The invention relates to anti-inflammatory peptides that are based on peptide regions 7-10, 11-14, and 57-64 of CD14.

FIELD OF THE INVENTION 
Generally, the invention relates to the field of peptides that have 
anti-inflammatory properties. These peptides were designed based on amino 
acids 57 to 64, 7 to 10 and 11 to 14, inclusive, of the cell-surface 
antigen CD14. Amino acids 57 to 64 of CD14 were found to correspond to the 
binding domain of CD14 to lipopolysacharride (LPS, also referred to as 
endotoxin). Amino acids 7 to 10 and 11 to 14 of CD14 were found to be an 
important CD14 domain for inflammatory responses in cells, including IL-6 
production. The peptides of the invention may be used to treat 
inflammatory conditions, such as sepsis, and to detect LPS in samples. 
BACKGROUND OF THE INVENTION 
Sepsis is a life-threatening medical condition that can be brought on by 
infection or trauma. The symptoms of sepsis can include chills, profuse 
sweating, fever, weakness, or hypotension, followed by leukopenia, 
intravascular coagulation, shock, adult respiratory distress syndrome, 
multiple organ failure, and often, death. R. Ulevitch, et al., J. Trauma 
30: S189-92 (1990). 
The symptoms of sepsis can be induced by certain released microbes during 
infection or trauma. Some pathogenic bacteria, viruses, and plants produce 
such sepsis-inducing substances. 
The lipopolysaccharides ("LPS"; also, "endotoxins") that are typically 
present on the outer membrane of all gram-negative bacteria are among the 
most studied and best understood sepsis-inducing substances. While the 
precise chemical structures of LPS molecules obtained from different 
bacteria may vary in a species-specific fashion, a region called the lipid 
A region is common to all LPS molecules. E. Rietschel et al., in Handbook 
of Endotoxins, 1: 187-214, eds. R. Proctor and E. Rietschel, Elsevier, 
Amsterdam (1984). This lipid A region is responsible for many, if not all, 
of the LPS-dependent pathophysiologic changes that characterize sepsis. 
LPS is believed to be a primary cause of death in humans afflicted with 
gram-negative sepsis. van Deventer et al., Lancet, 1: 605 (1988); Ziegler 
et al., J. Infect. Dis., 136: 19-28 (1987). Treatment of patients 
suffering from sepsis and gram-negative bacteraemia with a monoclonal 
antibody against LPS decreased their mortality rate. Ziegler et al., N. 
Eng. J. Med., 324: 429 (1991). 
Sepsis is also caused by gram-positive bacteria. Bone, R. C. Arch. Intern, 
Med., 154: 26-34 (1994). The activation of host cells can originate from 
gram-positive cell walls or purified cell components such as peptidoglycan 
and lipoteichoic acid. Such substances induce a similar pattern of 
inflammatory responses to those induced by LPS. Chin and Kostura, J. 
Immunol. 151: 5574-5585 (1993); Mattson et al., FEMS Immun. Med. 
Microbiol. 7: 281-288 (1993); and Rotta, J. Z. Immunol. Forsch. Bd.: 149: 
230-244 (1975).). 
LPS and gram-positive cell wall substances cause polymorphonuclear 
leukocytes, endothelial cells, and cells of the monocyte/macrophage 
lineage to rapidly produce and release a variety of cell products, 
including cytokines, which are capable of initiating, modulating or 
mediating humoral and cellular immune responses and processes. 
One particular cytokine, alpha-cachectin or tumor necrosis factor (TNF), is 
apparently a primary mediator of septic shock. Beutler et al., N. Eng. J. 
Med., 316: 379 (1987). Intravenous injection of LPS into experimental 
animals and man produces a rapid, transient release of TNF. Beutler et 
al,, J. Immunol., 135: 3972 (1985); Mathison et al., J. Clin. Invest. 81: 
1925 (1988). Pretreatment of animals with anti-TNF antibodies can modulate 
septic shock. Beutler et al., Science, 229: 869, (1985); Mathison et al., 
J. Clin. Invest. 81: 1925 (1988). 
Molecular receptors that can combine with sepsis inducing substances, and 
that once combined, initiate certain chemical reactions, play a critical 
role in the etiology of the symptoms of sepsis. CD14 is a 55-kD 
glycoprotein expressed strongly on the surface of monocytes and 
macrophages, and weakly on the surface of granulocytes, such as 
neutrophils. S. M. Goyert et al., J. Immunol. 137: 3909 (1986). A. Haziot 
et al., J. Immunol. 141: 547-552 (1988); S. M. Goyert et al., Science 239: 
497 (1988). CD14 is linked by a cleavable glycosyl phosphatidyl inositol 
tail A. Haziot et al., J. Immunol. 141: 547-552 (1988)! to the exoplasmic 
surface of mature monocytes, macrophages, granulocytes and dendritic 
reticulum cells, or renal nonglomerular endothelium, and of hepatocytes in 
rejected livers. A soluble form of CD14 is present in normal sera and in 
the urine of nephrotic patients. Bazil et al., Eur. J. Immunol. 16: 1583 
(1986). 
CD14 plays a crucial role in mediating responses of cells to LPS. Treatment 
of monocytes (Wright, S. D., et al., Science 90: 1431-1433 (1990)) or PMN 
(Wright, S. D., et al., J. Exp. Med. 173: 1281-1286 (1991)) with 
monoclonal antibodies against CD14 blocks their responses to LPS. Several 
cell types, such as endothelial cells and astrocytes, respond to LPS but 
do not express CD14. These cells nevertheless require CD14, and sCD14 from 
the plasma mediates this response (Frey, E. A., et al., J. Exp. Med. 176: 
1665-1671 (1992)). These observations have been confirmed with a wide 
number of cell types and animal species, and have been confirmed with 
assays of a large variety of in vitro responses to LPS. Importantly, 
animals injected with anti-CD14 become hyporesponsive to LPS and mice 
lacking CD14 fail to respond to LPS. 
CD14 mediates responses by binding to LPS. Complexes of LPS and sCD14 
exhibit a 1:1 stoichiometry (Hailman, E., et al., J. Exp. Med. 179: 
269-277 (1994)), and these complexes initiate TNF production in monocytes 
(Dentener, M. A., et al., J. Immunol. 7: 2885-2891 (1993)), IL-6 
production in astrocytes (Frey, E., et al., Ibid. (1992)), production of 
adhesion molecules in endothelial cells (Frey, E., et al., Ibid. (1992)) 
and activation of leukocyte integrins in PMN (Hailman, E., et al., Ibid. 
(1994)). Spontaneous binding of LPS to CD14 is slow, but this binding may 
be dramatically accelerated by lipopolysaccharide binding protein LBP. LBP 
acts in a catalytic fashion, with one molecule of LBP transferring 
hundreds of LPS molecules to hundreds of CD14 molecules. 
Other experiments have shown that cell activation can also be induced by 
interaction of CD14 with components of gram-positive bacteria such as B. 
subtilis, S. aureus, and S. mitus (Pugin et al., Immunity 1: 509-516 
(1994). Furthermore, interaction of CD14 with lipoarabinomannan from the 
cell wall of Mycobacterium tuberculosis also induces cellular activation 
in a CD14 dependent fashion (Zhang et al., J. Clin. Invest. 91: 2076-2083 
(1993); Pugin et al., Immunity 1: 509-516 (1994)). These studies suggest 
that CD14 is a receptor which recognizes a wide variety of bacterial 
structures. Interaction of CD14 with these structures initiates host 
inflammatory responses. 
From the preceding background, it is evident that preventing interaction of 
CD14 with microbial structures could reduce inflammatory responses in 
leukocytes, endothelial and epithelial cells. Indeed, neutralizing mAbs to 
CD14 antagonize cellular responses to LPS, lipoarabinomannan and 
gram-positive cell wall components in vitro (Pugin et al., Immunity 1: 
509-516 (1994)) and recent reports have shown that CD14 mAbs are also 
effective in vivo. These observations suggest that CD14 may be an 
important pharmacologic target for diseases mediated by LPS, 
lipoarabinomannan and gram-positive bacterial components. 
The cDNAs and the genes for human and murine CD14 have been cloned and 
sequenced. E. Ferrero and S. M. Goyert, Nuc. Acids Res. 16: 4173 (1988); 
S. M. Goyert et al., Science 239: 497 (1988); M. Setoguchi et al., 
Biochem. Biophys. Acta 1008: 213-22 (1989). The sequence analysis revealed 
that CD14 belongs to a family of leucine-rich membrane-bound and soluble 
proteins that have receptor and cell adhesive functions. M. Setoguchi et 
al., Biochem. Biophys. Acta 1008: 213-22 (1989); E. Ferrero, et al., J. 
Immunol. 145: 133 (1990). 
The human CD14 protein sequence contains five potential sites for N-linked 
glycosylation and contains a 10 fold repeat of a leucine rich motif 
(LXXLXLX). There is a 66% amino acid sequence identity between the murine 
and human CD14s. 
In situ chromosomal hybridization of the .sup.3 H-labeled cDNA probe to 
normal human metaphase cells resulted in specific labeling only of 
chromosome 5. S.M. Goyert et al., Science 239: 497 (1988). The labeled 
sites were clustered at regions 5q22-q32 of this chromosome. The largest 
cluster of grains was located at 5q23-q31. S. M. Goyert et al., Science 
239: 497 (1988). This region of human chromosome 5 is known to contain a 
cluster of genes that encode several myeloid-specific growth factors or 
growth factor receptors, as well as other growth factor and receptor 
genes. S. M. Goyert et al., Science 239: 497 (1988). The mapping of the 
CD14 gene to this region of chromosome 5, its expression preferentially by 
mature myeloid cells, and its deletion in the malignant cells of patients 
having myeloid leukemias and del(5q) suggest that the CD14 antigen may 
play a role in the pathogenesis of myeloid disorders. 
The murine gene is located on mouse chromosome 18, which like the human 
gene also contains at least five genes encoding receptors. M. Setoguchi, 
H. Nasu, S. Yoshida, Y. Higuchi, S. Akizuki, and S. Yamamoto, Biochem. 
Biophys. Acta 1008: 213-22 (1989); E. Ferrero, C. L. Hsieh, U. Francke and 
S. M. Goyert, J. Immunol. 145: 133 (1990). 
For the preceding reasons, it is an object of this invention to develop 
methods and therapies for the effective treatment, including prevention, 
for symptoms of inflammatory conditions, including sepsis. It is also an 
object of this invention to develop methods and therapies for the 
effective protection of individuals who are at risk of becoming afflicted 
by the symptoms of inflammation, including sepsis. 
It is another object of this invention to develop methods and therapies for 
the effective treatment, including prevention, of symptoms of diseases 
that are mediated by LPS, gram-negative bacteraemia, gram-positive cell 
components, gram-positive bacteraemia, mycobacterial lipoarabinomannan, 
mycobacterial infections and/or CD14. Such diseases include ARDS, acute 
pancreatitis, acute and chronic liver failure, intestinal or liver 
transplantation, inflammatory bowel disease, graft vs. host disease in 
bone marrow transplantation and tuberculosis. 
SUMMARY OF THE INVENTION 
The present inventors have discovered a first group of peptides that are 
capable of binding to lipopolysaccharide, resulting in inhibition of the 
binding of LPS or gram-positive cell components to CD14, thus reducing or 
eliminating CD14 mediated inflammatory responses. As used herein, 
inhibition of binding of LPS also means inhibition of binding of 
gram-positive cell components. This first group of peptides was designed 
by the inventors based on their important discovery, disclosed herein, of 
an LPS-binding domain in CD14 to LPS. This first group of peptides is 
capable of binding to LPS thereby preventing further binding of microbial 
cell components to CD14. If microbial cell interaction with CD14 is 
prevented, the cascade of events leading to inflammation, and especially 
sepsis, are reduced or prevented. Therefore, the peptides of this 
invention have anti-inflammatory properties. 
