WO1997000081A1 - A method for inhibiting bacteremia and bacterial dissemination - Google Patents

Abstract

A method of inhibiting the dissemination of bacteria into and from the bloodstream is described. The method is useful in the prevention of peritonitis and bacteremia.

Description

A METHOD FOR INHIBITING BACTEREMIA AND BACTERIAL DISSEMINATION

This application is a continuation-in-part of Serial No. 08/491,759, filed June 19, 1995, and incorporated by reference in its entirety. The following related applications are also incorporated by reference in their entirety: 08/165,583, filed December 13, 1993; 08/231,855, filed April 22, 1994; and 08/254,095, filed June 6, 1994.

Field of the . Invention This invention relates to a method of preventing or treating bacteremia, and subsequent dissemination of microorganisms from the blo dstream into other tissues.

BACKGROUND OF THE INVENTION Description of the Background Art

Bacterial infection occurs when a pathogenic bacterium enters the host and multiplies inside the body. Most infections begin on the mucous membranes of the respiratory, alimentary, or genitourinary tracts, or in tissues exposed by wounds. After initial entry, the bacteria may remain localized, or they may spread through the blood and lymph systems, resulting in the infection of diverse tissues.

This bacterial dissemination is undesirable. Dissemination may be inhibited either by interfering with the transfer of the bacteria from the local seat of infection to the systemic circulation, or by speeding the clearance of the bacteria from the blood and lymph systems.

If bacteria are allowed to disseminate into the circulation, and are not cleared quickly enough, the bacterial levels in the bloodstream will become higher than normal, a condition known as "bacteremia" .

Gram-negative bacteria produce lipopolysaccharides (LPS) as part of the outer layer of their cell walls. When released, such as by lysis of the bacteria, they have a toxic effect, and hence are referred to clinically as "endotoxins" .

If bacteremia persists, the patient may develop "septicemia". This is a life-threatening condition attributable to the body's reaction to high levels of bacterial endotoxins.

Cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor (TNF-o.) are known to promote the invasiveness of pathogenic bacteria, particularly gram-negative bacteria. It is known that IL-6 and TNF-α, as well as other inflammatory mediators, are released by the body in response to activation of the immune system by LPS endotoxins. Tracey, et al. , Ann. Rev. Med. 45: 491-503 (1994) ; Akira, et al. , Adv. Immunol. 54: 1-78 (1993) ; Bone, R.C., Ann. Intern. Med. 115: 457 (1991) . Activation of the immune system by endotoxin/LPS can result in a cascade of reactions including the production of pro-inflammatory cytokines such as IL-6 and TNF-a. Activation of the immune system by endotoxin/LPS can result in a cascade of reactions including the production of proinflammatory cytokines such as IL-6, IL-1 and TNF-o.. This initial production of cytokines may lead to a cascade of events which can includ some or all of the following: bacteremia and bacterial dissemination, procoagulant activity, acute respiratory distress syndrome and death. This activation is mediated in part by interactions of various molecular receptors with foreign antigen. Several monocyte/macrophage surface markers that possess receptor and signal transduction functions have been identified. Many of them are cell differentiation markers, i.e., characteristically present in defined stages of development, especially the end stages of cells of defined lineage and function.

One such marker, CD14, is a 55-kD glycoprotein expressed strongly on the surface of monocytes and weakly on the surface of neutrophils. Goyert, et al., J. Immunol. 137: 3909 (1986) ; Haziot, et al. , J. Immunol. 141: 547-552 (1988); Goyert, et al. , Science 239: 497 (1988) . CD14 is linked by a cleavable phosphinositol tail (Haziot, et al . , J. Immunol. 141: 547-552

(1988)) to the exoplasmic surface of mature monocytes, macrophages, granulocytes and dendritic reticulum cells of renal nonglomerular endothelium, and hepatocytes in rejected liver. A soluble form of CD14 is present in normal sera, i.e., about 2 to 4 μg/ml, and in the urine of nephrotic patients. Bazil, et al. , Eur. J. Immunol. 16: 1583 (1986) . CD14 has been shown to bind to LPS. Haziot, et al.,J. Immuno. 151: 1500 (1993) . The binding causes cells to become highly activated and release interleukins, TNF-o., and other substances that promote the growth and invasiveness of bacteria, enabling the dissemination of bacteria into the bloodstream or into the peritoneal cavity. Another mechanism by which bacteria can disseminate into the blood stream involves interference with the body's normal clearing mechanisms to remove bacteria. Some bacteria, such as E. coli and Salmonella spp; produce endotoxins that interfere with the body's bacterial clearance mechanisms that depend largely on fixed macrophages lining the sinusoids of a number of parenchymatous organs, especially the liver and spleen. Interference with cellular defenses that otherwise would destroy large numbers of bacteria in the bloodstream enables bacteria to rapidly disseminate, and can lead to death. The peritoneal cavity can be an important pathway for bacterial dissemination. The parietal peritoneum is a membrane lining the walls of the abdominal and pelvic cavities. The visceral peritoneum is a similar membrane investing the contained viscera. Together the two membranes define an enclosed space known as the peritoneal cavity.

Bacteria can enter the peritoneal cavity as a result of perforation of the GI tract, infection of an intraabdominal organ, and direct contamination from an external source, such as by trauma, burns or surgery. Once bacteria enter the peritoneal cavity, dissemination is rapid. Within 6 minutes of intraperitonal inoculation of bacteria in dogs, thoracic lymph is culture-positive; within 12 minutes, there is bacteremia (bacteria at elevated levels in the bloodstream) .

Peritonitis is an inflammation of the peritoneum, often attributable to a severe local infection. It may be caused by a number of pathogenic microorganisms, and can result from gastrointestinal trauma, including surgery or peritoneal dialysis. Typical medical treatments for the prevention of peritonitis include antibiotic therapy, especially prior to surgical procedures, radio therapy or chemotherapy. This approach is hindered, however by the multiple drug resistance of many of the bacteria known to cause peritonitis. Moreover, since peritonitis may be caused by both gram-positive and gram-negative microorganisms, the choice of antibiotic may not be sufficient. Also, antibiotic treatment is non-specific, eliminating many non-pathogenic indigenous microorganisms which normally prevent bacterial disease through bacterial antagonism, especially in the gastrointestinal tract. When broad spectrum antibiotics, such as tetracyclines, are given in large doses for many days, growth of most of the bacteria that thrive in the intestinal tract is suppressed. As a result, antibiotic resistant strains of pathogenic microorganisms, normally held in check by the antagonistic action of the coliforms and other organisms, multiply freely and can actually foster peritonitis, rather than prevent it.

There is, therefor, a need for a non-invasive, effective method for the prevention and treatment of bacteremia, particularly in the treatment of severe local infections, such as those associated with peritonitis, pneumonia, gastrointestinal colitis, dysentery, severe cellulitis, urinary tract infections, bacterial translocation from the gastrointestinal tract, inflammatory bowel disease, hemorrhagic shock, burn infections, etc.

SUMMARY OF THE INVENTION

The present invention provides a method for controlling severe local infections, interfering with bacterial dissemination from local infections to other parts of the body, and preventing and treating bacteremia and septicemia.

The patient is given a drug which acts as an antagonist to cellular CD14. The CD14 antagonist may be a substance which competes with cellular CD14 for CD14 ligands, such as LPS, or it may be a substance which binds to cellular CD14 and interferes with the latter's binding to relevant ligands, such as LPS. When the antagonist is one that competes with cellular CD14, it is preferably a soluble fragment of CD14, or a mutant thereof. When the antagonist is a substance which binds CD14, it may be an antibody, a peptide, or other CD14-binding molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure IA is a bar graph of percent survival of control and CD14-deficient mice following intraperitoneal (i.p.) inoculation of Salmonella minnescia LPS. Figure IB is a bar graph of the amount of TNF- a (ng/ml) in the blood stream of control and CD14-deficient mice following i.p. inoculation of S_j_ minnescia LPS.

Figure IC is a bar graph of the amount of serum IL-6 (ng/ml) in control and CD14-deficient mice following i.p. inoculation of S_j_ minnescia LPS.

Figure 2A is a bar graph of the per cent survival of control and CD14 deficient mice following i.p. inoculation of viable E. coli.

Figure 2B is a bar graph of the amount of serum TNF-o. (ng/ml) present in control and CD14-deficient mice following i.p. inoculation of viable E. coli.

Figure 2C is a bar graph of the amount of serum IL-6 (ng/ml) present in control and CD14-deficient mice following i.p. inoculation of viable E. coli. Figure 3A is a graph of serum concentrations of TNF-α (ng/ml) in E. coli infected control and CD14-deficient mice over a seven hour period.

Figure 3B is a graph of serum concentration of IL-6 (ng/ml) in E. coli infected control and CD14-deficient mice over a seven hour period.

Figure 4A is a bar graph of E. coli CFU/ml blood in control and CD14 deficient mice seven hours after i.p. inoculation of E. coli (IO⁷ CFU) .

Figure 4B is a bar graph of E. coli CFU/ml blood in control and CD14 deficient mice seven hours after i.p. inoculation of E. coli (10⁵ CFU) .

Figure 4C is a bar graph of E. coli CFU/ml lung in control and CD14 deficient mice seven hours after i.p. inoculation of E. coli (IO⁷ CFU) . DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

It is known that endotoxin/LPS, a component of the outer membrane of gram-negative bacteria activates the immune system. Several receptors for endotoxin/LPS have previously been identified and characterized, including CD14, the cell surface glycoprotein expressed on the surface of monocytes and neutrophils. However, the role of these receptors in bacteremia and particularly, peritonitis, was not heretofore known. Applicant has discovered that CD14 deficient mice are more resistant than normal mice to intraperitoneally administered purified LPS (Example 1) and gram-negative bacteria (Example 2) , and exhibit reduced serum levels of TNF-alpha, IL-6 and IL-1. Moreover, the blood and organ (lung) counts of bacteria were lower in CD14-deficient mice than in similarly challenged normal mice. (Example 5) . Finally, CD-14 deficient mice with simulated peritonitis are more likely to survive than normal mice (Example 6) .

