, they are useful as therapeutic agents in sequestering and promoting the clearance of endoto~ins and other endoto~in-related substances found in the circulation of a subject suffering from septic shock.
, The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the :
meaning and range of equivalency of the claims are therefore intended to be embraced therein.
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Claims (21)

I Claim:

1. A method for treating endotoxemia comprising:
administering to a patient in need of therapy an effective amount to bind endotoxin of a polypeptide fragment including the collagen binding region of the extracellular portion of a substantially pure macrophage scavenger receptor protein, the macrophage scavenger receptor protein being naturally present on the surface of macrophages where it is formed from three subunits each having an apparent molecular weight on SDS-polyacrylamide gels of about 77,000 daltons, the receptor protein when glycosylated having an apparent molecular weight on SDS-polyacrylamide gels of about 220,000 daltons, and a binding capacity for acetylated low density lipoprotein of at least 1.4 mg of Acetyl-LDL protein/mg receptor protein.

2. The method of claim 1 wherein said fragment of the scavenger receptor protein further includes the spacer domain.

3. The method of claim 1 wherein said fragment further comprises the .alpha.-helical coiled coil domain.

4. The method of claim 1 wherein said fragment further comprises the cysteine-rich domain.

5. The method of claim 1 further comprising cleaving said fragment by biochemical means from substantially purified native scavenger receptor protein.

6. The method of claim 1 further comprising expressing the fragment from an isolated DNA fragment encoding the scavenger receptor protein in an appropriate host expression system.

7. The method of claim 1 wherein said fragment has an affinity for acetylated low density lipoprotein of about 0.5 to 5.0 microgram(s) protein per milliliter.

8. The method of claim 1 wherein said fragment can bind chemically-modified low density lipoprotein.

9. The method of claim 1 wherein said fragment can bind a negatively-charged macromolecule selected from the group consisting of polyvinylsulfate, maleyl-BSA, fucoidan, and purine polynucleotides, and poly[G].

10. The method of claim 1 wherein said fragment binds the Lipid A portion of endotoxin.

11. The method of claim 1 wherein said fragment binds gram-negative bacteria.

12. A pharmaceutical composition for treating endotoxemia, comprising:
a polypeptide fragment including the collagen binding region of the extracellular portion of a substantially pure macrophage scavenger receptor protein, wherein the macrophage scavenger receptor protein is present on the surface of macrophages where it is formed from three subunits each having an apparent molecular weight on SDS-polyacrylamide gels of about 77,000 daltons, the receptor protein when glycosylated having an apparent molecular weight on SDS-polyacrylamide gels of about 220,000 daltons, and a binding capacity for acetylated low density lipoprotein of at least 1.4 mg of Acetyl-LDL protein/mg receptor protein; and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition of claim 12 wherein said fragment further comprises the spacer domain.

14. The pharmaceutical composition of claim 12 wherein said fragment further comprises the .alpha.-helical coiled coil domain.

15. The pharmaceutical composition of claim 12 wherein said fragment further comprises the cysteine-rich domain.

16. The pharmaceutical composition of claim 12 wherein said fragment is expressed from a DNA fragment encoding a protein consisting essentially of the scavenger receptor protein in an appropriate expression system.

17. The pharmaceutical composition of claim 12 further characterized by having an affinity for acetylated low density lipoprotein of about 0.5 to 5.0 microgram(s) protein per milliliter.

18. The pharmaceutical composition of claim 12 further characterized by the ability to bind a negatively-charged macromolecule selected from the group consisting of polyvinylsulfate, maleyl-BSA, fucoidan, and purine polynucleotides, and poly[G].

19. The pharmaceutical composition of claim 12 further characterized by the ability to bind chemically-modified low density lipoprotein.

20. The pharmaceutical composition of claim 12 further characterized by binding the Lipid A moiety of endotoxin.

21. The pharmaceutical composition of claim 12 further characterized by binding gram-negative bacteria.