A second group of peptides is expected to be capable of preventing LPS (or 
gram positive cell components) stimulation of inflammatory responses, such 
as IL-6 production. These peptides were designed based on the discovery 
that there are two additional important peptide regions in CD14: amino 
acids 7 to 10 and 11 to 14. The evidence provided herein shows, inter 
alia, that a CD14 mutant having the region from amino acids 7 to 10 in 
CD14 replaced with alanine residues mediates substantially reduced 
cellular production of the inflammatory cytokine IL-6 in response to LPS, 
as compared to native CD14 (See Example 10). Moreover, the regions 7-10 
and 11-14 in CD14 are recognized by the neutralizing anti-CD14 antibody 
3C10 (See Example 9). 
The first group of peptides of the present invention may be linear or 
cyclic. The linear first group peptides of this invention comprise the 
following amino acid sequence: 
EQU H.sub.2 N-X.sub.1 -X.sub.2 -X.sub.3 -X.sub.4 -X.sub.5 -X.sub.6 -X.sub.7 
-X.sub.8 -COOH (SEQ ID NO:46), 
wherein, 
X.sub.1 is selected from the group consisting of Asp and Glu; 
X.sub.2 is selected from the group consisting of Ala and Ser; 
X.sub.3 is selected from the group consisting of Asp and Glu; 
X.sub.4 is selected from the group consisting of Pro and Gly; 
X.sub.5 is selected from the group consisting of Arg and Lys; 
X.sub.6 is selected from the group consisting of Gln, Asn and His; 
X.sub.7 is selected from the group consisting of Tyr, Trp and Phe; 
X.sub.8 is selected from the group consisting of Ala and Ser; and 
physiologically acceptable salts thereof; and further wherein said peptide 
binds to lipopolysaccharide. 
A particularly preferred linear peptide is: 
EQU H.sub.2 N-Asp-Ala-Asp-Pro-Arg-Gln-Tyr-Ala-COOH (SEQ ID NO.1) 
which corresponds to amino acids 57-64 of native human CD14. These are the 
amino acids in CD14 primarily involved in binding to LPS. 
The cyclic first group peptides of this invention comprise any of the 
following amino acid sequences: 
##STR1## 
wherein, X.sub.1 is selected from the group consisting of Asp and Glu; 
X.sub.2 is selected from the group consisting of Pro and Gly; 
X.sub.3 is selected from the group consisting of Arg and Lys; 
X.sub.4 is selected from the group consisting of Gln, Asn and His; 
X.sub.5 is selected from the group consisting of Tyr, Trp and Phe; 
X.sub.6 is selected from the group consisting of Ala and Ser; 
n is from 1 to 3; and physiologically acceptable salts thereof; and further 
wherein said peptide binds to lipopolysaccharide. 
##STR2## 
wherein, X.sub.1 is selected from the group consisting of Ala and Ser; 
X.sub.2 is selected from the group consisting of Asp and Glu; 
X.sub.3 is selected from the group consisting of Pro and Gly; 
X.sub.4 is selected from the group consisting of Arg and Lys; 
X.sub.5 is selected from the group consisting of Gln, Asn and His; 
X.sub.6 is selected from the group consisting of Tyr, Trp and Phe; 
X.sub.7 is selected from the group consisting of Ala and Ser; 
X.sub.8 is selected from the group consisting of Asp and Glu; 
n is from 1 to 3; and physiologically acceptable salts thereof; and further 
wherein said peptide binds to lipopolysaccharide. 
##STR3## 
wherein, X.sub.1 is selected from the group consisting of Asp and Glu; 
X.sub.2 is selected from the group consisting of Ala and Ser; 
X.sub.3 is selected from the group consisting of Asp and Glu; 
X.sub.4 is selected from the group consisting of Pro and Gly; 
X.sub.5 is selected from the group consisting of Arg and Lys; 
X.sub.6 is selected from the group consisting of Gln, Asn and His; 
X.sub.7 is selected from the group consisting of Tyr, Trp and Phe; 
X.sub.8 is selected from the group consisting of Ala and Ser; 
X.sub.9 is selected from the group consisting of Asp and Glu; 
X.sub.10 is selected from the group consisting of Thr and Ser; 
n is from 1 to 3; and physiologically acceptable salts thereof; and further 
wherein said peptide binds to lipopolysaccharide. 
Specifically preferred cyclic first group peptides are the following: 
##STR4## 
and, in each case, physiologically acceptable salts thereof. 
The second group of peptides comprises the following amino acid sequences: 
##STR5## 
wherein n is from 1 to 3 (preferably 1); or 
##STR6## 
and, in each case, physiologically acceptable salts thereof. 
The peptides of this invention may be prepared by (a) standard synthetic 
methods, (b) derivation from CD14, (c) recombinant methods, (d) a 
combination of one or more of (a)-(c), or other methods of preparing 
peptides. 
The peptides of this invention may be used for therapeutic or prophylactic 
purposes by incorporating them into appropriate pharmaceutical carrier 
materials and administering an effective amount to a patient, such as a 
human (or other mammal) in need thereof. 
Since the peptides of this invention are capable of binding to LPS or 
gram-positive cell components, they may also be bound to a support 
material and used to remove LPS or gram-positive cell components from a 
sample, such as a body fluid. Further, in labeled form they may be used to 
detect and/or quantitate LPS or gram-positive cell components in a sample, 
such as a body fluid. Accordingly, kits containing one or more of these 
peptides may be supplied for diagnostic or purification purposes. 
The invention also relates to antibodies, including monoclonal antibodies, 
to the peptides of this invention, and to hybridoma cell lines that 
produce the monoclonal antibodies. Since the antibodies bind to the domain 
of CD14 that enables binding of CD14 to LPS or gram-positive cell 
components, the antibodies inhibit or prevent binding of cell-bound CD14 
to LPS or gram-positive cell components and hence, are useful to treat 
inflammatory conditions. Such antibodies may also be used to detect and/or 
quantitate LPS or gram positive cell components in a sample, such as a 
body fluid.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention is based on the discovery by the present inventors of 
(a) at least one portion of CD14 that is necessary for binding of CD14 to 
LPS or gram-positive cell components and (b) additional regions on CD14 
that are involved in inflammatory cellular responses mediated by CD14, 
such as production of IL-6 in response to gram negative or gram positive 
bacterial cell components (e.g., LPS from gram negative bacteria). The 
Examples below explain in detail the evidence supporting these 
discoveries. 
FIG. 1 shows a map of human CD14, including the LPS binding region, 
corresponding to amino acids 57-64, and the IL-6 inducing regions, 
corresponding to amino acids 7 to 10 and 11 to 14. 
The peptides of this invention may be linear or cyclic, of either or a 
mixture of D- or L-stereo chemistry (preferably all L-), chemically 
modified (as defined below), and in the form of physiologically acceptable 
salts (e.g., acetate or trifluoroacetate). 
The linear peptides comprise the following amino acid sequence: 
EQU H.sub.2 N-X.sub.1 -X.sub.2 -X.sub.3 -X.sub.4 -X.sub.5 -X.sub.6 -X.sub.7 
-X.sub.8 -COOH 
wherein, 
X.sub.1 is selected from the group consisting of Asp and Ser; 
X.sub.2 is selected from the group consisting of Ala, and Glu; 
X.sub.3 is selected from the group consisting of Asp and Glu; 
X.sub.4 is selected from the group consisting of Pro and Gly; 
X.sub.5 is selected from the group consisting of Arg and Lys; 
X.sub.6 is selected from the group consisting of Gln, Asn and His; 
X.sub.7 is selected from the group consisting of Tyr, Trp and Phe; 
X.sub.8 is selected from the group consisting of Ala and Ser; and 
physiologically acceptable salts thereof; and further wherein said peptide 
binds to lipopolysaccharide. 
As used herein "comprising" means that a peptide may include additional 
amino acids on either or both of the N- or C-termini of the given 
sequence. Preferably, these additional amino acids will correspond to the 
adjacent amino acids in CD14, as shown in such as FIG. 1. However, as long 
as the minimal structure such as X.sub.1 to X.sub.8, shown above, is 
present, the remaining chemical structure is relatively unimportant. Of 
course, any structure outside of the core, e.g., X.sub.1 to X.sub.8, 
structure should not significantly interfere with LPS or gram-positive 
cell component binding by the peptide. The peptides of this invention are 
generally from 8 to 60 amino acids in length, preferably 8 to 12 amino 
acids in length. 
Some exemplary linear peptides of this invention are: 
H.sub.2 N-Asp-Ala-Asp-Pro-Arg-Gln-Tyr-Ala-COOH (SEQ ID NO.7) 
H.sub.2 N-Asp-Ala-Glu-Pro-Arg-Gln-Tyr-Ala-COOH (SEQ ID NO.8) 
H.sub.2 N-Asp-Ala-Glu-Pro-Arg-Asn-Tyr-Ala-COOH (SEQ ID NO:9) 
H.sub.2 N-Asp-Ala-Glu-Pro-Arg-Gln-Phe-Ala-COOH (SEQ ID NO:10) 
H.sub.2 N-Asp-Ala-Glu-Pro-Arg-Asn-Phe-Ala-COOH (SEQ ID NO.11) 
H.sub.2 N-Asp-Ala-Asp-Pro-Arg-Asn-Tyr-Ala-COOH (SEQ ID NO.12) 
H.sub.2 N-Asp-Ala-Asp-Pro-Arg-Asn-Phe-Ala-COOH (SEQ ID NO.13) 
H.sub.2 N-Asp-Ala-Asp-Pro-Arg-Gln-Phe-Ala-COOH (SEQ ID NO.14) 
H.sub.2 N-Glu-Leu-Asp-Asp-Glu-Asp-Phe-Arg-COOH (SEQ ID NO.15) 
Additional linear peptides useful for the purposes of this invention is the 
peptide corresponding to amino acids 7 to 64, inclusive, of the amino acid 
sequence of CD14, as shown in FIG. 1, and physiologically acceptable salts 
thereof. 
The cyclic peptides of this invention comprise one of the following basic 
structures: 
##STR7## 
wherein, X.sub.1 is selected from the group consisting of Asp and Glu; 
X.sub.2 is selected from the group consisting of Pro and Gly; 
X.sub.3 is selected from the group consisting of Arg and Lys; 
X.sub.4 is selected from the group consisting of Gln, Asn and His; 
X.sub.5 is selected from the group consisting of Tyr, Trp and Phe; 
X.sub.6 is selected from the group consisting of Ala and Ser; 
n is from 1 to 3; and physiologically acceptable salts thereof; and further 
wherein said peptide binds to lipopolysaccharide. 
##STR8## 
wherein, X.sub.1 is selected from the group consisting of Ala and Ser; 
X.sub.2 is selected from the group consisting of Asp and Glu; 
X.sub.3 is selected from the group consisting of Pro and Gly; 
X.sub.4 is selected from the group consisting of Arg and Lys; 
X.sub.5 is selected from the group consisting of Gln, Asn and His; 
X.sub.6 is selected from the group consisting of Tyr, Trp and Phe; 
X.sub.7 is selected from the group consisting of Ala and Ser; 
X.sub.8 is selected from the group consisting of Asp and Glu; 
n is from 1 to 3; and physiologically acceptable salts thereof; and further 
wherein said peptide binds to lipopolysaccharide. 