This suggests that bacteremia can be prevented or treated by administering to a patient a CD14 "antagonist", that is, a substance that interferes with the ability of the bacteria to interact, directly or indirectly, with CD14 and thereby trigger a metabolic or immunologic reaction that leads (e.g., through cytokine release) to increased bacterial dissemination.

Soluble CD14 Analogues

In one embodiment, the CD14 antagonist is a soluble analogue (sCD14) of cellular CD14. The analogue acts as an antagonist by "decoying", that is, the bacterial LPS binds the analogue rather than cellular CD14. This analogue may be a soluble fragment of a naturally occurring cellular CD14, or a substantially homologous mutant or other functional derivative of CD14 (or a fragment thereof) . Preferably, the analogues retain at least 10% of the LPS (or other relevant ligand) -specific binding activity of cellular CD14. As contemplated herein, sCD14 may be naturally occurring human soluble CD14 purified by methods known in the art or it may be recombinant sCD14, made by the method described in Applicant's co-pending application Serial No. 08/254,095 or any other art-known method. By "naturally occurring" is meant any form of CD14 found in the human population that is biologically functional. Functional derivatives of sCD14 include any sCD14 molecules that have been modified by such means as, for example, alteration in amino acid sequence or alteration in the glycosylation pattern thereof, but which molecules retain the ability to bind to LPS.

Fragments of CD14 useful in the present invention include all soluble fragments of CD14 that retain the relevant biological properties of the parent molecule, e.g., ability to bind to LPS present on the outer membrane of bacteria. As used herein, the term "fragment" refers to an isolated polypeptide sequence corresponding to a portion of the CD14 amino acid sequence. The fragments may be generated by direct proteolytic digestion of CD14 or by expression of genetically engineered nucleic acid sequence that encodes a portion of the full size CD14 polypeptide. Alternatively, fragments of sCD14 may be synthesized from amino acids by art known methods of peptide synthesis. A preferred fragment of sCD14 is the peptide comprising the amino acid sequence:

ALA GLU LEU GLN GLN TRP LEU LYS PRO GLY LEU LYS VAL LEU SER ILE ALA GLN ALA HIS SER LEU ASN PHE SER CYS (SEQ ID NO:l) . This fragment corresponds to amino acids 143 to 168 of the sCD14 sequence.

CD14 is a glycophosphoinositol (GPI) anchored membrane protein. A soluble CD14 analogue will omit the GPI anchor. It may contain the full-length amino acid sequence of CD14, or only a portion thereof. It may also differ from the native amino acid sequence by substitution, insertion, or internal deletion mutations, or by fusion of additional amino acid sequences to either terminus.

The carboxy terminal of CD14 contains the signal (believed to be at least about 15 amino acids long) which directs mammalian cells to add on the GPI anchor after translation of CD14. The identity of the attachment signal is readily determined by systematically expressing truncation mutants of CD14 and testing for the presence of the GPI anchor. Some soluble CD14 occurs naturally, most likely as a result of proteolytic cleavage of the membrane CD14, leaving the GPI anchor behind and releasing a soluble fragment.

Soluble CD14 can be prepared by a number of means, including (1) nonbiological synthesis of CD14 (including full-length CD14) without the GPI anchor, (2) chemically or enzymatically removing the GPI anchor from GPI-linked CD14, e.g., with a glycosidase and/or lipidase; (3) semi-synthesis, wherein the carboxy terminal

(including the GPI anchor) is removed from GPI-linked CD14 and the fragment condensed with a replacement peptide restoring the carboxy terminal lacking the GPI anchor, (4) biological synthesis of a CD14 fragment lacking the GPI attachment signal, as a result of expression of a corresponding gene fragment; and (5) biological synthesis of CD14 in a biological system that does not recognize the GPI attachment signal, and hence does not link GPI to the polypeptide.

Functional soluble fragments are readily identified by systematically fragmenting CD14 and testing the fragments for LPS or other relevant CD14-like ligand-binding activity. It is not necessary that every possible fragment be prepared and tested. Rather, the skilled practitioner will designate candidate fragmentation points within the CD14 molecule. These fragmentation points may be spaced uniformly or positioned in the light of the known sequence. For example, the fragmentation points may be chosen to lie between conserved regions, regions of high (or low) hydrophilicity, or regions likely to have significant secondary structure. If none of the fragments tested has activity, the skilled practitioner will test larger fragments. Once a functional fragment has been identified, subfragments may be tested to more finely delineate the smallest fragment which is functional. Preferably, at least one of the fragments tested retains the sequence corresponding to amino acids 143-168, which applicant has found to inhibit LPS binding, by CD14, and/or 57-64, a putative LPS-binding site of CD14 identified by Juan, et al . , J. Biol. Chem., 270:5219-24 (1995) . If desired, instead of starting with the full length sequence, and fragmenting, one may start -.'ith the putative LPS-binding site, and extend in either the N- or C- direction, or both, until a functional fragment is obtained.

Any desired fragment may be made by expressing a corresponding gene. If the fragments are generated by treating a full length CD14 with proteolytic enzymes, the fragments obtained will be a function of the enzyme selected. The enzymes vary in site specificity, and hence in the number and position of the fragments generated.

The analogues are not limited to derivatives of human, or even mammalian, CD14s. The CD14 sequence may be mutated provided the mutant substantially retains the desired solubility and binding activity. Preferably, the amino acid sequence is at least 50%, more preferably at least 80%, identical with the corresponding CD14 sequence, the compared sequences being at least 20 amino acids long. A protein is more likely to tolerate a mutation which

(a) is a substitution rather than an insertion or deletion;

(b) an insertion or deletion at the terminus, rather than internally; or, if at an internal residue, at a domain boundary or at a "turn" or "loop";

(c) affects a surface residue rather than an interior residue;

(d) affects a part of the molecule distal to the binding site; (e) is a substitution of one amino acid for another of similar size, charge, and/or hydrophobicity; and (f) is at a site which is subject to substantial variation among a family of homologous proteins to which the protein of interest belongs . These considerations can be used to design functional mutants.

Surface vs . Interior Residues

Charged residues almost always lie on the surface of the protein. For uncharged residues, there is less certainty, but in general, hydrophilic residues are partitioned to the surface and hydrophobic residues to the interior.

Surface residues may be identified experimentally by various labeling techniques, or by 3-D structure mapping techniques like X-ray diffraction and NMR. A 3-D model of a homologous protein can be helpful.

Binding Si te Residues

Residues forming the binding site may be identified by (1) comparing the effects of labeling the surface residues before and after complexing the protein to its target, (2) labeling the binding site directly with affinity ligands, (3) fragmenting the protein and testing the fragments for binding activity, and (4) systematic mutagenesis (e.g., alanine-scanning mutagenesis) to determine which mutants destroy binding. If the binding site of a homologous protein is known, the binding site may be postulated by analogy.

Protein libraries may be constructed and screened that a large family (e.g., 10⁸) of related mutants may be evaluated simultaneously.

Conserved and Unconserved Residues

If several proteins are known which have the desired binding activity, that activity is likely to be attributable to conserved segments of the proteins, i.e., segments which are similar or identical.

In the case of CD14, one may compare homologous sequences, such as human and mouse CD14, to identify regions likely to be involved in LPS binding.

It is well known that certain amino acid substitutions are more often tolerated than others, and these are often correlatable with similarities in size, charge, etc. between the original amino acid and its replacement.

Conservative Substi tutions

Conservative substitutions are herein defined as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly

II. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gin III. Polar, positively charged residues: His , Arg , Lys

IV. Large, aliphatic, nonpolar residues: Met, Leu, Ile, Val, Cys

V. Large aromatic residues Phe, Tyr, Trp

Within the foregoing groups, the following substitutions are considered "highly conservative" : Asp/Glu His/Arg/Lys Phe/Tyr/Trp Met/Leu/Val

Semi-conservative substitutions are defined to be exchanges between two of groups (I) - (V) above which are limited to supergroup (A) , comprising (I) , (II) , and (III) , above, or to supergroup (B) , comprising (IV_ and (V) above. Also, Gly and Ala are considered to be semi-conservative substitutions for any other residue. Non-conservative substitutions are those which are neither conservative nor semi-conservative.

It will be appreciated that highly conservative substitutions are less likely to affect activity than other conservative substitutions, conservative substitutions are less likely to affect activity than merely semi-conservative substitutions, and semi-conservative substitutions less so than non-conservative substitutions. Although a substitution mutant, either single or multiple, of the peptides of interest may not have quite the potency of the original peptide, such a mutant may well be useful. If desired, the effects of many different mutations on any binding activity may be explored simultaneously by constructing and testing a combinatorial library.

Use of Non -Genet ically Encoded Amino Acids

Substitutions are not limited to the naturally occurring amino acids. When the peptides are prepared by peptide synthesis, the desired amino acid may be used directly. Alternatively, a genetically encoded amino acid may be modified by reacting it with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Aromatic amino acids may be replaced with D- or L- naphthylalanine, D- or L-phenylglycine, D- or L-thieneylalanine, D- or L-1-, 2- 3-, or 4-pyreneylalanine, D- or L-3- thieneylalanine, D- or L- (2-pyridinyl) -alanine, D- or L-(3- pyridinyl) -alanine, D- or L- (2-pyrazinyl) -alanine, D- or L-(4- isopropyl) -phenylglycine, D- (trifluoromethyl) -phenylglycine, D- trifluoromethyl) -phenylalanine, D-p-fluorophenylalanine, D- or L- p - b ipheny1 pheny1 a 1 an i ne , D- or L-p- methoxybiphenylphenylalanine, D- or L-2-indole- (alkyl)alanines, and D- or L-alkylalanines where alkyl may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isobutyl, isopentyl, non-acidic amino acids of 1-20 carbon atoms.

Acidic amino acids can be substituted with non- carboxylate amino acids while maintaining a negative charge, and derivatives or analogs thereof, such as the non-limiting examples of

(photophone) -alanine, glycine, leucine, isoleucine, threonine, or serine; or sulfated (e.g., -S0₃H) threonine, serine, tyrosine.

Other substitutions include unnatural hydroxylated amino acids made by combining "alkyl" (as defined and exemplified herein) with any natural amino acid. Basic amino acids may be substituted with alkyl groups at any position of the naturally occurring amino acids lysine, arginine, ornithine, citrulline, or (guanidino) -acetic acid, or other (guanidino)alkyl-acetic acids, where "alkyl" is as defined above. Nitrile derivatives

(i.e., containing the CN moiety in place of COOH) may also be substituted for asparagine or glutamine, and methionine sulfoxide may be substituted for methionine. Methods of preparation of such peptide derivatives are well known to one skilled in this art. In addition, any amide linkage can be replaced by a ketomethylene moiety, e.g., (-C(=0) -CH2-) for (- (C=0) -NH-) . Such derivatives are expected to have the property of increased stability to degradation by enzymes, and therefore possess advantages for the formulation of compounds which may have increased in vivo half lives when administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.

In addition, any amino acid can be replaced by the same amino acid but of the opposite chirality. Thus, any amino acid which occurs naturally in the L-configuration (which may also be referred to as the R or S configuration, depending upon the structure of the chemical entity) may be replaced with an amino acid of the same chemical structural type, but of the opposite chirality, generally referred to as the D-amino acid but which can additionally be referred to as the R- or S-, depending upon its composition and chemical configuration. Such derivatives have the property of greatly increased stability to degradation by enzymes, and therefore are advantageous in the formulation of compounds which have longer in vivo half lives when administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.

Additional amino acid modifications of amino acids may include the following: Cysteinyl residues may be reacted with alpha-haloacetates (and corresponding amines) , such as 2- chloroacetic acid or chloroacetamine, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues may also be derivatized by reaction with compounds such as bromotrifluoroacetone, alpha-bromo-beta- (5-imidazoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2- pyridyl; disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-r-nitrophenol, or chloro- 7- nitrobenzo-2-oxa-l,3-diazole. Histidyl residues may be derivatized by reaction with compounds such as diethylprocarbonate, e.g., at pH 5.5-7.0, because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used, e.g., where the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides. Derivatization with these agents is expected to have the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include compounds such as imidoester, e.g., methyl picolinimidate pyridoxal phosphate; pyridoxal chloroborohydride trinitrobenzenesulfonic acid; O-methylisourea; 2,3-pentanedione and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3- butanedione, 1,2-cyclohexandedione and ninhydrin according to known method steps. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon- mino group. The specific modification of tyrosyl residues per se is well known, such as for introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. N-acetylimidazol and tetranitromethane may be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides, (R'-N-C-N- R' ) suchas 1-cyclohexyl-3- (2-morpholinyl) - (4-ethyl)carbodiimide or 1- ethyl-3- (4-azonia-4,4-dimethyIpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be readily deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues may be deamidated under mildly acidic conditions. Either form of these residues falls within the scope of the present invention.

Derivatization with bifunctional agents is useful for crosslinking the peptide to a water-insoluble support matrix or to other macromolecular carriers, according to known method steps. Commonly used crosslinking agents include, e.g., 1,1- bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, for example, esters with 4- azidosalicylic acid, homobifunctional imidoesters, including di succ inimidyl esters such as 3 , 3 ' - dithiobis(succinimidylpropionate) , and bifunctional maleimides such as bis-N-maleimido-1, 9-octane. Derivatizing agents such as methyl-3- [ (p-azidophenyl)dithio]pro ioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices can be used, such as cyanogen bromide- activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440, all of which are hereby incorporated in their entirety by reference.

Other modifications include hydroxylation of proline and lysine phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (Creighton, T.E., Proteins: Structure and Molecular Properties. W.H. Freeman & Col, San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, methylation of main chain amide residues (or substitution with N- methyl amino acids) and, in some instances, amidation of the C- terminal carboxyl groups, according to known method steps. Glycosylation is also possible.

Derivatized moieties may improve the solubility, absorption, biological half life, and the like or eliminate or attenuate any possible undesirable side effects of the molecules. Moieties capable of mediating such effects are disclosed, for example in Remington's Pharmaceutical Sciences, 16th ed. , Mack Publishing Co., Easton, PA (1980).

Modifications are not limited to the side chains of the amino acids. One may also modify the peptidyl linkage itself, e.g., -NRCO- (where R is alkyl or aryl), instead of -NHCO-, as in the so-called "peptoids."

The peptides may also comprise isoteres of two or more residues in the original peptide. An isotere as defined here is a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence. The term specifically includes peptide backbone modifications well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the α-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. See, generally, Spatola, Chemistry and Biochemistry of Amino Acids, peptides and Proteins, Vol. VII (Weinstein ed. , 1983) .

It is also possible to construct and use so-called peptide mimetics whose conformation is similar to that of a peptide but do not have a peptide-like molecular formula. In effect, in a mimetic, all of the residues of the peptide are replaced by one or more isoteres as defined above.

Chimeras form a special class of mutants. The chimeras may be of e.g., different mammalian CD14s, or of CD14 and another endotoxin-binding protein, such as bacterial permeability- increasing protein. See Marra, et al. , Critical Care Medicine 22:559 (1994); Fisher, Jr., et al. , Critical Care Medicine, 22:553 (1994) .

Anti-idiotype antibodies may also be used as CD14 analogues when the parental antibody occludes the LPS binding site of CD14. The comments made herein regarding mutation and derivatization of CD14 analogues should be taken to apply, mutatis mutandis, to other CD14 antagonists. CD14 Binding Molecules

The CD14 antagonist may also be a molecule which binds CD14. In this case, the antagonistic effect is attributable to the drug occupying, or sterically hindering access to, the LPS (or other relevant ligand) binding site of cellular CD14. One class of such CD14 antagonists are anti-CD14 antibodies. The antibodies of interest include both whole antibodies, and antigen-binding derivatives of whole antibodies, such as F(ab)₂, Fab', Fab, Fv, V_H and V_L. In some cases it may be advantageous to construct a hybrid antibody that binds two different epitopes.

Anti-CD14 antibodies, whether polyclonal or monoclonal, can be made by any art-known method. For example, polyclonal antibodies may be prepared in a conventional manner, by using the subject polypeptide, e.g. CD14, as an immunogen and injecting the polypeptide into a mammalian host, e.g.,mouse, cow, goat, sheep, rabbit, etc., particularly with an adjuvant, such as, for example, complete Freund 's adjuvant, aluminum hydroxide gel, or the like. The host may then be bled and the blood used for isolation of polyclonal antibodies. Alternatively, monoclonal antibodies may be made by fusing peripheral blood lymphocytes (B-cells) of the immunized host with an appropriate myeloma cell to produce an immortalized cell line that secretes monoclonal antibodies specific to CD14.

Additional (yet different) monoclonal antibodies which bind to the same antigen, and, if desired, the same epitope of that antigen, as bound by a lead antibody may be obtained by one or more of the following procedures.

A. Immunizing animals (of the same or different species) with the same preparation used initially to obtain the lead antibody. B. Immunopurifying the antigen recognized by the lead antibody and using the immunopurified antigen to immunize animals and obtain a new round of antibodies.

C. Characterizing the epitope recognized by the lead antibody and incorporating the epitope into a synthetic immunogen, which is then used to immunize animals.

D. Producing conservatively mutated antibodies by recombinant DNA techniques.

In a preferred embodiment, messenger RNA is extracted from the hybridoma. The mRNA is sequenced directly, see Griffiths and Mulstein, Hybridoma Technology in the Biosciences and Medicine, Chap. 6, pp. 103-115 (1985) to determine both the light and heavy variable domain encoding sequences. Oligonucleotides hybridizing in the constant domains of the kappa and gamma chains are used as primers to synthesize first strand cDNAs, which are then used as templates for PCR amplification of the variable domain. Suitable PCR primers are chosen based on study of the mRNA sequence.

These light and heavy chain-encoding sequences may now be mutated to produce new antibodies of similar but different reactivity relative to the parental antibody. The mutated sequences may be introduced into full-length (or deliberately truncated) heavy and light chain genes by cassette mutagenesis. Vectors carrying these sequences may be used to transform suitable host cells. The heavy and light chains may be co- expressed, or expressed separately and combined in vitro. The constant region of the heavy and light chains may be replaced, if desired, by the corresponding portions of human heavy and light chains. Such chimeric antibodies are likely to elicit a smaller HAMA response than the original mouse antibody. Framework residues in the variable regions may likewise be "humanized." Merck, EP 438,310; Winter, EP 239,400; Queen, WO92/11018; Celltech, WO91/09967 and WO91/09968; Wellcome Foundation, WO92/07075; Gorman, WO92/05274. General references on recombinant production of monoclonal antibodies include Schering, EP 88,494; Genentech, Inc., EP 125,023; Intl. Gen. Eng'g, WO87/02671; Gillies, USP 4,663,281; Kudo, USP 5,101, 024; Moore, USP 4,642,334; Kudo, USP 4,786,719. In one embodiment, a set of single substitution mutants are prepared by site-specific mutagenesis, so that each of the hypervariable residues is conservatively mutated in one of the mutants.

In another embodiment, a V_H or V_L gene is randomly mutated at predetermined "variable" codons and the mutant domain is displayed on the surface of phage. These "antibody phage" libraries are then screened for binding activity. At any given variable codon, the allowed amino acids may be any of 2-20 genetically encoded amino acids, and may (and preferably do) include the wild-type amino acid. The frequence of occurrence may be, e.g., such that all allowed amino acids occur with substantially equal frequency, or such that the wild-type amino acid (or some class of amino acid) predominates. In a preferred embodiment, at least 20 residues are mutated so each variable residue may be wild-type, or a single possible conservative substitution, and the frequencies of occurrence are chosen so single substitution mutants will predominate. General references on preparation of "antibody phage" include McCafferty, et al., Nature, 348:552-54 (1990) ; Garrard, et al. , Bio/Technology, 9:1373-77 (1991) ; Huse, et al. , J. Immunol., 149:3914-20 (1992) ; Kang, et al. , PNAS, 88:4363-66 (1991) ; Hoogenboom, et al. , Nucleic Acids Res., 19:4133-37 (1991) ; Marks, et al. , J. Mol. Biol., 222:581-97 (1991) ; Burton et al. , PNAS, 88:1934-37 (1991) ; Clackson et al . , Nature, 352:624-628 (1991) ; Barbas, et al. , PNAS, 89:9339-43 (1992) ; Zebedee, et al. , PNAS, 89:3175-79 (1992) ; Gram, et al . , PNAS, 89:3576-80 (1992) . Ladner, USP 5,223,409 is of interest for its discussion of mutagenesis.