##STR9## 
wherein, X.sub.1 is selected from the group consisting of Asp and Glu; 
X.sub.2 is selected from the group consisting of Ala and Ser; 
X.sub.3 is selected from the group consisting of Asp and Glu; 
X.sub.4 is selected from the group consisting of Pro and Gly; 
X.sub.5 is selected from the group consisting of Arg and Lys; 
X.sub.6 is selected from the group consisting of Gln, Asn and His; 
X.sub.7 is selected from the group consisting of Tyr, Trp and Phe; 
X.sub.8 is selected from the group consisting of Ala and Ser; 
X.sub.9 is selected from the group consisting of Asp and Glu; 
X.sub.10 is selected from the group consisting of Thr and Ser; 
n is from 1 to 3; and physiologically acceptable salts thereof; and further 
wherein said peptide binds to lipopolysaccharide. 
Some exemplary cyclic peptides of this invention are: 
##STR10## 
and, in each case, physiologically acceptable salts thereof. 
For the above structures, it will be understood that 
##STR11## 
refers to the structure 
##STR12## 
The second group of peptides comprises the following amino acid sequences: 
##STR13## 
wherein n is from 1 to 3; or 
##STR14## 
and, in each case, physiologically acceptable salts thereof. These 
peptides are expected to have the ability to inhibit inflammatory 
responses in cells, which may conveniently be measured by reduction of 
IL-6 production by such cells using, e.g., the method described in Example 
10 below. Preferably the amount of IL-6 reduction will be at least 5-fold, 
particularly preferably, at least 10-fold (compared to when no peptide is 
present; see Example 10). 
In each of the above cases, n is preferably 1. Also, in each case, the 
amino acids may be chemically derivatized as long as LPS binding or IL-6 
inducing activity is not destroyed. Thus, "chemical derivatives" of the 
present peptides are included within the scope of the term "peptide" as 
used herein. These chemical derivatives contain additional chemical 
moieties not part of the unmodified peptide. 
Covalent modifications of the peptide are included within the scope of this 
invention. Such modifications may be introduced into the molecule by 
reacting targeted amino acid residues of the peptide with an organic 
derivatizing agent that is capable of reacting with selected side chains 
or terminal residues. 
Histidyl residues are derivatized by reaction with diethylpyrocarbonate at 
pH 5.5-7.0 because this agent is relatively specific for the histidyl side 
chain. Para-bromophenacyl bromide also is useful; the reaction is 
preferably performed in 0.1M sodium cacodylate at pH 6.0. 
Lysinyl and amino terminal residues are reacted with succinic or other 
carboxylic acid anhydrides. Derivatization with these agents has the 
effect of reversing the charge of the lysinyl residues. Other suitable 
reagents for derivatizing alpha-amino-containing residues include 
imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; 
chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 
pentanedione; and transaminase-catalyzed reaction with glyoxylate. 
Arginyl residues are modified by reaction with one or several conventional 
reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, 
and ninhydrin. Derivatization of arginine residues requires that the 
reaction be performed in alkaline conditions because of the high pK a of 
the guanidine functional group. Furthermore, these reagents may react with 
the groups of lysine as well as the arginine epsilon-amino group. 
The specific modification of tyrosyl residues per se has been studied 
extensively, with particular interest in introducing spectral labels into 
tyrosyl residues by reaction with aromatic diazonium compounds or 
tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane 
are used to form O-acetyl tyrosyl species and 3-nitro derivatives, 
respectively. 
Carboxyl side groups (aspartyl or glutamyl) are selectively modified by 
reaction with carbodiimides (R'--N--C--N--R') such as 
1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 
1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, 
aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl 
residues by reaction with ammonium ions. 
Glutaminyl and asparaginyl residues are frequently deamidated to the 
corresponding glutamyl and aspartyl residues. Alternatively, these 
residues are deamidated under mildly acidic conditions. Either form of 
these residues falls within the scope of this invention. 
Derivatization with bifunctional agents is useful for cross-linking the 
peptides or their functional derivatives to a water-insoluble support 
matrix or to other macromolecular carriers. Commonly used cross-linking 
agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, 
N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic 
acid, homobifunctional imidoesters, including disuccinimidyl esters such 
as 3,3'-dithiobis (succinimidylpropionate), and bifunctional maleimides 
such as bis-N-maleimido-1,8-octane. Derivatizing agents such as 
methyl-3-(p-azidophenyl)dithio!propioimidate yield photoactivatable 
intermediates that are capable of forming crosslinks in the presence of 
light. Alternatively, reactive water-insoluble matrices such as cyanogen 
bromide-activated carbohydrates and the reactive substrates described in 
U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 
4,330,440 are employed for protein immobilization. 
Other modifications include hydroxylation of proline and lysine, 
phosphorylation of hydroxyl groups of seryl or threonyl residues, 
oxidation of the sulfur atom in Cys, methylation of the alpha-amino groups 
of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: 
Structure and Molecule Properties, W. H. Freeman & Co., San Francisco, pp. 
79-86 (1983)), acetylation of the N-terminal amine, and, in some 
instances, amidation of the C-terminal carboxyl groups. 
The activity of the peptide variant can be screened in a suitable screening 
assay for the desired characteristic. Biological activity is screened in 
an appropriate bioassay, as described herein. For example, binding of LPS 
to CD14 may be measured in a standard competitive binding assay. Activity 
to reduce cellular inflammatory responses may be measured in terms of 
reduction of IL-6 production by cells (e.g., U373 cells) as described 
herein. 
Modifications of such peptide properties as redox or thermal stability, 
hydrophobicity, susceptibility to proteolytic degradation or the tendency 
to aggregate with carriers or into multimers are assayed by methods well 
known to the ordinarily skilled artisan. 
Such derivatized moieties may improve the solubility, absorption, 
biological half life, and the like. The moieties may alternatively 
eliminate or attenuate any undesirable side effect of the protein and the 
like. Moieties capable of mediating such effects are disclosed, for 
example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing 
Co., Easton, Pa. (1980). 
The peptides of the invention may also be covalently or noncovalently 
associated with a carrier molecule, such as a peptide or non-CD14 protein, 
a linear polymer (such as polyethylene glycol, polylysine, etc.), a 
branched-chain polymer (see, for example, U.S. Pat. No. 4,289,872 to 
Denkenwalter et al., issued Sep. 15, 1981; U.S. Pat. No. 5,229,490 to Tam, 
issued Jul. 20, 1993; WO 93/21259 by Frechet et al., published 28 Oct. 
1993); a lipid; a cholesterol group (such as a steroid; or a carbohydrate 
or oligosaccharide. 
The peptides of the first group (and also possibly the second group) of 
this invention are expected to have the ability to bind to LPS. This 
binding renders LPS unable to bind to CD14 and therefore produces an 
anti-inflammatory responses response in a mammal. They are also expected 
to bind to cellular components of gram positive cells that cause 
inflammatory (analogous to LPS; however, the structure(s) in gram positive 
bacteria that cause inflammatory responses to cells is (are) not yet 
known). "Binding" to LPS means that in a standard competition assay, the 
peptide is capable of inhibiting 50% binding of CD14 to LPS between 1 mM 
and 1 nM, preferably 100 .mu.m to 10 nM (IC.sub.50 values). A binding 
assay such as that in FIG. 4 may be carried out as is well known in the 
art. 
The peptides of this invention may be made in a variety of ways. For 
example, solid phase synthesis techniques may be used. Suitable techniques 
are well known in the art, and include those described in Merrifield, in 
Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds. 1973); 
Merrifield, J. Am. Chem. Soc., 85, 2149 (1963); Davis et al., Biochem. 
Int'l, 10, 394-414 (1985); Stewart and Young, Solid Phase Peptide 
Synthesis (1969); U.S. Pat. No. 3,941,763; Finn et al., in The Proteins, 
3rd ed., vol. 2, pp. 105-253 (1976); and Erickson et al. in The Proteins, 
3rd ed., vol. 2, pp. 257-527 (1976). Solid phase synthesis is the 
preferred technique of making individual peptides since it is the most 
cost-effective method of making small peptides. 
The peptide may also be made in transformed host cells using recombinant 
DNA techniques. To do so, a recombinant DNA molecule coding for the 
peptide is prepared. Methods of preparing such DNA molecules are well 
known in the art. For instance, sequences coding for the peptides could be 
excised from DNA using suitable restriction enzymes. Alternatively, the 
DNA molecule could be synthesized using chemical synthesis techniques, 
such as the phosphoramidite method. Also, a combination of these 
techniques could be used. 
The invention also includes a vector capable of expressing the peptides in 
an appropriate host. The vector comprises the DNA molecule that codes for 
the peptides operatively linked to appropriate expression control 
sequences. Methods of effecting this operative linking, either before or 
after the DNA molecule is inserted into the vector, are well known. 
Expression control sequences include promoters, activators, enhancers, 
operators, ribosomal binding sites, start signals, stop signals, cap 
signals, polyadenylation signals, and other signals involved with the 
control of transcription or translation. 
The resulting vector having the DNA molecule thereon is used to transform 
an appropriate host. This transformation may be performed using methods 
well known in the art. 
Any of a large number of available and well-known host cells may be used in 
the practice of this invention. The selection of a particular host is 
dependent upon a number of factors recognized by the art. These include, 
for example, compatibility with the chosen expression vector, toxicity to 
it of the peptides encoded for by the DNA molecule, rate of 
transformation, ease of recovery of the peptides, expression 
characteristics, bio-safety and costs. A balance of these factors must be 
struck with the understanding that not all hosts may be equally effective 
for the expression of a particular DNA sequence. 
Within these general guidelines, useful microbial hosts include bacteria 
(such as E. coli sp.), yeast (such as Saccharomyces sp.) and other fungi, 
insects, plants, mammalian (including human) cells in culture, or other 
hosts known in the art. 
Next, the transformed host is cultured under conventional fermentation 
conditions so that the desired peptides are expressed. Such fermentation 
conditions are well known in the art. 
Finally, the peptides are purified from the culture. These purification 
methods are also well known in the art. 
To synthesize the cyclic peptides, the procedure set forth in Example 8 may 
be used. 
The peptides of this invention may be used in any of a number of situations 
where LPS/gram positive cell component binding is required. For example, 
therapeutically and prophylactically, the peptides may be used for 
inflammatory bowel disease, acute and chronic liver failure, graft vs. 
host disease (bone marrow transplant), intestinal or liver transplant, 
ARDS, acute pancreatitis and tuberculosis. Septic shock is a particularly 
preferred target condition. 
The novel peptides are useful for the prophylaxis or treatment of septic 
shock in mammals, including humans, at doses of about 0.1 to 100 mg/kg of 
body weight, preferably at a level of about 1 to 50 mg/kg of body weight, 
and the amount may be administered, e.g., in divided doses on daily basis. 
The peptides may be administered prophylactically to patients who may be 
exposed to or have been exposed to organisms which may cause septic shock 
or to detoxify LPS (bacterial endotoxins) by the use of the same dose set 
forth above in vivo, In vitro detoxification or prevention of endotoxin 
contamination may be carried out at a level which is effective to achieve 
the desired result. The amount may be based on routine experimentation 
based on the premise that about 1 mole of endotoxin is bound by 1 mole of 
peptide. The particular dose of a particular peptide may be varied within 
or without the range that is specified herein depending on the particular 
application or severity of a disease and the condition of the host. Those 
who are skilled in the art may ascertain the proper dose using standard 
procedures. 
The protein or pharmaceutical compositions of the present invention may be 
administered by any means that achieve their intended purpose. For 
example, administration may be by parenteral routes, including 
subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, 
intrathecal, transdermal, or buccal routes. Alternatively, or 
concurrently, administration may be by the oral or rectal route. The 
proteins and pharmaceutical compositions can be administered parenterally 
by bolus injection or by gradual perfusion over time. 