E. Producing anti-anti-idiotype antibodies (Ab₃) which bind to the same epitope as the parental antibody.

A parental antibody (Ab_j) or its specific antigen binding fragments (e.g., F(ab) '₂, Fab', Fab, V_H or V_L, or CDRs) may be used to raise anti-idiotype antibodies (Ab₂) which bear the internal image of the epitope recognized by the parental antibody. These antibodies (Ab₂) or fragments therefore, may then be used to raise anti-anti-idiotype antibodies (Ab₃) which complement that internal image and therefore are cross-reactive with the original epitope. Kennedy, et al. , J. Clin. Invest., 80:1217-24 (1987) ; Chapman, et al. , Biologic Therapy of Cancer Updates, 2:1-9 (May 1992) .

The ability to bind CD14 is not limited to antibodies, or even to proteins, as is apparent from the fact that bacterial LPS bind CD14. Therefore, the antagonist may be a CD14-binding molecule other than an antibody. Such molecules may be obtained by determining the 3D-structure of at least the binding site of CD14 and modeling a molecule that theoretically would block that interaction, by modifying a known CD14-binding molecule (such as an antibody or portion thereof) or LPS by preparing and screening a library of potential CD14-binding molecules.

Other Binding Molecules

In a similar manner, one may identify a molecule which acts as an antagonist by binding to LPS, or to an LPS: LPS binding protein complex, rather than to CD14.

Identification of Binding Molecules by Screening a Combinational Library of Potential Binding Molecules

While candidate binding molecules can be individually prepared and tested, it is convenient to prepare and screen, essentially simultaneously, a combinatorial library of potential binding molecules. The term "combinatorial library" implies that the binding molecules are polymers or oligomers constructed by the at least partially random combination of monomeric units provided during synthesis. This random combination imparts diversity to the library. For example, if there are six randomized positions, and each unit is chosen from a set of twenty possible monomers, the library will have a diversity of 20⁶ different molecules.

In some libraries, all positions of the oligomer or monomer are randomized. In other libraries, some positions are held constant, while others are randomized. Typically, libraries of oligomers (e.g., hexapeptides) are fully randomized, while libraries of polymers are randomly varied at selected sites. However, the number of variable positions is limited only by one's ability to construct a library in which each theoretical sequence is reasonably likely to be represented in the library in amount sufficient for its binding to the target, if at an acceptable level, to be detectable.

The range of possibilities may differ from one variable position to another, and the library may be prepared so that the frequency of occurrence of the allowed monomers differs from one variable position to another, even when the possibilities are the same. It is not necessary that all possible monomers at a given position be equally likely. The choice of which positions to vary, which monomers to allow at each variable positions, and what proportions to allow, will typically be guided by knowledge of one or more reference molecules of known binding activity.

The library may be static (its sequences do not change after initial synthesis) or dynamic. In a dynamic library, the unbound molecules are exposed, intermittently or continuously, to conditions causing degradation and resynthesis (thus scrambling their sequences) , while the bound molecules are protected from further scrambling. See Venton, USP 5,366,862.

In one embodiment, the building blocks are amino acids, and the library is composed of peptides or proteins. The peptides or proteins may be composed solely of genetically encoded amino acids, in which case the library may be prepared by recombinant DNA techniques, or it may include other amino acids (including ones which do not occur in nature) , in which case the library is prepared by in vitro chemical or enzymatic synthesis (or possibly by in vitro chemical or enzymatic library) . When recombinant DNA techniques are used, the peptide or protein may be displayed on the surface of a virus, (e.g., a Mlamentous phage), a cell (especially a bacterial or yeast cell) , or a bacterial spore. In this manner, it can interact with a target, while remaining physically associated with the encoding DNA.

Amino acids are the basic building blocks with which peptides and proteins are constructed. Amino acids possess both an amino group (-NH₂) and a carboxylic acid group (-COOH) . Many amino acids, but not all, have the structure NH₂-CHR-COOH, where

R is hydrogen, or any of a variety of functional groups.

Twenty amino acids are genetically encoded: Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamic Acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, and Valine. Of these, all save Glycine are optically isomeric, however, only the L-form is found in humans. Nevertheless, the D-forms of these amino acids do have biological significance; D-Phe, for example, is a known analgesic. Many other amino acids are also known, including: 2- Aminoadipic acid; 3-Aminoadipic acid; beta-Aminopropionic acid; 2-Aminobutyric acid; 4-Aminobutyric acid (Piperidinic acid) ; 6-Ammocaproic acid; 2-Aminoheptanoic acid; 2-Aminoisobutyric acid, 3-Aminoisobutyric acid; 2-Aminopimelic acid; 2,4-Diaminobutyric acid; Desmosine; 2, 2 ' -Diaminopimelic acid;

2,3-Diaminopropionic acid; N-Ethylglycine; N-Ethylasparagine;

Hydroxylysine; allo-Hydroxylysine; 3-Hydroxyproline;

4-Hydroxyproline; Isodesmosine; allo-Isoleucine; N-Methylglycine

(Sarcosine) ; N-Methylisoleucine; N-Methylvaline; Norvaline; Norleucine; and Ornithine.

Peptides are constructed by condensation of amino acids and/or smaller peptides. The amino group of one amino acid (or peptide) reacts with the carboxylic acid group of a second amino acid (or peptide) to form a peptide (-NHCO-) bond, releasing one molecule of water. Therefore, when an amino acid is incorporated into a peptide, it should, technically speaking, be referred to as an amino acid residue.

In a standard "Merrifield" synthesis, a side chain-protected amino acid is coupled by its carboxy terminal to a support material, such as a resin. A side chain and amino terminal protected amino acid reagent is adde _r , and its carboxy terminal reacts with the exposed amino terminal of the insolubilized amino acid to form a peptide bond. The amino terminal of the resulting peptide is then deprotected, and a new amino acid reagent is added. The cycle is repeated until the desired peptide has been synthesized. For an overview of techniques, see Geisaw, Trends. Biotechnol., 9:294-95 (1991). In the conventional application of this procedure, the amino acid reagent is made as pure as possible. However, if a mixture of peptides is desired, the amino acid reagent employed in one or more of the cycles may be a mixture of amino acids, and this mixture may be the same or different, from cycle to cycle. Thus, if Ala were coupled to the resin, and a mixture of Glu, Cys, His and Phe were added, the dipeptides Ala-Glu, Ala-Cys, Ala-His and Ala-Phe will be formed.

When, during a synthetic cycle, a pure amino acid is added, the resulting residue in the peptide molecules being synthesized is called a constant residue. If a mixture of amino acids is employed, the added residue is called a variable residue. The component amino acids of the mixture, which are the only amino acids which can occupy that variable residue position, are called the "set" of that variable residue. The set for one variable residue may be different from that of the next one. When any of the residues added during the synthesis of a peptide is a variable residue, so that the synthesis deliberately produces a mixture of peptides, the mixture is termed a peptide library. The differences among the peptide molecules of the library will lie, essentially at, and only at, the predetermined variable residue positions.

A peptide library may consist essentially only of peptides of the same length, or it may include peptides of different length. The peptides of the library may include, at any variable residue position, any desired amino acid. Possible sets include, but are not limited to: (a) all of the genetically encoded amino acids, (b) all of the genetically encoded amino acids except cysteine (because of its ability to form disulfide crosslinks) ,

(c) all of the genetically encoded amino acids, as well as their D-forms; (d) all naturally occurring amino acids (including, e.g., hydroxyproline); (e) all hydrophilic amino acids; (f) all hydrophobic amino acids; (g) all charged amino acids; (h) all uncharged amino acids; etc. The peptide library may include branched and/or cyclic peptides.

The current trend in peptide synthesis, especially in the approach of irrational drug design calls for the incorporation of many different types of building blocks. Many of the new building blocks used are amino acids which are not genetically encoded, e.g., glycosylated amino acids, lipidated amino acids, or various unnatural amino acids. The references cited below indicate some of these recent efforts :

1. Bielfeldt, T. , Peters, S., Meldal, M. , Bock, K. and Paulsen, N.A. new strategy for solid-phase synthesis of Oglycopeptides.

Angew. Chem . (Engl ) 31:857-859, 1992.

2. Gurjar, M.K. and Saha, U.K. Synthesis of the glycopeptide-O- (3,4-di-O-methyl-2-0- [3,4-di-O-methyl-α-L-rhamnopyranosyl] -α-L- rhamnophyranosyl) -L-alanilol: An unusual part structure in the glycopeptidolipid of Mycobacterium fortuitum. Tetrahedron 48:4039-4044, 1992.

3. Kessler, H., Wittmann, V., Kock, M. and Kottenhahn, M. Synthesis of C-glycopeptides via free radical addition of glycosyl bromides to dehydroalanine derivatives. Angew. Chem. (Engl . ) 31:902-904, 1992.^'

4. Kraus, J.L. and Attardo, G. Synthesis and biological activities of new N- formylated methionyl peptides containing an α-substituted glycine residue. European Journal of Medicinal Chemistry 27:19-26, 1992. 5. Mhaskar, S.Y. Synthesis of Ν-lauroyl dipeptides and correlation of their structure with surfactant and antibacterial properties. J. Am . Oil Chem. Soc .69 :647-652, 1992.

6. Moree, W.J., Van der Marel, G.A. and Liskamp, R.M.J. Synthesis of peptides containing the 3-substituted aminoethane sulfinamide or sulfonamide transition-state isostere derived from amino acids. Tetrahedron Lett . 33:69-6392, 1992.

7. Paquet, A. Further studies on the use of 2,2,2- trichloroethyl groups for phosphate protection in phosphoserine peptide synthesis. International Journal of Peptide and Protein Research 39:82-86, 1992.

8. Sewald, Ν. , Riede, J. , Bissinger, P. and Burger, K. A new convenient synthesis of 2-trifluoromethyl substituted aspartic acid and its isopeptides. Part 11. Journal of the Chemical Society. Perkin Transactions 1 1992:267-274, 1992.

9. Simon, R.J., Kania, R.S., Zuckermann, R.N. , Huebner, V.D., Jewell, D.A., Banville, S., Ng, S., Wang, L. , Rosenberg, S., Marlowe, C.K., Spellmeyer, D.C, Tan, R., Frankel, A.D., Santi, D.V., Cohen, F.E. and Bartlett, P.A. Peptoids: A modular approach to drug discovery. Proc. Natl , Acad. Sci . USA 89:9367-9371, 1992.

10. Tung, C.-H., Zhu, T. , Lackland, H. and Stein, S. An acridine amino acid derivative for use in Fmoc peptide synthesis. Peptide Research 5:115-118, 1992.

11. Elofsson, M. Building blocks for glycopeptide synthesis: Glycosylation of 3-mercaptopropionic acid and Fmoc amino acids with unprotected carboxyl groups. Tetrahedron Lett. 32:7613- 7616, 1991. 12. McMurray, J.S. Solid phase synthesis of a cyclic peptide using Fmoc chemistry. Tetrahedron Letters 32:7679-7682, 1991. 13. Nunami, K.-I., Yamazaki, T. and Goodman, M. Cyclic retro- inverso dipeptides with two aromatic side chains. I. Synthesis. Biopolymers 31:1503-1512, 1991. 14. Rovero, P, Synthesis of cyclic peptides on solid support. Tetrahedron Letters 32:2639-2642, 1991.

15. Elofsson, M. , Walse, B. and Kihlberg, J. Building blocks for glycopeptide synthesis: Glycosylation of 3-mercaptopropionic acid and Fmoc amino acids with unprotected carboxyl groups . Tetrahedron Letter, 32:7613-7616, 1991.

16. Bielfeldt, T., Peter, S., Meldal, M. , Bock, K. and Paulsen, H. A new strategy for solid-phase synthesis of O-glycopeptides. Agnew. Chem (Engl ) 31:857-859, 1992.

17. Luning, B., Norberg, T. and Tejbrant, J. Synthesis of glycosylated amino acids for use in solid phase glycopeptide synthesis, par 2 :N- (9-fluorenylmethyloxycarbonyl) -3-0- [2,4, 6-tri- O-acetyl-α-D-sylopyranosyl) -J-D-glucopyranosyl] -L-serine. J. Carbohydr. Chem . 11:933-943, 1992.

18. Peters, S., Bielfeldt, T., Meldal, M. , Bock, K. and Paulsen, H. Solid phase peptide synthesis of mucin glycopeptides.

Tetrahedron Lett . 33:6445-6448, 1992.

19. Urge, L., Otvos, L., Jr., Lang, E., Wroblewski, K. , Laczko,!. and Hollosi, M. Fmoc-protected, glycosylated asparagines potentially useful as reagents in the solid-phase synthesis of N-glycopeptides, Carbohydr. Res . 235:83-93, 1992. 20. Gerz, M., Matter, H. and Kessler, H., S-glycosylated cyclic peptides, Angew. Chem. (Engl . ) 32:269-271, 1993. The linkages between the amino acid residues may be peptide bonds (-NHC0-) , as in a classical peptide or protein, or another type of linkage, such as a peptide bond analogue (e.g., -CH₂CH₂-, -NRC0-, -NHCS-) . In the latter case, the library will usually be described as a peptoid library. See Simon, et al., P.A., "Peptoids: A modular approach to drug discovery." Proc. Natl . Acad. Sci . USA 89:9367-9371, 1992, and cf. Gilon, et al, "Backbone cyclization: A new method for conferring conformation constraint on peptides." Biopolymers 31:745-750, 1991.

The peptides or peptoids may be linear, branched, or cyclic. One of the advantages of the chemical approach to peptide libraries (as opposed to libraries expressed by biological means, e.g. filamentous phages) is the ability to produce and test cyclic and branching peptides. Early examples in the literature for such structures are found in the use of multiple antigenic peptides (MAP) as immunogens, in MAPs, peptide haptens are attached to a branching "tree" of lysines.

1. Baleux, F. and Dubois, P. Novel version of Multiple Antigenic Peptide allowing incorporation on a Cysteine functionalized lysine tree. Int. J^". Pept. Protein Res . 40:7-12, 1992.

2. Munesinghe, D.Y., Clavijo, P., Calle, M.C, Nussenzweig, R.S. and Nardin, E. Immunogenicity of multiple antigen peptides

(MAP) containing T and B cell epitopes of the repeat region of the P. falciparum circumsporozoite protein. Bur. J. Immunol . 21:3015-3020, 1991.

In the current MAP system identical peptide sequences are repeated several time in the MAP structure. Such an arrangement apparently stabilizes the peptide conformation, allowing for better presentation of the antigenic structure and hence better immunogenicity. The use of approaches similar to the MAP could enable better biological activity of peptides due to stabilization of conformation.

The interaction of short linear peptides with their targets occurs along the peptide length. Formation of branching peptides may enable interaction of the peptide with the target throughout a surface and thus mimic the type of interaction of some antibodies with their target antigens (as observed by X-ray crystalography and analysis of antibody-antigen complexes) . This type of interaction opens up new possibilities for small peptide- ligand interactions which are non-existent for linear peptides but existent for protein-ligand interactions.

Many naturally occurring peptide are cyclic. Cyclization is a common mechanism for stabilization of peptide conformation thereby achieving improved association of the peptide with its ligand and hence improved biological activity. Cyclization is usually achieved by intra-chain Cystine formation, by formation of peptide bond between side chains or between N- and C- terminals. Cyclization was usually achieved by peptides in solution, but several publications have appeared recently that describe cyclization of peptides on beads (see references below) . These published techniques may be directly applicable to our library approach. 1. Spatola, A.F., Anwer, M.K. and Rao, M.N. Phase transfer catalysis in solid phase peptide synthesis. Preparation of cycle [Xxx-Pro-Gly-Yyy-Pro-Gly] model peptides and their conformational analysis. Int. J. Pept . Protein Res . 40:322-332, 1992

2. Tromelin, A., Fulachier, M.-H., Mourier, G. and Menez, A. Solid phase synthesis of a cyclic peptide derived from a curaremimetic toxin. Tetrahedron Lett . 33:5197-5200, 1992.

3. Trzeciak, A. Synthesis of 'head-to-tail' cyclized peptides on solid supports by Fmoc chemistry. Tetrahedron Lett . 33:4557- 45560, 1992. 4. Wood, S. J. and Wetzel, R. Novel cyclization chemistry especially suited for biologically derived, unprotected peptides, Int. J. Pept . Protein Res . 39:533-539, 1992.

5. Gilon, C, Halle, D., Chorev, M. , Selinger, Z. and Byk, G. Backbone cyclization: A new method for conferring conformational constraint on peptides. Biopolymers 31:745-750, 1991.

6. McMurray, J. S. Solid phase synthesis of a cyclic peptide using Fmoc chemistry. Tetrahedron Letters 32:7679-7682, 1991.

7. Rovero, P. Synthesis of cyclic peptides on solid support. Tetrahedron Letters 32:2639-2642, 1991.

8. Yajima, X. Cyclization on the bead via following Cys Acm deprotection. Tetrahedron 44:805, 1988.

For protein libraries, see Ladner, USP 5,223,409; Swimmer, et al., Proc. Nat. Acad. Sci., 89:3756-60 (1992) ; Bass, et al. ,

Proteins:Structure, Function and Genetics, 8:309-14 (1990);

Lowman, et al. , Biochemistry, 30:10832-8 (1991) ; Ixsys,

WO92/06204. For peptide libraries, see Rutter, WO89/10931;

Scott, Proc. Nat. Acad. Sci., 89:5398 (1992) ; Devlin, Science, 249:404 (1990); Cwirla, Proc. Nat. Acad. Sci. USA, 87:6378-82

(1990) ; Scott, Science, 249:386 (1990) ; Barrett, et al. , Anal.

Biochem., 204:357-64 (1992) ; O'Neil, Proteins: Structure Function and Genetics, 14:509-15 (1992) ; Christian, et al., J. Mol. Biol.,

227:711-18 (1992) ; Affymax, W091/19818; Houghten, et al. , Biol. Techniques, 13:412-21 (1992) .

In a second embodiment, the polymer or oligomer is composed of nucleic acids. The usual bases are the purines adenine and guanine and the pyrimidines thymidine (uracil for RNA) and cytosine. Such bases may be combined by conventional DNA and RNA synthesis methods. Unusual bases, such as those listed below, may be incorporated into the synthesis or produced by post synthetic treatment with mutagenic agents.

4-acetylcytidine.

5- (carboxyhydroxylmethyl)uridine. 2 ' -O-methylcytidine.

5-carboxymethylaminomethyl-2-thioridine.

5-carboxymethylaminomethyluridine. dihydrouridine.

2 ' -O-methylpseudouridine. beta,D-galactosylqueosine

2 ' -O-methylguanosine. inosine.

N6-isopentenyladenosine.

1-methyladenosine. 1-methylpseudouridine.

1-methylguanosine.

1-methylinosine.

2, 2-dimethylguanosine.

2-methyladenosine. 2-methylguanosine.

3-methylcytidine.

5-methylcytidine.

N6-methyladenosine.

7-methylguanosine. 5-methylaminomethyluridine. 5-methoxyaminomethy1-2-thiouridine. beta,D-mannosylqueosine.

5-methoxycarbonylmethyluridine.

5-methoxyuridine. 2-methylthio-N6-isopentenyladenosine.

N- ( (9-beta-D-ribofuranosyl-2-mehtylthiopurine-6- yl) carbamoyl) threonine.

N- ( (9-beta-D-ribofuranosylpurine-6-yl)N-methyl- carbamoyl) threonine. uridine-5-oxyacetic acid methylester. uridine-5-oxyacetic acid (v) . wybutoxosine. pseudouridine. queosine. 2-thiocytidine.

5-methyl-2-thiouridine.

2-thiouridine.

4-thiouridine.

5-methyluridine. N- ( (9-beta-D-ribofuranosylpurine-6-yl) carbamoyl) threonine.2 ' -0- methyl-5-methyluridine.