In addition to the peptide, these pharmaceutical compositions may contain 
suitable pharmaceutically acceptable carriers comprising excipients and 
auxiliaries which facilitate processing of the active compounds into 
preparations which can be used pharmaceutically. Preferably, the 
preparations, particularly those which can be administered orally and 
which can be used for the preferred type of administration, such as 
tablets, dragees, and capsules, and also preparations which can be 
administered rectally, such as suppositories, as well as suitable 
solutions for administration by injection or orally, contain from about 
0.1 to about 99 percent, preferably from about 25-85 percent, of active 
compound(s), together with the excipient. 
Suitable excipients are, in particular, fillers such as sugars, such as 
lactose, sucrose, mannitol, or sorbitol; cellulose preparations and/or 
calcium phosphates, such as tricalcium phosphate or calcium hydrogen 
phosphate; as well as binders such as starch paste made using, for 
example, maize starch, wheat starch, rice starch, potato starch, gelatin, 
gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium 
carboxymethyl cellulose, and/or polyvinylpyrrolidone. If desired, 
disintegrating agents may also be added, such as the above-mentioned 
starches as well as carboxymethyl starch, cross-linked polyvinyl 
pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium 
alginate. Auxiliaries which can be used in the compositions according to 
the present invention include flow-regulating agents and lubricants such 
as silica, talc, stearic acid or salts thereof, and/or polyethylene 
glycol. 
The peptides of this invention are also useful to detect the presence of 
inflammatory gram positive or gram negative bacterial cell components, 
such as LPS from gram negative bacteria, in a sample such as a body fluid 
(i.e., blood, urine, CSF, saliva, etc.) or any other sample that might 
contain LPS or a gram positive cell component. Standard methodology can be 
used to carry out such a test. 
Also included within the scope of the present invention is an antibody 
specific for the peptides disclosed herein, or specific for a functional 
derivative thereof. 
The term "antibody" refers both to monoclonal antibodies which are a 
substantially homogeneous population and to polyclonal antibodies which 
are heterogeneous populations. Polyclonal antibodies are derived from the 
sera of animals immunized with an antigen. Monoclonal antibodies (mAbs) to 
specific antigens may be obtained by methods known to those skilled in the 
art. See, for example Kohler and Milstein, Nature 256:495-497 (1975) and 
U.S. Pat. No. 4,376,110. Such antibodies may be of any immunoglobulin 
class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. 
The term "antibody" is also meant to include both intact molecules as well 
as fragments thereof, such as, for example, Fab and F(ab')2, which are 
capable of binding antigen. Fab and F(ab')2 fragments lack the Fc fragment 
of intact antibody, clear more rapidly from the circulation, and may have 
less non-specific tissue binding than an intact antibody (Wahl et al., J. 
Nucl. Med. 24:316-325 (1983)). 
It will be appreciated that Fab and F(ab')2 and other fragments of the 
antibodies useful in the present invention may be used for the detection 
and quantitation of the protein of the present invention in the same 
manner as an intact antibody. Such fragments are typically produced by 
proteolytic cleavage, using enzymes such as papain (to produce Fab 
fragments) or pepsin (to produce F(ab')2 fragments). 
An antibody is said to be "capable of binding" a molecule if it is capable 
of specifically reacting with the molecule to thereby bind the molecule to 
the antibody. The term "epitope" is meant to refer to that portion of any 
molecule capable of being bound by an antibody which can also be 
recognized by that antibody. Epitopic determinants usually consist of 
chemically active surface groupings of molecules such as amino acids or 
sugar side chains and have specific three dimensional structural 
characteristics as well as specific charge characteristics. 
An "antigen" is a molecule or a portion of a molecule capable of being 
bound by an antibody which is additionally capable of inducing an animal 
to produce antibody capable of binding to an epitope of that antigen. An 
antigen may have one, or more than one epitope. The specific reaction 
referred to above is meant to indicate that the antigen will react, in a 
highly selective manner, with its corresponding antibody and not with the 
multitude of other antibodies which may be evoked by other antigens. 
One group of antibodies of this invention are those that bind specifically 
to the epitopes consisting at most of amino acids 7-14 or 57-64 of CD14. 
Although the existence and importance of these epitopic regions has not 
been appreciated prior to this invention, the inventors recognize that the 
MEM18 antibody (see Examples section) has as its epitope, amino acids 
57-64, and antibody 3C10 has as its epitope(s) amino acids 7-10 and 11-14 
of CD14. Thus, these antibodies are excluded from the claims herein. 
Nevertheless, until the present invention, it was not appreciated whether 
and how to make any additional antibodies against these epitopes. For 
example, prior to this invention, anti-CD14 antibodies were generally made 
by employing full-length or soluble CD14 as the antigen. The present 
invention makes it possible to generate additional antibodies against 
these important epitopes by using the peptides of this invention as 
antigens, thus insuring that substantially all of the generated antibodies 
will be against the desired epitope(s). 
The antibodies may be useful therapeutically in the same manner as the 
peptides described herein, and, accordingly, that disclosure is to be read 
as including the antibodies also. 
The antibodies, or fragments of antibodies, of the present invention may be 
further used to quantitatively or qualitatively detect the presence of the 
peptides disclosed herein, or of intact CD14 containing the epitope 
recognized by the antibody. For example, it would be of benefit to monitor 
the level of a peptide in the circulation or in the tissues of a subject 
receiving therapeutic doses of the peptide. 
An assay for the peptides disclosed herein typically comprises incubating a 
biological sample from the subject in the presence of a detectably labeled 
antibody or antibody fragment capable of identifying the protein and 
detecting the antibody which is bound in the sample. 
Thus, in this aspect of the invention, a biological sample may be treated 
with nitrocellulose, or other solid support which is capable of 
immobilizing cells, cell particles or soluble proteins. The support may 
then be washed with suitable buffers followed by treatment with the 
detectably labeled specific antibody. The solid phase support may then be 
washed with the buffer a second time to remove unbound antibody. The 
amount of bound label on said solid support may then be detected by 
conventional means. 
By "solid phase support" or "carrier" is intended any support capable of 
binding antigen (i.e., the peptide) or antibodies. Well-known supports, or 
carriers, include glass, polystyrene, polypropylene, polyethylene, 
dextran, nylon, amylases, natural and modified celluloses, 
polyacrylamides, agaroses, and magnetite. The nature of the carrier can be 
either soluble to some extent or insoluble for the purposes of the present 
invention. The support material may have virtually any possible structural 
configuration so long as the coupled molecule is capable of binding to the 
antibody. These support materials may also be used to immobilize the 
peptides for uses such as detection or removal of LPS from samples. 
The binding activity of an antibody specific for the peptides disclosed 
herein may be determined according to well known methods, such as enzyme 
immunoassay (EIA) or radioimmunoassay (RIA). Those skilled in the art will 
be able to determine operative and optimal assay conditions for each 
determination by employing routine experimentation. 
For EIA, the antibody is detectably labeled by linking to an enzyme. This 
enzyme, in turn, when later exposed to its substrate, will react with the 
substrate in such a manner as to produce a chemical moiety which can be 
detected, for example, by spectrophotometric, fluorometric or by visual 
means. Enzymes which can be used to detectably label the antibody include, 
but are not limited to, malate dehydrogenase, staphylococcal nuclease, 
delta-V-steroid isomerase, yeast alcohol dehydrogenase, 
alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, 
horseradish peroxidase, alkaline phosphatase, asparaginase, glucose 
oxidase, beta-galactosidase, ribonuclease, urease, catalase, 
glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. 
By radioactively labeling the antibody or peptide, it is possible to detect 
binding through the use of a RIA. See, for example: Weintraub, B., 
Principles of Radioimmunoassays, Seventh Training Course on Radioligand 
Assay Techniques, The Endocrine Society, March, 1986, pp. 1-5, 46-49 and 
68-78; Work, T. S. et al., Laboratory Techniques and Biochemistry in 
Molecular Biology, North Holland Publishing Company, NY, 1978. The 
radioactive isotope can be detected by such means as the use of a gamma 
counter or a scintillation counter or by autoradiography. 
It is also possible to label the antibody or peptide with a fluorescent 
compound. When the fluorescently labeled species is exposed to light of 
the proper wavelength, its presence can then be detected due to 
fluorescence. Among the most commonly used fluorescent labeling compounds 
are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, 
allophycocyanin, o-phthaldehyde and fluorescamine. 
The antibody or peptide can also be detectably labeled using fluorescence 
emitting metals such as &lt;152&gt; Eu, or others of the lanthanide series. 
These metals can be attached to the antibody or peptide using such metal 
chelating groups as diethylenetriaminepentaacetic acid (DTPA) or 
ethylenediaminetetraacetic acid (EDTA). 
The antibody or peptide can also be detectably labeled by coupling it to a 
chemiluminescent compound. The presence of the chemiluminescent-tagged 
species is then determined by detecting the presence of luminescence that 
arises during the course of a chemical reaction. Examples of particularly 
useful chemiluminescent labeling compounds are luminol, isoluminol, the 
aromatic acridinium ester, imidazole, acridinium salt and oxalate ester. 
Likewise, a bioluminescent compound may be used to label the antibody or 
peptide of the present invention. Bioluminescence is a type of 
chemiluminescence found in biological systems in which a catalytic protein 
increases the efficiency of the chemiluminescent reaction. The presence of 
a bioluminescent protein is determined by detecting the presence of 
luminescence. Important bioluminescent compounds for purposes of labeling 
are luciferin, luciferase and aequorin. 
It is understood that the application of the teachings of the present 
invention to a specific problem or situation will be within the 
capabilities of one having ordinary skill in the art in light of the 
teachings contained herein. Examples of the products of the present 
invention and representative processes for their isolation, use, and 
manufacture appear below. 
EXAMPLES 
Materials and Methods 
Reagents 
Recombinant soluble CD14 (rsCD14) and recombinant LBP (rLBP) were 
constructed and purified as described (Hailman, E., et al. J. Exp. Med. 
179, 269-277 (1994). Concentrations of all purified proteins were 
determined with a Micro BCA protein kit (Pierce, Rockford, Ill.) according 
to manufacturer's specification. Since full-length rsCD14 terminates at 
position 348 of the mature protein (Hailman et al., Ibid. (1994)), we 
herein refer to it as sCD14.sub.1-348. The anti-CD14 mAbs used were 3C10 
purified by chromatography on Protein G from the conditioned medium (CM) 
of ATCC TIB 228!, MEM-18 (SANBIO, The Netherlands), My4 (Coulter 
Immunology, Hialeah, Fla.), and 60b (Todd, R. F., et al., Hybridoma 1, 
329-337 (1982). Rabbit polyclonal anti-human CD14 antiserum was raised 
against sCD14.sub.1-348 and was prepared by Antibodies, Inc. (Davis, 
Calif.). Enzymes for DNA manipulation and polymerase chain reaction (PCR) 
were purchased from Boehringer Mannheim (Indianapolis, Ind.). p-nitro blue 
tetrazolium chloride (NBT), 5-bromo-4-chloro-3-indolyl phosphate salt 
(BCIP), and alkaline phosphatase-conjugated goat anti-rabbit IgG were 
purchased from BioRad (Richmond, Calif.). 