2 ' -O-methyluridine. wybutosine.

3- (3-amino-3-carboxypropyl)uridine.

DNA may be synthesized by the stepwise addition of nucleotides to a nascent chain. The first step of the synthesis may be the coupling of a nucleoside, via a succinyl linkage, to a suitable support, such as cellulose. This nucleoside represents the 3' end. Chain elongation proceeds from 3' to 5 ' ; each cycle being composed (in one conventional method) of the following steps :

(1) Selective deprotection

For example, if the 5 '-hydroxyl is protected by a dimethoxytrityl group, it is removed with acid. (2) Condensation

A protected nucleotide is coupled to the exposed 5 ' end. The protected nucleotides may be 5 ' -O- dimethoxytrityl-N⁶- (benzoyl) -2 ' -deoxyadenosine, 5 ' - dimethoxytrityl-N⁴- (anisoyl) -2 ' -deoxycytidine, 5 ' -0- dimethoxytrityl-N⁶- (N' ,N' , -di-n-butyl formadine) -2 ' - deoxyadenosine, and 5 ' -O-dimethoxytrityl-N²-

(propionyl) -0⁶- (diphenylcarbamoyl) -2 ' -deoxyguanosine.

(3) Capping

Unreacted 5 ' -hydroxyl groups are protected, e.g., by acylation. The traditional method for DNA sequencing by chemical cleavage depends on the parallel execution of four base-specific or base-selective modification protocols and the parallel electrophoretic resolution of the hydrolysates in four lanes. It is also possible to analyze DNA based on a single base modification procedure, if it produces some degree of backbone cleavage at all bases in the DNA but the rates of cleavage at the four canonical bases (A, T, G, C) are clearly different. See Ambrose and Pless, Meth. Enzymol., 152:522 (1987) (modification with 0.5M aqueous piperidine, 0.3M NaCl, 90°C , pH > 12, 5 hrs) . The single reagent method is faster but less accurate.

In a third embodiment, the building blocks are monosaccharides. Polysaccharides are larger polymers of monosaccharides in a branched or unbranched chain. Oligosaccharides are shorter polymers of monosaccharides, such as di-, tri-, tetra-, penta-, and hexasaccharides. For the sake of convenience, the term "polymeric carbohydrate" will be used to cover both poly- and oligosaccharides.

Monosaccharides in a polymeric carbohydrate library may be aldoses, ketoses, or derivatives. They may be tetroses, pentoses, hexoses or more complex sugars. They may be in the D- or the L-form. Suitable D-sugars include D-glyceraldehyde, D- erythrose, D-threose, D-arabinose, D-ribose, D-lyxose, D-xylose, D-glucose, D-mannose, D-altrose, D-allose, D-talose, D-galactose, D-idose, D-gulose, D-rhamnose, and D-fucose. Suitable L-sugars include the L-forms of the aforementioned D-sugars.

A sugar hemiacetal may be reacted with a hydroxyl group of another sugar to form a disaccharide, and the reaction may be repeated. For carbohydrate synthesis methods, see Kanie, 0. and Hindsgaul, 0., "Synthesis of Oligosaccharides, Glycolipids and Glycopeptides," Curr. Opin Struc. Bio. , 2:674-681, (1992) . For sequencing, see Y.C Lee, "Review: High-Performance Anion- exchange Chromatography for Carbohydrate Analysis," Anal . Biochem. 189:151-162, (1990) ; Maley, F., Trimble, R.B., Tarentino, A.L., Plummer, T.H. , "Review: Characterization of Glycoproteins and Their Associated Oligosaccharides Through the Use of Endoglycosidases, " Anal. Biochem. , 180:195-204, (1989) ; and Spellman, M.W. , "Carbohydrate Characterization of Recombinant Glycoproteins of Pharmaceutical Interest," Anal. Chem. 62:1714- 1722, (1990) .

Building blocks of particular interest are those found in bacterial LPS, since that is known to bind CD14. However, the libraries of the present invention are not limited to the foregoing types. Any set of organic compounds which can be, essentially interchangeably, ligated to form an oligomer or polymer in reasonable yields can be used to construct a combinatorial library. In some cases, it is advantageous to first screen a peptide library, and then construct a library of peptide mimetics. For further insights, see the preceedings of the Fourth Approval Program on Development of Small molecule Mimetic Drugs (Cambridge Healthtech Institute; May 2-3, 1996) and the Fifth Annual International Conference on High-Throughput Screening (Intermembrane Business Communications; April 25-26, 1996) .

Once the library is constructed, it must be screened for binding to the target. In some embodiments, the target is insolubilized, and unbound library molecules are removed. This is analogous to a heterogeneous affinity assay. In other embodiments, the target is labeled, and either the detectability of the label is altered by binding (as in a homogeneous assay) or the library molecules are immobilized, so that the target molecules "marks" the successful members of the library. After successful binding molecules are recovered, it is necessary to characterize them. This may be done by sequencing the molecule; this is a standard practice with oligopeptide and nucleic acid libraries. The primary sequence of amino acids in a peptide or protein is commonly determined by a stepwise chemical degradation process in which amino acids are removed one-by-one from one end of the peptide, and identified. In the

Edman degradation, the N-terminal amino acid of the peptide is coupled to phenylisothiocyanate to form the phenylthiocarbamyl

(PTC) derivative of the peptide. The PTC peptide is then treated with strong acid, cyclizing the PTC _L -tptide at the first peptide bond and releasing the N-terminal amino acid as the anilino- thiozolinoe (ATZ) derivative. The ATZ amino acid, which is highly unstable, is extracted and converted into the more stable phenylthiohydantoin (PTH) derivative and identified by chromatography. The residual peptide is then subjected to further stepwise degradation. For carboxy terminal sequencing (of peptides synthesized with their amino terminal coupled to a support) , the cleavage reagent may be a carboxypeptidase.

The present invention is not limited to any particular method of sequencing. With protein libraries, it is common to express the protein so that it remains physically associated with the corresponding DNA (e.g., the protein is integrated into the coat of a phage encapsulating DNA encoding the protein) . In this case, it is possible to sequence the DNA rather than the protein. It is also possible, during synthesis, to attach a specific tag (not expected to participate in binding) which is easier to identify than the binding molecule itself. Altematively, the library may be synthesized so that the spatial position of the binding molecule is fixed, and assists in identifying the binding molecule.

It should be noted that if a particular type of polymer cannot be sequenced readily, it can be studied indirectly by means of an "encoding" strategy in which the beads carry both peptides and the non-peptide polymer. Cf. Brenner and Lerner, "Encoded Combinatorial Chemistry," PNAS (USA) . 89:5381-83, (1992) , who disclose chemically linking a "genetic tag" (amplifiable by PCR) to a polymer which is not itself genetically encodable. The peptide need not, however, be chemically linked to the non-peptide polymer in the present method. For example, a library may be structured so that on a given bead, there is a single peptide sequence, and a family of related nucleic acid sequences. Sequencing the peptide then identifies the nucleic acid family.

After successful binding molecules are characterized, a new library may be designed which takes advantage of the knowledge of the structure-activity relations! _,.ps gained from the previous library.

Thus, in another embodiment of the present invention the compound that is used to block or neutralize CD14 is a random peptide that binds to LPS, LPS-binding protein or LPS-LPS binding protein complex and thereby blocks or neutralizes the interaction of LPS with CD14. Random peptides may be generated by art-known methods of peptide synthesis. Generally, the random peptides contain about four to ten amino acids, preferably five to eight amino acids, and most preferably six amino acids. Most preferred random peptides useful in the invention method include six amino acid long peptides having the following combinations of amino acids at the first two positions of the peptide, i.e., at the amino terminal end of the peptide: ALA-HIS, ARG-TRP, ALA-TRP, ALA-GLN, VAL-TRP, THR-TRP, ARG-TRP, ILE-TRP, SER-ARG, TYR-TRP, PHE-TRP, HIS-TRP, CYS-TRP, THR-CYS, ARG-CYS, ILE-CYS, TYR-CYS, HIS-CYS,^' CYS-CYS, and CYS-ARG.

Pharmaceutical Compositions and Methods

The pharmaceutical composition of the present invention comprise one or more drugs as previously defined, and are effective, when administered according to an effective pharmacological schedule to a patient, of providing "protection". "Protection", as used herein, is intended to include prevention, suppression, and treatment. Prevention involves administration of the protective composition prior to the induction of the disease. Treatment involves administration of the protective composition after the appearance of the disease. It will be understood that in medicine, it is not always possible to distinguish between preventing and suppressing, since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, it is common to use the term protection as distinct from treatment to encompass both preventing and suppressing as defined herein. The term protection, as used herein, is meant to include prophylaxis.

It should also be understood that to be useful, the protection provided need not be absolute, provided that it is sufficient to carry clinical value. An agent which provides protection to a lesser degree than do competitive agents may still be of value if the other agents are ineffective for a particular individual, if it can be used in combination with other agents to enhance the overall level of protection, or if it is safer than competitive agents.

The treatment method may be applied prior to, during or after other medical procedures, such as, for example, surgery, especially gastrointestinal surgery, radiotherapy, chemotherapy or peritoneal dialysis. The present method may also be applied to a patient thought to be at risk of bacteremia to thereby prevent dissemination of bacteria. The composition may be administered parentally or orally, and, if parentally, either systemically or topically. Parenteral routes include subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes. One or more such routes may be employed. Parenteral administration can be, e.g., by bolus injection or by gradual perfusion over time. Alternatively, or concurrently, administration may be by the oral route.

It is understood that the suitable dose of a composition according to the present invention will depend upon the age, sex, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. However, the most preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. This typically involves adjustment of a standard dose, e.g., reduction of the dose if the patient has a low body weight.

Prior to use in humans, a drug is first evaluated for safety and efficacy in laboratory animals. In human clinical trials, one begins with a dose expected to be safe in humans, based on the preclinical data for the drug in question, and on customary doses for analogous drugs, if any. If this dose is effective, the dosage may be decreased to determine the minimum effective dose, if desired. If this dose is ineffective, it will be cautiously increased, with the patients monitored for signs of side effects. See, e.g., Berkow et al. , eds., The Merck Manual. 15th edition, Merck and Co., Rahway, N.J., 1987; Goodman et al. , eds, Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y. (1990) ; Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, MD (1987) ; Ebadi, Pharmacology, Little, Brown and Co., Boston (1985) , which references and references cited therein are entirely incorporated herein by reference.