Site-directed Mutagenesis 
A cDNA which encodes mutant sCD14 lacking amino acids 57-64 
(sCD14.sub..DELTA.57-64) was constructed using a Transformer site-directed 
mutagenesis kit (Clontech, Palo Alto, Calif.) according to the protocol 
specified by the manufacturer. Briefly, mutation primer 
(5'-TAAAGCGCGTCGATGCGGACACGGTCAAGGCTC-TCC-3') (SEQ ID NO.16) and selection 
primer (Trans oligo Ssp 1/EcoR V, Clontech) were annealed to a mammalian 
expression vector (pDSR.alpha.2) containing the CDNA for sCD14.sub.1-348 
(Hailman et al., Ibid. (1994)). Primers were extended and ligated using T4 
DNA polymerase/T4 DNA ligase for 2 h. The reaction was digested with Ssp I 
to linearize un-mutated wild-type plasmids and undigested circular 
plasmids which contained mutagenized DNA were transformed into E. coli 
strain DH5.alpha.. Plasmid DNA was isolated from transformants and DNA 
sequence analysis verified the presence of the deletion. 
The Transformer site-directed mutagenesis kit was also used to generate 
mutant cDNAs encoding sCD14 having alanine substituted at various position 
between amino acids 59 and 65. For these experiments, the following mutant 
primers were used: 
5'-GATGCGGACGCCGCCCCTAGGCAGTATGCTGACACG-3' (SEQ ID NO.17) for 
sCD14.sub.D59A, 
5'-GATGCGGACGCCGACGCGCGGCAGTATGCTGAC-3' (SEQ ID NO.18) for sCD14.sub.P60A, 
5'-GCGGACGCCGACCCTGCGCAGTATGCTGACAC-3' (SEQ ID NO.19) for sCD14.sub.R61A, 
5'-GACGCCGACCCGCGAGCGTATGCTGACACGGTC-3' (SEQ ID NO.20) for sCD14.sub.Q62A, 
5'-CGCCGACCCGCGTCAGGCTGCTGACACGGTTCAAG-3' (SEQ ID NO.21) for 
sCD14.sub.Y63A, 
5'-CCGCGGCAGTATGCTGCCACGGTCAAGGCTCTCC-3' (SEQ ID NO.22) for sCD14.sub.D65A, 
and 
5'-GTCGATGCGGACGCCGCCGCGGCGGCGGCTGCTGCCACGGTCAAGGCTCTCCGC-3' (SEQ ID NO.23) 
for sCD14.sub.(59-65)A. Introduction of the appropriate mutation in all 
cDNAs was confirmed by DNA sequencing. 
Transient Expression of Mutant sCD14 Proteins in COS-7 Cells 
To express mutant sCD14 proteins, mammalian expression vectors containing 
mutant sCD14, cDNAs were introduced into COS-7 (ATCC CRL 1651) cells by 
electroporation. Expression of mutant sCD14 was analyzed by Western blot 
and the concentration of mutant proteins was determined with the aid of a 
BIAcore biosensor instrument (Pharmacia Biosensor, Piscataway, N.J.) using 
protocols described by the manufacturer. 
Purification of sCD14.sub..DELTA.57-64 
The expression vector containing the cDNA encoding sCD14.sub..DELTA.57-64 
was stably transfected into Chinese hamster ovary (CHO) cells deficient in 
dihydrofolate reductase as described (Hailman, E., et al., J. Exp. Med. 
179, 269-277 (1994)). A single clone was grown without serum to generate 
CM containing sCD14.sub..DELTA.57-64. Mutant protein was purified by 
immunoaffinity chromatography on a column to which mAb 3C10 was coupled to 
Sepharose 4B (Pharmacia, Piscataway, N.J.). Briefly, CM was concentrated 
20.times. using a S10Y10 spiral-wound cartridge (Amicon, Beverly, Mass.). 
Concentrated CM was passed over the column pre-equilibrated with 
phosphate-buffered saline (PBS, GIBCO-BRL) and protein was monitored by 
following the absorbance at 280 nm. The column was washed with PBS until 
the absorbance reached baseline. The protein was then eluted with 0.1M 
glycine-HCl, pH 2.5 into collection tubes containing 0.5M phosphate, pH 
8.0. sCD14.sub..DELTA.57-64 -containing fractions were pooled, 
concentrated, and diafiltered into PBS in a Centriprep-10 (Amicon, 
Beverly, Mass.) concentrator. Purity of the sample was checked by 
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver 
staining or Coomassie Blue staining. 
Native PAGE Assay 
Variations of a previously described native PAGE assay (Hailman, et al., 
Ibid. (1994) and the description of FIG. 2) were used to assess whether 
unpurified or purified sCD14 preparations bound LPS. For unpurified sCD14 
expressed in CM of COS-7 cells, 30 .mu.l of CM was incubated at 37.degree. 
C. with various amounts of LPS (Salmonella minnesota type Re595, List 
Biological Laboratories, Campbell, Calif.) for 30 min and mixtures were 
resolved by native PAGE on 10% gels (Noval Experimental Technologies). 
Proteins were then transferred to nitrocellulose membranes and Western 
blot analysis was performed as previously described (Hailman et al., Ibid. 
(1994)) using anti-human CD14 polyclonal antibody. 
To assess LPS-binding of purified sCD14 preparations, sCD14.sub.1-348 or 
sCD14.sub..DELTA.57-64 were incubated at various concentrations (0, 101, 
303, and 909 nM) with 3 .mu.g/ml of .sup.3 H-LPS prepared from E. coli K12 
strain LCD25 (List Biological Laboratories, Menlo Park, Calif.) in the 
presence or absence of 16.7 nM rLBP. The reaction was incubated at 
37.degree. C. for 30 min and then electrophoresed on native 4-20% 
polyacrylamide gels. Gels were prepared for fluorography as previously 
described (Hailman, et al., Ibid. (1994)). 
Experiments were also performed to determine whether various CD14 mAbs 
could compete with LPS for binding to sCD14. In these studies, .sup.3 
H-LPS-sCD14 complexes were formed by incubating 130 .mu.g/ml 
sCD14.sub.1-348 with 10 .mu.g/ml .sup.3 H-LPS for 15 h at 37.degree. C. in 
PBS with 1 mM EDTA. Complexes were then diluted 10-fold and incubated 20 
min at 37.degree. C. with 200 .mu.g/ml various mAbs in a total volume of 
10 .mu.l. Mixtures were then electrophoresed on 8-16% native gels and 
processed for fluorography as above. 
In other experiments, we examined whether rLBP could lower the effective 
dose of LPS required to competitively inhibit binding of MEM-18 or 3C10 to 
sCD14. MEM-18 or 3C10 (40 .mu.g/ml) was incubated with sCD14.sub.1-348 
(2.6 .mu.g/ml) for 10 min at 37.degree. C. Various concentrations of LPS 
(from S. minnesota strain R60, List Biological Laboratories) were then 
added in the presence or absence of rLBP (1 .mu.g/ml) for 20 min at 
37.degree. C. in a total volume of 10 .mu.l. Mixtures were then 
electrophoresed on 8-16% native gels and transferred to nitrocellulose in 
Tris-glycine buffer with 20% methanol. The nitrocellulose was blocked in 
PBS with 10% dry milk, and incubated with polyclonal antibodies in PBS 
with 0.1% dry milk. CD14 was detected using a rabbit polyclonal antibody 
(generous gift of Dr. Pat Detmers) raised against sCD14.sub.1-348 and an 
alkaline phosphatase-conjugated secondary antibody. Bound antibody was 
detected using NBT and BCIP according to the manufacturer's instruction. 
Activation of Polymorphonuclear Leukocytes (PMN) by LPS and sCD14 
The ability of rLBP and sCD14.sub..DELTA.57-64 or sCD14.sub.1-348 to enable 
PMN adhesion to fibrinogen-coated plates was assessed by previously 
established protocols (Hailman, et al., Ibid. (1994) and Detmers et al., 
J. Immunol. 152:2137-2145 (1994)). Briefly, PMN were incubated for 10 min 
with LPS, LBP, and sCD14.sub.1-348, washed and adhesion to 
fibrinogen-coated surfaces was measured. 
U373 Bioassays 
Growth of U373 cells, activation by COS-7 CM containing sCD14 or by 
purified sCD14 preparations, and quantitation of IL-6 were performed 
exactly as described (Frey, et al., J. Exp. Med. 176:1665-1671 (1992)). 
Briefly, mixtures of sCD14.sub.1-348 and LPS were added to monolayers of 
U373 cells in serum-free medium and incubated for 24 h. IL-6 in the 
supernatant was then measured by ELISA. 
BIAcore Analyses of Interactions Between sCD14 and anti-CD14 mAbs 
Recognition of sCD14 preparations by anti-CD14 mAbs was performed with a 
BIAcore biosensor instrument. The instrument, CM5 sensor chips, and amine 
coupling kit were purchased from Pharmacia Biosensor (Piscataway, N.J.). 
Briefly, mAb 3C10 (200 .mu.g/ml in 20 mM sodium acetate, pH 3.4) was 
immobilized to a CM5 sensor chip by amine coupling according to 
manufacturer's specifications. The flow cell immobilized with 3C10 was 
then incubated in succession with 4 solutions as detailed in the following 
steps: Step 1, COS-7 CM or 10 .mu.g/ml purified sCD14.sub..DELTA.57-64 for 
2 min; Step 2, HBS buffer 10 mM 
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.5, 0.15M NaCl, 
3.4 mM EDTA, 0.005% (V/V) surfactant P20 (Pharmacia Biosensor)! for 2 min; 
Step 3, 50 .mu.g/ml MEM-18 (in HBS buffer) for 2-3 min; Step 4, HBS buffer 
for 2 min. All solutions were injected at a flow rate of 5 .mu.l/min. To 
quantitate the binding of MEM-18 to sCD14 mutants in COS-7 CM, we 
calculated a relative response unit (RRU). RRU was obtained by subtracting 
the response unit (RU) recorded just before injection of MEM-18 from the 
RU recorded after injection of MEM-18 and a 2 min wash. Since there are 
slight differences in the concentrations of sCD14 proteins expressed in 
CM, and since the signal is linearly related to the concentration of sCD14 
under the condition employed, we present binding data as RRU per nM sCD14 
mutants. 
Example 1 
LPS Does Not Bind to sCD14.sub..DELTA.57-64 Expressed in COS-7 CM 
We have found that endoproteinase AspN cleaves sCD14 before aspartic acid 
residues 57, 59, 64 and that these cleavage sites are masked when LPS is 
complexed to sCD14 (data not shown). This observation suggested that the 
region between amino acids 57 and 65 could be important for LPS binding. 
To test this hypothesis, we utilized the technique of site-directed 
mutagenesis to construct a cDNA which encodes mutant sCD14 
(sCD14.sub..DELTA.57-64) lacking amino acids 57-64. A mammalian expression 
vector containing sCD14.sub..DELTA.57-64 was transiently transfected into 
COS-7 cells and serum-free CM was collected. Western blot analysis (data 
not shown) revealed that expression of sCD14.sub..DELTA.57-64 was 
comparable to expression of sCD14.sub.1-348 in COS-7 transfected cells. 
We then used a native PAGE assay to assess whether sCD14.sub..DELTA.57-64 
present in COS-7 CM binds LPS. CM containing sCD14.sub.1-348 or 
sCD14.sub..DELTA.57-64 were incubated with increasing amounts of LPS and 
the mixtures were electrophoretically transferred to nitrocellulose 
membranes. sCD14 or sCD14-LPS complexes were then detected with anti-CD14 
polyclonal antiserum. FIG. 2 shows that LPS caused a shift in the 
electrophoretic mobility of sCD14.sub.1-348, and this shift is caused by 
binding of LPS to CD14 (Hailman, et al., Ibid. (1994)). In contrast, no 
shift was observed in CM containing sCD14.sub..DELTA.57-64 even at an LPS 
concentration 5-fold higher then that needed to completely shift 
sCD14.sub.1-348. These results are consistent with the notion that amino 
acids 57-64 in sCD14 are necessary for LPS binding. 