The total dose required for each treatment may be administered in multiple doses (which may be the same or different) or in a single dose, according to a pharmacological schedule, which may be predetermined or ad hoc. The schedule is selected so as to be pharmaceutically effective, i.e., so as to be sufficient to elicit a response which protective is in itself or which enhances the protection provided by other agents. The doses adequate to accomplish this are defined as "therapeutically effective doses." (Note that a schedule may be effective even though an individual dose, if administered by itself, would not be effective, and the meaning of "therapeutically effective dose" is best interpreted in the context of the schedule. ) Amounts effective for this use will depend on, e.g., the peptide composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.

The appropriate dosage form depends on the status of the disease, the composition administered, and the route of administration. Dosage forms include tablets, capsules, lozenges, dental pastes, suppositories, inhalants, solutions, ointments, and parenteral depots. See, e.g., Berker, supra, Goodman, supra, Avery, supra and Ebadi, supra, which are entirely incorporated herein by reference, including all references cited therein.

In one embodiment, the drug is dissolved or suspended in an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.

For aerosol administration, the drugs are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of drugs are 0.01%-20% by weight, preferably 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. the balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.

In addition to the drugs of the invention, a pharmaceutical composition may contain suitable pharmaceutically acceptable carriers, such as excipients, carriers and/or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. As an alternative to a pharmaceutical composition comprising the drug of the present invention, per se, when the drug is a peptide or protein, the pharmaceutical composition may instead comprise a vector comprising an expressible gene encoding such drug. In the case of genes encoding naturally occurring proteins, or peptide fragments thereof, one may, but need not, use the DNA sequence which encodes the proteins or peptides in nature.

The pharmaceutical composition and method would then be chosen so that the vector was delivered to suitable cells of the subject, so that the gene would be expressed and the drug produced in such a manner as to elicit a protective effect. A preferred vector would be a Vaccinia virus. Alternatively, a nonpathogenic bacterium could be genetically engineered to express the drug. The drug must, of course, either be secreted, or displayed on the outer membrane of the bacterium (or the coat of a virus) in such a manner that it can interact with bacterial LPS or cellular CD14, as appropriate.

The amount of CD14 antagonist used in the present method may vary depending on the type of antagonist. For example, in the case where sCD14 or fragment thereof is administered to the patient the amount administered is generally in the range of from about 1 to about 50-fold excess of the amount of SCD14 found in normal healthy individuals, i.e., about 2 to about 4 μg/ml sCD14 is found in blood serum of healthy humans. Thus, an amount of from about 6 to about 100 μg/ml, preferably, about 10 to about 90 μg/ml and most preferably about 40 to about 80 μg/ml are administered to the patient. In the case where an anti-CD14 monoclonal or polyclonal antibody is administered to the patient the dosage is generally in the range of from about 10 to about 70 μg/ml, preferably from about 15 to about 40 μg/ml and most preferably, from about 20 to about 30 μg/ml. This range of amount of anti-CD14 antibody has been demonstrated to block binding of endotoxin to CD14 in vitro. Similarly, the amount of a peptide administered to a patient to effect binding to LPS, LPS-binding protein or LPS-LPS-binding protein complex is in the range of from about 1 to about 50-fold excess over the amount of sCD14 normally found in the blood of healthy individuals and preferably, in the range of from about 1 to about 20-fold excess over the amount of sCD14 normally found in the blood of healthy individuals.

It is believed that these levels of CD14 neutralizing or blocking compounds are sufficient to decrease the risk of bacteremia. However, the specific amount of CD14 neutralizing or blocking compound administered for the prevention of bacteremia or peritonitis can be determined readily for any particular patient according to recognized procedures and based on the expertise and experience of the skilled practitioner. Precise dosing for a patient can be determined according to routine medical practice.

The term "patient" is used herein to mean an animal, including humans and other mammals. Thus, the present method is useful in veterinary medicine as well as in the treatment of humans.

Treatment of a patient with a pharmaceutically effective amount of a CD14 antagonist invention is carried out for a period of time required to prevent bacteremia, subsequent or the systemic dissemination of bacteria from the bloodstream to tissues such as the lung. The treatment regimen will vary depending on such factors as the particular condition to be treated, e.g. ,peritonitis or gastroenteritis, the medical condition underlying the risk of bacteremia ,e.g.,the presence of a localized bacterial infection or trauma, such as an invasive medical treatment or surgery, particularly to the area of the gastrointestinal or respiratory tract, or other predisposing medical condition, the overall health of the patient, the route of administration, etc. For example, the present method may be applied to a patient thought to be at risk of bacterial dissemination into or from the blood stream from such underlying medical conditions as peritonitis, physical injury resulting in intestinal perforation, diverticulitis, appendicitis, acute pancreatitis, other trauma (incl. surgery, i.v. lines, or invasive diagnostic procedures) immunosuppression as a result of chemotherapy, preparation for transplant, or HIV infection, pneumonia, gastroenteritis, colitis, dysentery, severe cellulitis, urinary tract infection, inflammatory bowel disease, hemorrhagic shock, burn infection, endocarditis, meningitis, tuberculosis, etc. Typically, use of the present method to prevent bacteremia includes application of the method at least once per day until the risk of bacteremia is assessed to be over. The skilled medical practitioner can assess the relative risk of bacteremia according to routine medical practice and procedure.

EXAMPLES

The invention is further described by way of examples set forth below. The examples are not intended in any way to be limiting, as it should be readily apparent to those skilled in the art how alternative means might be used to achieve the intended results.

EXAMPLE 1

Resistance of CD14-deficient mice to LPS-induced shock. To assess the relative role of CD14 in the rapid dissemination of bacteria into the blood stream and in the development of peritonitis CD14-deficient mice were examined for their sensitivity to gram-negative bacteria and LPS. CD14 deficient mice were produced by homologous recombination in embryonic stem cells as described by Thomas, et al . , Cell 51: 503-12 (1987) . The human CD14 gene, whose isolation and characterization are described in Applicant 's co-pending application Serial No. 08/254,095 filed June 6, 1995, incorporated in its entirety herein by reference thereto, was disrupted in its first and second exons with a neo gene construct. The defective gene was transfected into W9.5 embryonic stem cells by electroporation. Cells carrying the disrupted CD14 gene were injected into C57BL/6 mouse blastocytes. Male chimeras were bred with C57BL6 mice and the offspring were interbred to produce homozygous CD14-negative mice. The absence of expression of CD14 was confirmed by staining of peritoneal macrophages with polyclonal and monoclonal anti-murine CD14 antibodies. CD14-deficient mice have no obvious abnormalities and are fertile and healthy ( > 1 year) when housed in a clean, non-pathogen-free environment. The role of CD14 in the in vivo response to dissemination of bacteria into the bloodstream was analyzed by injecting CD14-deficient and control mice intraperitoneally (i.p.) with a dose of LPS purified from wild type Salmonella minnesota corresponding to an LD100 for control mice or with a dose corresponding to ten times the LD100. Mice were monitored for seven days. The results are shown in Figure IA.

A significant difference in the response of control and CD14-deficient mice was observed. With an LD100 dose (20 mg/kg body weight) , CD14-deficient mice showed almost no sign of toxicity while all the control mice died within 48 hours. CD14 mice that received an LD100 of LPS (200 mg/kg) showed only minor signs of response to LPS, such as eye exudate and diarrhea during the first 24 hours but remained fully active and no death was observed.

The decreased sensitivity of CD14-deficient mice to LPS was confirmed by measuring the levels of cytokines normally associated with endotoxin shock in the serum of control and CD14-deficient mice after exposure to LPS. LPS (20 mg/kg) was injected i.p. into control and CD14-deficient mice and the amount of serum levels of cytokines were measured two hours after injection by bioassay (TNF-α) (Haziot, et al. , J. Immuno. 150: 5556-65 (1993) and Elisa assay (IL-6) . The results are shown in Figure IB and IC

CD14-deficient mice had a serum level of TNF-α that was ten times lower than that of control mice. Furthermore, there was no additional release of TNF when CD14-deficient mice were exposed to a ten-fold higher dose of LPS (200 mg/kg) . Similarly, the serum level of IL-6 in CD14-deficient mice was at least ten times lower than in control mice. However, at a ten-fold higher dose of LPS there was a slightly stronger IL-6 response. The IL-6 measurements are shown in Figure IC

EXAMPLE 2

Resistance of CD14-deficient mice to bacterial-induced shock. To assess the role of CD14 in the response to intact gram-negative bacteria, CD14-deficient mice and control mice were inoculated with E. coli (5 x 10° CFU) that was lethal to control mice. E. coli 0111 :B4 were grown on tryptic soy broth (Difco) agar plates and single colonies were inoculated in tryptic soy broth medium. Bacteria in mid-logarithmic phase were collected, chilled on ice, the concentration of bacteria determined on the basis of absorbance at 620 nm using a pre-determined calibration curve. For injections bacteria were washed once in non-pyrogenic saline, and serial delusions were prepared in saline. The volume injected was 0.2 ml. Mice were injected i.p. The condition of surviving mice was monitored for 21 days. The results are shown in Figure 2A. Numbers above the bars indicate number of survivors/number of mice per group. Cytokine levels were determined as in Example 1. Results are represented as mean +/- SE in each group, (p < 0.05) (Mann-Whitney test) .

Normal control mice died with 36 hours. In contrast, 100% of the CD14 deficient mice survived and none showed signs of shock. When the dose of E. cold was increased six-fold (30 x 10° CFU) the CD14-deficient mice appeared healthy and alert for a period of 12 to 16 hours before their condition deteriorated and they began to die. In contrast, control mice were adversely affected within four hours of receiving the inoculum of bacteria and showed typical signs of endotoxic shock such as diarrhea, eye exudate and ruffled fur. All of the control mice died within 24 hours at the increased dose.

The serum levels of TNF-α and IL-6 in control and CD14-deficient mice were measured two hours after the injection of gram-negative bacteria. The results are shown in Figure 2B. The mean serum concentrations of TNF-α in CD14-deficient mice infected i.p. with gram-negative bacteria was ten-fold lower than in control mice. Similarly, the IL-6 levels after infection were 48-fold lower in CD14-deficient mice than in control mice (Figure 2C) . Serum concentrations of IL-1 were also depressed in infected CD14-deficient mice.

EXAMPLE 3

Serum concentrations of TNF-α and IL-6 in E. coli infected mice.

Mice were injected intravenously with E. coli ( (3 x IO⁷ CFU/mouse, n=3) . Cytokines were measured in peripheral blood as described in Example 1. The results are shown in Figure 3A (TNF- e) and Figure 3B (IL-6) . Open circles represent cytokine levels in peripheral blood of control mice; blackened squares represent peripheral blood cytokine levels in CD14-deficient mice.

EXAMPLE 4

Blood and organ counts of bacteria in infected mice. Control and CD14 mice were also examined to determine the level of viable bacteria in the blood, which is a direct measurement of bacteremia. Mice were injected i.p. with (a) 3 x IO⁷ CFU (4 mice per group) , (b) 5 x 10° CFU (4 mice per group) and (c) 3 x 10⁷ CFU (3 mice per group) of E. coli 0111 :B4. Seven hours after injection mice were sacrificed by C02 inhalation, bled by heart puncture and lungs were aseptically recovered and homogenized. Bacterial counts were determined by plating 10-fold serial dilutions of blood or homogenized lungs on tryptic soy broth agar plates. The results are shown in Figure 4A, 4B and 4C. Results are represented as a mean +/- SE in each group. (p< 0.05) (Mann- Whitney test) .

CD14-deficient mice had 35-fold fewer bacteria in the blood than control mice after a lethal i.p. injection of viable E. coli (3 x IO⁷ CFU per mouse) (Figure 4A) . Similarly, when a dose that was lethal only to control mice was used as inoculum (5 x IO⁶ CFU per mouse) a 27-fold lower level of bacteremia was observed in the CD14-deficient mice (Figure 4B) . The number of live bacteria in the lungs of mice injected with 3 x IO⁷ bacteria was also similarly reduced in CD14-deficient mice (Figure 4C) . Overall, these data demonstrate that the inability to activate monocytes through CD14 in CD14-deficient mice is associated with a greatly reduced bacteremia and bacterial load in organs . These data demonstrate the prominent role for CD14 for bacterial dissemination into the blood stream and into the organs.

HYPOTHETICAL EXAMPLE 5

A patient suffering from an underlying medical condition requiring surgery within the gastrointestinal tract, e.g. ,colonostomy, is administered 100 mg to 800 mg of SCD14 in a sterile saline solution by intravenous inoculation once per day beginning two days prior to surgery, and again the day of surgery and for five days following surgery. The level of viable bacteria in the blood is measured on the day following surgery and at three days following surgery to determine whether there is a bacteremic condition.

EXAMPLE 6

We have analyzed the effect of CD14 expression in a peritonitis model called "cecal ligation and puncture". In this model the cecum was ligated and punctured with an 18 gauge needle; the normal bacterial flora of the gut leak out of the puncture site and cause a peritonitis. In our initial experiments we tested the effects of this model on survival using 5 normal control mice and 7 CD14-deficient mice and found that the 5 control mice died with 48 hours while 4 out of 7 CD14- deficient mice survived.

EXAMPLE 7

1. Antagonists will be defined by screening combinatorial libraries of small organic compounds obtained from a variety of commercial sources by a combination of three methods. a. Plate binding assay (ref. 1) in which human sCD14 prepared by us (ref. 2) is bound to plate, compounds are added, and their ability to inhibit binding of biotinylated-LPS is measured by ELISA. b. Positive compounds in assay (a) are then rescreened for their ability to inhibit activation (as measured by release of cytokines, TNF-α, IL-1, IL-6) of human PBMNC (peripheral blood mononuclear cells) , by LPS or organism (bacterial or fungal) (whole blood assay, ref. 2) . c Positive compounds in assay (b) , which do not activate PBMNC spontaneously, are then rescreened in an in vivo model consisting of genetically engineered mice deficient in mouse CD14 but expressing human CD14 on the surface of PBMNC. These mice have been made by crossing CD14-deficient mice (3) with mice expressing human CD14 (4) and selecting, by standard blotting or PCR procedures, for mice homozygous for inactive mouse CD14 gene and expressing human CD14. This allows for identification of antagonists that are selective for human CD14. Functional assays will be death of mice and/or activation of PBMNC (1-4) .

2. Define new antagonists by continuing screening of combinatorial hexamer peptide library. a. Take positive hits (based on assays (a) and/or (b) above) , in which amino acids at first two positions are known and the remaining four positions consist of mixture of all 20 amino acids, and proceed with identification of amino acids at these four positions that provide optimal activity. Identification will be performed by binding soluble human CD14 (1) to 96-well plate, incubate separately with each of 400 pools of combinatorial hexamer peptide library (prepared as described by Houghton) , wash, elute peptides with weak acid, filter out soluble CD14 by membrane filter which separates molecules on basis of size or, by affinity chromatography using anti-human CD14 antibody coupled to resin, and determine amino acid sequence of peptide(s) preferentially bound and compare to sequence of peptides in pool to identify optimal amino acids at positions 3- 6. Alternatively, instead of using plate-binding assay to identify peptides preferentially bound to soluble human CD14, use soluble human CD14 covalently bound to resin and select for peptides preferentially bound to soluble human CD14 by conventional affinity chromatography. b. Peptides with optimal sequence based on 2(a) will be synthesized and rescreened in assays 1(a), (b) , and (c) as described above. c Additional antagonists will be identified on the basis of this information by making D-amino acid peptides with the same sequence or by making D-amino acid peptides with the same sequence but in reverse order. d. Additional antagonists will also be identified by solving NMR structure of peptides identified in (a) and (b) above and designing small organics with similar 3D structure and structure-activity analyses.

References for Example 7 1. Haziot, A., et al. , (1993), Recombinant soluble CD14 mediates the activation of endothelial cells by lipopolysaccharide, J. Immunol.. 151:1500-1507. 2. Haziot, A., et al . , (1994) , Recombinant soluble CD14 inhibits LPS-induced Tumor Necrosis Factor-α production by cells in whole blood, J. Immunol.. 152:5868-5876.

3. Haziot, et al. , (1996) Resistance to endotoxin shock and reduced dissemination of gram-negative bacteria in CD14-deficient mice, Immunity, 4:407-414.

4. Ferrero, et al. , (1993) , Transgenic mice expressing human CD14 are hypersensitive to lipopolysaccharide, Proc. Natl. Acad. Sci. USA, 90:2380-2384. All references ci ted herein, including journal articles or abstracts, abandoned or pending (whether or not published) U. S. or foreign patent applications, issued U. S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the ci ted references . Addi tionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by reference.

Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge wi thin the skill of the art (including the contents of the references ci ted herein) , readily modify and/or adapt for various applications such specific embodiments , wi thout undue experimentation, wi thout departing from the general concept of the present invention . Therefore, such adaptations and modifications are intended to be wi thin the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein . It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limi tation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination wi th the knowledge of one of ordinary skill in the art . Any description of a class or range as being useful or preferred in the practice of the invention shall be deemed a description of any subclass or subrange contained therein, as well as a separate description of each individual member or value in said class or range.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT:

(A) NAME: GOYERT, Sanna M. (B) STREET: 350 Community Drive

(C) CITY: Manhasset

(D) STATE: New York

(E) COUNTRY: United States of America

(F) POSTAL CODE (ZIP) : 11030 (G) TELEPHONE: (516) 562-1115

(H) TELEFAX: (516) 562-2866

(ii) TITLE OF INVENTION: A METHOD FOR INHIBITING BACTEREMIA AND

BACTERIAL DISSEMINATION

(iii) NUMBER OF SEQUENCES: 1

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/491,759

(B) FILING DATE: 19-JUN-1995

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Ala Glu Leu Gin Gin Trp Leu Lys Pro Gly Leu Lys Val Leu Ser Ile 1 5 10 15

Ala Gin Ala His Ser Leu Asn Phe Ser Cys 20 25

Claims

What is claimed is:

I. A method of preventing or treating bacteremia in a patient comprising administering to said patient a pharmaceutically effective amount of a CD14 antagonist . 2. The method according to claim 1 wherein said antagonist is a soluble CD14 analogue.

3. The method according to claim 2 wherein the patient is administered an amount of said antagonist in the range of from about 1 to 20-fold excess over the amount of sCD14 in serum of a healthy individual.

4. The method according to claim 1 wherein said antagonist binds CD14.

5. The method according to claim 4 wherein said antagonist is a CD14 monoclonal antibody. 6. The method according to claim 4 wherein the antagonist is a peptide.

7. The method according to claim 6 wherein the peptide comprises a dipeptide sequence selected from the group consisting of: ALA-HIS, ARG-TRP, ALA-TRP, ALA-GLN, VAL-TRP, THR-TRP, ARG-TRP, ILE-TRP, SER-ARG, TYR-TRP, PHE-TRP, HIS-TRP, CYS-TRP, THR-CYS, ARG-CYS, ILE-CYS, TYR-CYS, HIS-CYS, CYS-CYS, and CYS-ARG.

8. The method according to claim 1 wherein the antagonist binds to LPS, LPS-binding protein or the LPS-LPS binding protein complex. 9. The method according to claim 1 wherein said compound is administered to the patient prior to performance of an invasive medical treatment of the patient, during treatment, following treatment or a combination thereof. IO*. The method according to claim 9 wherein the medical treatment is peritoneal dialysis.

II. The method according to claim 9 wherein the medical treatment is surgery.

12. The method according to claim 1 wherein the antagonist is administered to a patient suffering from a localized bacterial infection.

13. The method according to claim 12 wherein the antagonist is administered to a patient suffering from a localized gram-negative bacterial infection. 14. A method for preventing the translocation of bacteria from the gastrointestinal tract comprising administering to a patient in need thereof a therapeutically effective amount of a CD14 antagonist.

15. A transgenic mouse which expresses human CD14 but not mouse CD14.