Example 2 
Purification and Characterization of sCD14.sub..DELTA.57-64 
To further characterize the LPS-binding and biological activities of 
sCD14.sub..DELTA.57-64, it was necessary to purify large quantities of the 
mutant protein. Therefore, a stable CHO cell line expressing 
sCD14.sub..DELTA.57-64 was constructed and mutant protein was purified 
from the serum-free CM of this cell line. FIG. 3 shows that purified 
sCD14.sub..DELTA.57-64 migrated with an apparent Mr of 55,000 when 
analyzed by reducing SDS-PAGE. In order to determine whether the deletion 
in sCD14.sub..DELTA.57-64 affected protein structure, we analyzed purified 
sCD14.sub..DELTA.57-64 and sCD14.sub.1-348 by CD. Both the far and near UV 
spectra of the sCD14.sub..DELTA.57-64 were within experimental error of 
sCD14.sub.1-348, as was the thermal stability determined by change in CD 
signal with temperature (data not shown). This result suggests that the 
deletion does not significantly interfere with the folding structure or 
stability of sCD14. Furthermore, the data also imply that Tyr at position 
63 does not contribute to the spectrum of the native protein and that this 
residue therefore is located in a flexible or symmetrical environment. 
This is consistent with the theory that the region between amino acids 
57-64 is a flexible bridge connecting two compactly folded domains. 
Example 3 
Purified sCD14.sub..DELTA.57-64 Does Not Form a Stable Complex with LPS 
To directly address whether sCD14.sub..DELTA.57-64 is capable of binding 
LPS, we used the native PAGE assay to detect stable complexes between 
sCD14.sub.1-348 or sCD14.sub..DELTA.57-64 and .sup.3 H-LPS. Incubation of 
sCD14.sub.1-348 with LPS for 30 min lead to stable complexes (FIG. 4A), 
and addition of rLBP dramatically accelerated this process (FIG. 4B). In 
contrast, even the highest concentration of sCD14.sub..DELTA.57-64 did not 
support complex formation with .sup.3 H-LPS in the absence (FIG. 4A, lanes 
5-7) or presence (FIG. 4B, lanes 5-7) of rLBP. These data confirm that 
sCD14.sub..DELTA.57-64 fails to bind LPS. 
Example 4 
sCD14.sub..DELTA.57-64 Does not Enable Cellular Responses to LPS 
The lack of interaction between sCD14.sub..DELTA.57-64 and LPS suggested 
that the mutant protein would be impaired in its ability to enable 
cellular responses to LPS. We tested this hypothesis by using two 
previously characterized assays (Hailman, E., et al., J. Exp. Med. 179, 
269-277 (1994); Juan, T. S., et al., J. Biol. Chem. in press; Frey, E. A., 
et al., J. Exp. Med. 176, 1665-1671 (1992)) which measure sCD14 function. 
In the first assay, we examined whether sCD14.sub..DELTA.57-64 could 
enable LPS-induced adhesion of PMN to fibrinogen. FIG. 5 shows that 100 
ng/ml sCD14.sub.1-348 enabled a strong adhesive response of PMN to smooth 
LPS and rLBP. However, no response was seen even when 10,000 ng/ml 
sCD14.sub..DELTA.57-64 was added. 
We also examined the ability of sCD14.sub..DELTA.57-64 to support responses 
of U373 cells to LPS. Addition of as little as 5 ng/ml sCD14.sub.1-348 
enabled strong IL-6 production in response to LPS (FIG. 6), confirming 
previous reports (Frey, E. A., et al., J. Exp. Med. 176, 1665-1671 
(1992)). In contrast, sCD14.sub..DELTA.57-64 failed to support 
LPS-dependent IL-6 production even at a concentration of 80 ng/ml. These 
findings confirm that residues 57-64 are crucial to the biological 
function of sCD14.sub.1-348. 
Example 5 
An Epitope for mAb MEM-18 is Localized to Amino Acids 57-64 
The importance of amino acids 57-64 for LPS-binding suggested that this 
domain could be the site of interaction between neutralizing mAbs against 
CD14. Since mAb 3C10 was used to withdraw sCD14.sub..DELTA.57-64 from CM 
(FIG. 3), we reasoned that 3C10 must recognize an epitope outside amino 
acids 57-64. This was confirmed in a BIAcore analysis in which we observed 
a signal indicative of binding upon adding sCD14.sub..DELTA.57-64 to 
immobilized 3C10 (FIG. 7A, compare signal at 0 second to that at 480 sec). 
We used further BIAcore analysis to test whether a different neutralizing 
mAb, MEM-18, could interact with the bound sCD14.sub..DELTA.57-64. 
However, no binding of MEM-18 was observed to sCD14.sub..DELTA.57-64 (FIG. 
7A, compare signal at 480 sec to that at 800 sec). To confirm this result, 
we used Western blot analysis. While MEM-18 bound immobilized 
sCD14.sub.1-348, it failed to bind sCD14.sub..DELTA.57-64 (FIG. 7B). 
Parallel studies performed with polyclonal anti-CD14 confirmed the 
presence of sCD14.sub..DELTA.57-64 on the nitrocellulose membrane. These 
results suggest that MEM-18 recognizes an epitope in the region of amino 
acids 57-64. 
In an attempt to further characterize the MEM-18 epitope, we constructed a 
series of cDNAs encoding sCD14 having alanine substituted at various 
positions between amino acids 59-65. FIG. 8 summarizes the corresponding 
amino acid changes in each mutant construct. Mammalian expression vectors 
containing each mutant cDNA were transiently transfected into COS-7 cells 
and expression of the mutant protein in CM was monitored by Western blot 
analysis. No differences in expression of mutant sCD14 proteins were 
observed in COS-7 CM (data not shown). Therefore, we performed BIAcore 
analyses to test the ability of each CM containing mutant sCD14 to bind 
MEM-18. Immobilized mAb 3C10 recognized each of the constructs but 
sCD14.sub.D59A, sCD14.sub.Q62A, sCD14.sub.Y63A, and sCD14.sub.(59-65)A 
were not recognized by MEM-18 (FIG. 9). Binding of MEM-18 was not affected 
if Arg.sup.61 or Asp.sup.65 was mutated and substitution of alanine at 
Pro.sup.60 partially inhibited MEM-18 binding. In summary, we have 
demonstrated that MEM-18 recognizes an epitope which is minimally 
comprised of residues Asp.sup.59, Gln.sup.62, and Tyr.sup.63. 
Example 6 
LPS Competes for the Same Site on CD14 as MEM-18 
The localization of the MEM-18 epitope to a region we have implicated in 
LPS-binding suggests that LPS should compete with MEM-18 for binding to 
sCD14.sub.1-348. To demonstrate this, we measured the ability of MEM-18 to 
bind pre-formed LPS-sCD14.sub.1-348 complexes (FIG. 10A) and the ability 
of LPS to bind pre-formed sCD14.sub.1-348 -MEM-18 complexes (FIG. 10B). 
sCD14.sub.1-348 (FIG. 10B) and complexes of .sup.3 H-LPS and 
sCD14.sub.1-348 (FIG. 10A, lane 2) showed mobility characteristic of a 
50-kDa protein and this mobility was not affected by an irrelevant 
antibody (anti-CD18, FIG. 10A, lane 7). Addition of anti-CD14 mAbs MEM-18, 
My4, 60b, or 3C10 each caused a quantative "supershift" in the mobility of 
sCD14.sub.1-348 to a position consistent with a 250-kDa complex of IgG 
with two molecules of sCD14.sub.1-348 (FIG. 10B and data not shown). We 
further observed that a subset of these mAbs (MY4, 60b, and 3C10) also 
shifted the mobility of the .sup.3 H-LPS-sCD14.sub.1-348 complexes (FIG. 
10A). These observations indicate that MY4, 60b, and 3C10 bind to 
LPS-sCD14.sub.1-348 complexes and therefore do not compete with LPS for a 
binding site. In contrast, MEM-18 failed to shift the mobility of .sup.3 
H-LPS in LPS-sCD14.sub.1-348 complexes (FIG. 10A, lane 6) using conditions 
that caused complete shifting of sCD14 to the higher molecular weight 
position in the absence of LPS (FIG. 10B and data not shown). This 
observation indicates that sCD14.sub.1-348 cannot simultaneously bind 
MEM-18 and LPS. 
To confirm and extend this observation, complexes of MEM-18 and 
sCD14.sub.1-348 were first formed. These complexes showed a mobility 
characteristic of a 250-kDa protein, confirming the efficacy of MEM-18 in 
the "supershift" assay (FIG. 10B, lane 3). Addition of increasing doses of 
LPS to these complexes caused dissociation of the sCD14.sub.1-348 from the 
MEM-18 in a dose-dependent fashion. Moreover, the efficacy of LPS in 
disassociating sCD14.sub.1-348 from MEM-18 was enhanced by rLBP, a protein 
that catalytically hastens the binding of LPS to sCD14.sub.1-348 (Hailman, 
et al., Ibid. (1994)). LPS did not cause disassociation of sCD14.sub.1-348 
from 3C10 (FIG. 10B), MY4 or 60b (data not shown), confirming that these 
mAbs do not compete with LPS for binding to sCD14.sub.1-348. These results 
further confirm that MEM-18 and LPS bind sCD14.sub.1-348 in a competitive 
fashion and may thus recognize overlapping sites. 
Example 7 
CD14 has an Amphipathic Domain Between Amino Acids 53 and 63 
It has been hypothesized (Hoess, A., et al., EMBO J. 12, 3351-3356 (1993)) 
that Limulus anti-LPS factor (LALF, (Warren, H. S., et al., Infect. Immun. 
60, 2506-2513 (1992)), LBP and bactericidal/permeability-increasing (BPI, 
Gazzano-Santoro, H., et al, Infect. Immun. 60, 4754-4761 (1992)) proteins 
possess amphipathic domains which are involved in binding LPS. Since the 
hydrophobic moment (m, Eisenberg, D., Ann. Rev. Biochem. 53, 595-623 
(1984)) is directly proportional to amphipathicity, we calculated m 
throughout CD14 and identified the region having the highest m. Table 1 
(see below) compares this region to analogous regions in LALF, LBP, and 
BPI. The region (amino acids 53-63) having the highest m in CD14 overlaps 
the site we have identified as being critical for LPS-binding. This region 
was similar to LALF, LBP, and BPI with respect to its overall pattern of 
alternating hydrophilic and hydrophobic residues. However, the amphipathic 
domain in CD14 did differ significantly from the other proteins with 
respect to its net charge. 
TABLE 1 
______________________________________ 
Comparison of region of highest amphipathicity in 
CD14 and other LPS-binding proteins. 
Amino acid Hydrophobic 
Net 
Protein Sequence.sup.1 Moment.sup.2 
Change.sup.3 
______________________________________ 
CD14 .sup.53 RVDADADPRQY.sup.63 
0.83 -1.09 
(SEQ ID NO.24) 
LALF .sup.32 RLKWKYKGKFW.sup.50 
1.05 +4.90 
(SEQ ID NO.25) 
LBP .sup.86 SIRVQGRWKVR.sup.104 
1.36 +3.91 
(SEQ ID NO.26) 
BPI .sup.86 NIKISGKWKAQ.sup.104 
1.00 +2.91 
(SEQ ID NO.27) 
______________________________________ 
.sup.1 Sequences are numbered beginning with the first amino acid of the 
mature protein. Large, boldfaced letters indicate hydrophilic amino acids 
.sup.2 Hydrophobic moments (for an 11residue window) were calculated with 
d = 100.degree. as described (Hoess, A., et al., EMBO J. 12, 3351-3356 
(1993)) using a computer program obtained from the laboratory of D. 
Eisenberg. 
.sup.3 Net charge was calculated using a Protean program (DNASTAR, 
Madison, WI). 
Discussion of Examples 1-7 
In Examples 1-7, the inventors provide compelling evidence that the region 
between amino acids 57 and 64 of sCD14 is essential for proper binding of 
LPS. Deletion of this region abolished the ability of sCD14 to bind LPS in 
the presence or absence of rLBP. Furthermore, an epitope recognized by 
neutralizing mAb MEM-18 was mapped to this region and we showed that this 
mAb competes with LPS for binding to sCD14. 
The data also demonstrate the biological consequences of impairing 
LPS-binding to sCD14. sCD14.sub..DELTA.57-64 was inactive in enabling PMN 
and U373 responses to LPS. These results suggest that binding of LPS to 
sCD14 is a prerequisite for the biological activity of CD14. This 
conclusion is consistent with the finding (Hailman, et al., J. Exp. Med. 
179:269-277 (1994)) that binding of LPS to sCD14.sub.1-348 is temporally 
correlated with biological activity. 
Example 8 
Preparation of the Peptides of the Invention 
The linear polypeptides were synthesized by the solid phase method using 
either the original t-Boc/benzyl protocol of Merrifield (R. B. Merrifield; 
J. Am. Chem. Soc. (1963), 85, 2149-2154) or using the Fmoc 
(fluorenyloxycarbonyl)/t-Bu method (L. A. Carpino and G. Y. Hahn; 
J.Org.Chem (1972), 37,3404). 
The syntheses were carried out by automated technology using either the 
Applied Biosystems Inc. 430A or 431A instruments which were programmed 
with the manufacturer's standard single coupling Fmoc or t-Boc protocols. 
Preloaded resins, Fmoc and t-Boc protected amino acids and other 
prepackaged reagents were purchased from Applied Biosystems Inc. (Foster 
City, Calif.). Cleavage from the resin support and simultaneous side chain 
deprotection was accomplished by one of two methods depending on which 
synthetic protocol was used. 
Fmoc synthesis: Four hour treatment with 90% trifluoroacetic acid, 2.5% 
thioanisole, 2.5% 2-mercaptoethanol, 2.5% phenol, 2.5% H.sub.2 O, followed 
by concentration and ether precipitation. 
T-Boc synthesis: One hour treatment with 95% liquid HF and 5% m-cresol for 
one hour at 0 degrees Celsius. The HF was removed under reduced pressure 
and the peptide precipitated with ether. 
The crude peptides were purified by high pressure liquid chromatography. 
Characterization consisted of analytical HPLC, amino acid analysis and 
electrospray mass spectroscopy. 
In addition, synthesis of the following cyclic structure is detailed below 
to illustrate cyclic peptide synthesis: 
EQU cyclo-S-Ac-Asp-Pro-Arg-Gln-Tyr-Ala-Cys-COOH! 
The method used has been previously described by Barker et.al. (P. L. 
Barker et.al. J. Med. Chem (1992), 35, 2040) and Robey and Fields (F. A. 
Robey and R. L. Fields Anal.Biochem. (1989), 177,373) 
Synthesis of cyclo-S-Ac-Asp-Pro-Arg-Gln-Tyr-Ala-Cys-OH! 
The sequence H.sub.2 N-Asp-Pro-Arg-Gln-Tyr-Ala-Cys was assembled by 
stepwise Fmoc chemistry (as described above) and derivatized with 
bromoacetic acid and DCC (dicyclohexylcarbodiimide) to form 
BrAc-Asp-Pro-Arg-Gln-Tyr-Ala-Cys-COOH. The peptide was cleaved from the 
resin (as above) and cyclized under basic (pH 8) aqueous conditions to 
form the thioether cyclo-S-Ac-Asp-Pro-Arg-Gln-Tyr-Ala-Cys-COOH!. The 
material was purified by preparative HPLC. Mass spectral data 
(electrospray): expected mass=892 observed: m/e 891 and 892. Analytical 
HPLC: C-18 column, 5-50% B over 35 mins. Solvent A: 0.1% TFA; solventB 50% 
0.1% TFA, 50% acetonitrile) Elution time of 
cyclo-S-Ac-Asp-Pro-Arg-Gln-Tyr-Ala-Cys-COOH!=9.78 min. 
Example 9 
Amino Acid Regions 7-10 and 11-14 are Recognized by Monoclonal Antibody 
3C10 as its Epitope on CD14 
We have demonstrated that the epitope for the blocking monoclonal antibody 
3C10 is located within the first 152 amino acid of CD14. To determine the 
binding epitope of this monoclonal antibody, we used site-directed 
mutagenesis to generate a series of CD14 mutants. Each mutant construct 
has 3 or 4 amino acid residues substituted with alanine. FIG. 11 
summarizes the site-directed mutants. The cDNAs encoding these mutants 
were transfected into COS-7 cells and conditioned media were collected and 
tested for expression of CD14 mutants. Except for the mutant construct 
sCD14.sub.(18-21)A, which was expressed approximately 20-fold less, all 
the mutant proteins were expressed at similar levels as determined by 
Western immunoblot. 
We tested the ability of these mutant proteins to bind 3C10 by using the 
BIAcore biosensor instrument, as described above. Monoclonal antibody was 
immobilized onto a sensor chip in the BIAcore biosensor. Conditioned media 
containing wildtype sCD14 as well as mutant sCD14 proteins were then 
injected onto this sensor chip. Binding of sCD14 proteins to immobilized 
3C10 will cause a change in the light reflection from the sensor chip and 
this change was recorded as relative response unit. FIG. 12 shows the 
relative response units of various constructs. Conditioned media 
containing no sCD14 (MOCK) shows very little relative response unit. In 
contrast, injection of conditioned media with sCD14.sub.1-348 (WT sCD14) 
resulted in a relative response unit of about 150. Interestingly, 
injection of conditioned media with mutants sCD14.sub.(7-10)A or 
SCD14.sub.(11-14)A did not generate an increase in the relative response 
unit, suggesting that 3C10 antibody failed to recognize these two mutant 
proteins. This antibody is capable of binding other mutant constructs as 
illustrated by FIG. 12. Therefore, we conclude that CD14 amino acid 
regions 7-10 and 11-14 (i.e., 7-14, inclusive) are recognized by 
monoclonal antibody 3C10. 
Example 10 
sCD14.sub.(7-10)A is Impaired in Transducing LPS Signal 
To further determine whether sCD14.sub.(7-10)A is capable of transducing 
LPS signal, we performed massive transient transfections in COS-7 cells 
and purified this sCD14 mutant to homogeneity from conditioned medium of 
transfected COS-7 cells. Various concentrations of purified 
sCD14.sub.(7-10)A were used to treat U373 cells in the presence or absence 
of LPS, and the data are shown in FIG. 13. The ability of inducing IL-6 
production by this protein in response to LPS treatment is dramatically 
decreased as compared to that of wildtype sCD14. This observation strongly 
suggests that the region of from amino acids 7 to 10 is important for LPS 
signalling. 
We also determined whether mutant sCD14.sub.(7-10)A is capable of 
activating transcription factor NF-.kappa.B in U373 cells in response to 
LPS stimulation. Without sCD14, LPS caused a minor NF-.kappa.B activation 
(compare lanes 2 and 3). Activation of NF-.kappa.B is greatly increased 
when sCD14.sub.1-348 or sCD14.sub.1-152 was added in the presence of LPS 
(lanes 5 and 7). However, this strong induction of NF-.kappa.B was not 
observed when cells were treated with sCD14.sub..DELTA.57-64 or 
sCD14.sub.(7-10)A (lanes 9 and 11). This example further shows the 
critical role in LPS of the region between amino acids 7 to 10 in 
transducing LPS signal into cells. 
Example 11 
sCD14(7-10)A is Able to Form A Stable Complex with .sup.3 H-LPS 
To determine whether mutant sCD14(7-10)A is capable of binding LPS and 
further confirm that the region from amino acids 7 to 10 is important for 
transducing LPS signal but not involved in LPS binding, we performed a 
native PAGE assay to detect a stable complex between sCD14.sub.1-348 or 
SCD14.sub.(7-10)A and .sup.3 H-LPS. Incubation of sCD14.sub.1-348 or 
SCD14.sub.(7-10)A with .sup.3 H-LPS leads to the formation of stable 
complexes of sCD14-.sup.3 H-LPS (FIG. 15). This data demonstrates that 
sCD14.sub.(7-10)A is able to bind LPS and that the region from amino acids 
7 to 10, which is important for LPS signalling, is not involved in LPS 
binding. 
Example 12 
Gram Positive Cell Components Compete with LPS for Binding to sCD14 
FIG. 16 presents the evidence that a gram-positive molecule present in the 
phenol extract of S. aureus (SACE) can bind to sCD14 and compete with LPS 
for a binding site. Other data (not shown) indicates that SACE strongly 
stimulates cells in a CD14-dependent fashion. The binding site(s) now 
defined on CD14 may be relevant not only to responses initiated by 
gram-negative but also by gram-positive bacteria. 
Abbreviations 
The abbreviations used in the Examples section above are: BCIP, 
5-bromo-4-chloro-3-indoyl phosphate-toluidine salt; BPI, 
bactericidal/permeability-increasing protein; CHO, Chinese hamster ovary; 
CD, circular dichroism; CM, conditioned medium; HBSS, Hank's balanced salt 
solution; IL-6, interleukin-6; LALF, Limulus anti-LPS factor; LBP, 
LPS-binding protein; LPS, lipopolysaccharide; NBT, p-nitro blue 
tetrazolium chloride; PAGE, polyacrylamide gel electrophoresis; PBS, 
phosphate-buffered saline; PMN, polymorphonuclear leukocyte; r, 
recombinant; RU, response unit; sCD14, soluble CD14; ELISA, enzyme linked 
immunosorbant assay. 
The invention now being fully described, it will be apparent to one of 
ordinary skill in the art that many changes and modifications can be made 
thereto, without departing from the spirit and scope of the invention as 
set forth herein. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 49 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AspAlaAspProArgGlnTyrAla 
15 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..7 
(D) OTHER INFORMATION: /note= "Amino acids 1 and 7 are 
linked via - CO-CH2-S-" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
AspProArgGlnTyrAlaCys 
15 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..9 
(D) OTHER INFORMATION: /note= "Amino acids 1 and 9 are 
linked together via -CO-CH2-S-" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
AlaAspProArgGlnTyrAlaAspCys 
15 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..11 
(D) OTHER INFORMATION: /note= "Amino acids 1 and 11 are 
linked together via -CO-CH2-S-" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
AspAlaAspProArgGlnTyrAlaAspThrCys 
1510 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..9 
(D) OTHER INFORMATION: /note= "Amino acids 1 and 9 are 
linked together via -CO-(CH2)n-S-" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GluLeuAspAspGluAspPheArgCys 
15 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
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(ix) FEATURE: 
(A) NAME/KEY: Disulfide-bond 
(B) LOCATION: 1..10 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
CysGluLeuAspAspGluAspPheArgCys 
1510 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
AspAlaAspProArgGlnTyrAla 
15 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
AspAlaGluProArgGlnTyrAla 
15 
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AspAlaGluProArgAsnTyrAla 
15 
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(A) LENGTH: 8 amino acids 
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AspAlaGluProArgGlnPheAla 
15 
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AspAlaGluProArgAsnPheAla 
15 
(2) INFORMATION FOR SEQ ID NO:12: 
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(A) LENGTH: 8 amino acids 
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AspAlaAspProArgAsnTyrAla 
15 
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(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
AspAlaAspProArgAsnPheAla 
15 
(2) INFORMATION FOR SEQ ID NO:14: 
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(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
AspAlaAspProArgGlnPheAla 
15 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
GluLeuAspAspGluAspPheArg 
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(2) INFORMATION FOR SEQ ID NO:16: 
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(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
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TAAAGCGCGTCGATGCGGACACGGTCAAGGCTCTCC36 
(2) INFORMATION FOR SEQ ID NO:17: 
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(A) LENGTH: 36 base pairs 
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GATGCGGACGCCGCCCCTAGGCAGTATGCTGACACG36 
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GATGCGGACGCCGACGCGCGGCAGTATGCTGAC33 
(2) INFORMATION FOR SEQ ID NO:19: 
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(A) LENGTH: 32 base pairs 
(B) TYPE: nucleic acid 
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GCGGACGCCGACCCTGCGCAGTATGCTGACAC32 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
GACGCCGACCCGCGAGCGTATGCTGACACGGTC33 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
CGCCGACCCGCGTCAGGCTGCTGACACGGTTCAAG35 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
CCGCGGCAGTATGCTGCCACGGTCAAGGCTCTCC34 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 54 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
GTCGATGCGGACGCCGCCGCGGCGGCGGCTGCTGCCACGGTCAAGGCTCTCCGC54 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
ArgValAspAlaAspAlaAspProArgGlnTyr 
1510 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
ArgLeuLysTrpLysTyrLysGlyLysPheTrp 
1510 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
SerIleArgValGlnGlyArgTrpLysValArg 
1510 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
AsnIleLysIleSerGlyLysTrpLysAlaGln 
1510 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
ArgValAspAlaAspAlaAspProArgGlnTyrAlaAspThrValLys 
151015 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
ArgValAspAlaAspAlaAlaProArgGlnTyrAlaAspThrValLys 
151015 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
ArgValAspAlaAspAlaAspAlaArgGlnTyrAlaAspThrValLys 
151015 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
ArgValAspAlaAspAlaAspProAlaGlnTyrAlaAspThrValLys 
151015 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
ArgValAspAlaAspAlaAspProArgGlnAlaAlaAspThrValLys 
151015 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
ArgValAspAlaAspAlaAspProArgGlnAlaAlaAspThrValLys 
151015 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
ArgValAspAlaAspAlaAspProArgGlnTyrAlaAlaThrValLys 
151015 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
ArgValAspAlaAspAlaAlaAlaAlaAlaAlaAlaAlaThrValLys 
151015 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 55 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
ThrThrProGluProCysGluLeuAspAspGluAspPheArgCysVal 
151015 
CysAsnPheSerGluProGlnProAspTrpSerGluAlaPheGlnCys 
202530 
ValSerAlaValGluValGluIleHisAlaGlyGlyLeuAsnLeuGlu 
354045 
ProPheLeuLysArgValAsp 
5055 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 55 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
ThrThrProGluProCysAlaAlaAlaAlaGluAspPheArgCysVal 
151015 
CysAsnPheSerGluProGlnProAspTrpSerGluAlaPheGlnCys 
202530 
ValSerAlaValGluValGluIleHisAlaGlyGlyLeuAsnLeuGlu 
354045 
ProPheLeuLysArgValAsp 
5055 
(2) INFORMATION FOR SEQ ID NO:38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 55 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
ThrThrProGluProCysGluLeuAspAspAlaAlaAlaAlaCysVal 
151015 
CysAsnPheSerGluProGlnProAspTrpSerGluAlaPheGlnCys 
202530 
ValSerAlaValGluValGluIleHisAlaGlyGlyLeuAsnLeuGlu 
354045 
ProPheLeuLysArgValAsp 
5055 
(2) INFORMATION FOR SEQ ID NO:39: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 55 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
ThrThrProGluProCysGluLeuAspAspGluAspPheArgCysVal 
151015 
CysAlaAlaAlaAlaProGlnProAspTrpSerGluAlaPheGlnCys 
202530 
ValSerAlaValGluValGluIleHisAlaGlyGlyLeuAsnLeuGlu 
354045 
ProPheLeuLysArgValAsp 
5055 
(2) INFORMATION FOR SEQ ID NO:40: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 55 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
ThrThrProGluProCysGluLeuAspAspGluAspPheArgCysVal 
151015 
CysAsnPheSerGluAlaAlaAlaAlaTrpSerGluAlaPheGlnCys 
202530 
ValSerAlaValGluValGluIleHisAlaGlyGlyLeuAsnLeuGlu 
354045 
ProPheLeuLysArgValAsp 
5055 
(2) INFORMATION FOR SEQ ID NO:41: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 55 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
ThrThrProGluProCysGluLeuAspAspGluAspPheArgCysVal 
151015 
CysAsnPheSerGluProGlnProAspAlaAlaAlaAlaPheGlnCys 
202530 
ValSerAlaValGluValGluIleHisAlaGlyGlyLeuAsnLeuGlu 
354045 
ProPheLeuLysArgValAsp 
5055 
(2) INFORMATION FOR SEQ ID NO:42: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 55 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
ThrThrProGluProCysGluLeuAspAspGluAspPheArgCysVal 
151015 
CysAsnPheSerGluProGlnProAspTrpSerGluAlaPheGlnCys 
202530 
ValSerAlaValGluValGluIleHisAlaGlyGlyAlaAlaAlaAla 
354045 
ProPheLeuLysArgValAsp 
5055 
(2) INFORMATION FOR SEQ ID NO:43: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 55 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
ThrThrProGluProCysGluLeuAspAspGluAspPheArgCysVal 
151015 
CysAsnPheSerGluProGlnProAspTrpSerGluAlaPheGlnCys 
202530 
ValSerAlaValGluValGluIleHisAlaGlyGlyAlaAlaAlaAla 
354045 
ProPheLeuLysArgValAsp 
5055 
(2) INFORMATION FOR SEQ ID NO:44: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 55 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
ThrThrProGluProCysGluLeuAspAspGluAspPheArgCysVal 
151015 
CysAsnPheSerGluProGlnProAspTrpSerGluAlaPheGlnCys 
202530 
ValSerAlaValGluValGluIleHisAlaGlyGlyLeuAsnLeuGlu 
354045 
AlaAlaAlaAlaArgValAsp 
5055 
(2) INFORMATION FOR SEQ ID NO:45: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 348 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
ThrThrProGluProCysGluLeuAspAspGluAspPheArgCysVal 
151015 
CysAsnPheSerGluProGlnProAspTrpSerGluAlaPheGlnCys 
202530 
ValSerAlaValGluValGluIleHisAlaGlyGlyLeuAsnLeuGlu 
354045 
ProPheLeuLysArgValAspAlaAspAlaAspProArgGlnTyrAla 
505560 
AspThrValLysAlaLeuArgValArgArgLeuThrValGlyAlaAla 
65707580 
GlnValProAlaGlnLeuLeuValGlyAlaLeuArgValLeuAlaTyr 
859095 
SerArgLeuLysGluLeuThrLeuGluAspLeuLysIleThrGlyThr 
100105110 
MetProProLeuProLeuGluAlaThrGlyLeuAlaLeuSerSerLeu 
115120125 
ArgLeuArgAsnValSerTrpAlaThrGlyArgSerTrpLeuAlaGlu 
130135140 
LeuGlnGlnTrpLeuLysProGlyLeuLysValLeuSerIleAlaGln 
145150155160 
AlaHisSerProAlaPheSerCysGluGlnValArgAlaPheProAla 
165170175 
LeuThrSerLeuAspLeuSerAspAsnProGlyLeuGlyGluArgGly 
180185190 
LeuMetAlaAlaLeuCysProHisLysPheProAlaIleGlnAsnLeu 
195200205 
AlaLeuArgAsnThrGlyMetGluThrProThrGlyValCysAlaAla 
210215220 
LeuAlaAlaAlaGlyValGlnProHisSerLeuAspLeuSerHisAsn 
225230235240 
SerLeuArgAlaThrValAsnProSerAlaProArgCysMetTrpSer 
245250255 
SerAlaLeuAsnSerLeuAsnLeuSerPheAlaGlyLeuGluGlnVal 
260265270 
ProLysGlyLeuProAlaLysLeuArgValLeuAspLeuSerCysAsn 
275280285 
ArgLeuAsnArgAlaProGlnProAspGluLeuProGluValAspAsn 
290295300 
LeuThrLeuAspGlyAsnProPheLeuValProGlyThrAlaLeuPro 
305310315320 
HisGluGlySerMetAsnSerGlyValValProAlaCysAlaArgSer 
325330335 
ThrLeuSerValGlyValSerGlyThrLeuValLeu 
340345 
(2) INFORMATION FOR SEQ ID NO:46: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..4 
(D) OTHER INFORMATION: /note= "Xaa at position 1 is Asp or 
Glu;Xaa at position 2 is Ala or Ser;Xaa at position 3 
is Asp or Glu;Xaa at position 4 is Pro or Gly" 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 5..8 
(D) OTHER INFORMATION: /note= "Xaa at position 5 is Arg or 
Lys;Xaa at position 6 is Gln,Asn or His;Xaa at position 
7 is Tyr, Trp or Phe;Xaa at position 8 is Ala or Ser" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
XaaXaaXaaXaaXaaXaaXaaXaa 
15 
(2) INFORMATION FOR SEQ ID NO:47: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..6 
(D) OTHER INFORMATION: /note= "Xaa at position 1 is Asp or 
Glu;Xaa at position 2 is Pro or Gly;Xaa at position 3 is 
Arg or Lys;Xaa at position 4 is Gln, Asn or His;Xaa at 
position 5 is Tyr,Trp or Phe;Xaa at position 6 is Ala 
or Ser" 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..7 
(D) OTHER INFORMATION: /note= "amino acids 1 and 7 are 
linked together by -CO-(CH2)n-S-, wherein n=1 to 3" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: 
XaaXaaXaaXaaXaaXaaCys 
15 
(2) INFORMATION FOR SEQ ID NO:48: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..4 
(D) OTHER INFORMATION: /note= "Xaa at position 1 is Ala or 
Ser;Xaa at position 2 is Asp or Glu;Xaa at position 3 
is Pro or Gly;Xaa at position 4 is Arg or Lys" 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 5..8 
(D) OTHER INFORMATION: /note= "Xaa at position 5 is 
Gln,Asn or His;Xaa at position 6 is Tyr, Trp or Phe;Xaa 
at 7 is Ala or Ser;Xaa at position 8 is Asp or Glu" 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..9 
(D) OTHER INFORMATION: /note= "amino acids 1 and 9 are 
linked together by -CO-(CH2)n-S-, wherein n= 1 to 3" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
XaaXaaXaaXaaXaaXaaXaaXaaCys 
15 
(2) INFORMATION FOR SEQ ID NO:49: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 1..5 
(D) OTHER INFORMATION: /note= "Xaa at position 1 is Asp or 
Glu;Xaa at position 2 is Ala or Ser;Xaa at position 3 is 
Asp or Glu;Xaa at position 4 is Pro or Gly;Xaa at 
position 5 is Arg or Lys " 
(ix) FEATURE: 
(A) NAME/KEY: Protein 
(B) LOCATION: 6..10 
(D) OTHER INFORMATION: /note= "Xaa at position 6 is Gln, 
Asn or His;Xaa at position 7 is Tyr, Trp or Phe;Xaa at 
position 8 is Ala or Ser;Xaa at position 9 is Asp or 
Glu;Xaa at position 10 is Thr or Ser" 
(ix) FEATURE: 
(A) NAME/KEY: Modified-site 
(B) LOCATION: 1..11 
(D) OTHER INFORMATION: /note= "amino acids 1 and 11 are 
linked together by -CO-(CH2)n-S-,wherein n = 1 to 3" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaCys 
1510 
__________________________________________________________________________